U.S. patent application number 17/525101 was filed with the patent office on 2022-03-03 for bispecific t cell activating antigen binding molecules.
The applicant listed for this patent is Roche Glycart AG. Invention is credited to Marina BACAC, Thomas HOFER, Ralf HOSSE, Christian KLEIN, Ekkehard MOESSNER, Christiane NEUMANN, Pablo UMANA, Tina WEINZIERL.
Application Number | 20220064296 17/525101 |
Document ID | / |
Family ID | 1000005960442 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064296 |
Kind Code |
A1 |
BACAC; Marina ; et
al. |
March 3, 2022 |
BISPECIFIC T CELL ACTIVATING ANTIGEN BINDING MOLECULES
Abstract
The present invention generally relates to novel bispecific
antigen binding molecules for T cell activation and re-direction to
specific target cells. In addition, the present invention relates
to polynucleotides encoding such bispecific antigen binding
molecules, and vectors and host cells comprising such
polynucleotides. The invention further relates to methods for
producing the bispecific antigen binding molecules of the
invention, and to methods of using these bispecific antigen binding
molecules in the treatment of disease.
Inventors: |
BACAC; Marina; (Zuerich,
CH) ; HOFER; Thomas; (Zuerich, CH) ; HOSSE;
Ralf; (Cham, CH) ; NEUMANN; Christiane;
(Niederweningen, CH) ; KLEIN; Christian;
(Bonstetten, CH) ; MOESSNER; Ekkehard;
(Kreuzlingen, CH) ; UMANA; Pablo; (Wollerau,
CH) ; WEINZIERL; Tina; (Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Glycart AG |
Schlieren |
|
CH |
|
|
Family ID: |
1000005960442 |
Appl. No.: |
17/525101 |
Filed: |
November 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17306068 |
May 3, 2021 |
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17525101 |
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17127291 |
Dec 18, 2020 |
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17306068 |
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16871573 |
May 11, 2020 |
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17127291 |
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16592565 |
Oct 3, 2019 |
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16871573 |
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16282708 |
Feb 22, 2019 |
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16592565 |
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16034012 |
Jul 12, 2018 |
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16282708 |
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14188486 |
Feb 24, 2014 |
10155815 |
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16034012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2809 20130101;
A61K 2039/505 20130101; C07K 16/3007 20130101; C07K 2317/94
20130101; C07K 2317/66 20130101; C07K 16/3053 20130101; C07K
2317/33 20130101; C07K 2317/55 20130101; C07K 2317/73 20130101;
C07K 2319/00 20130101; C07K 2317/71 20130101; C07K 2317/64
20130101; C07K 2317/526 20130101; C07K 2317/31 20130101; C07K
2317/53 20130101; C07K 2317/92 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 26, 2013 |
EP |
13156686.1 |
Claims
1. A T cell activating bispecific antigen binding molecule
comprising (i) a first antigen binding moiety which is a Fab
molecule capable of specific binding to CD3, comprising at least
one heavy chain complementarity determining region (CDR) selected
from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID
NO: 6 and at least one light chain CDR selected from the group of
SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; (ii) a second antigen
binding moiety which is a Fab molecule capable of specific binding
to a target cell antigen.
2. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the first antigen binding moiety comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO:
32 and SEQ ID NO: 33 and a light chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence selected from the
group of: SEQ ID NO: 7 and SEQ ID NO: 31.
3. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the first antigen binding moiety comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 3 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 7.
4. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the second antigen binding moiety is capable of
specific binding to Carcinoembryonic Antigen (CEA) and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25
and SEQ ID NO: 26 and at least one light chain CDR selected from
the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
5. The T cell activating bispecific antigen binding molecule of
claim 4, wherein the second antigen binding moiety comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 23 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 27.
6. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the second antigen binding moiety is capable of
specific binding to Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP) and comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least
one light chain CDR selected from the group of SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:
48, SEQ ID NO: 49 and SEQ ID NO: 50.
7. The T cell activating bispecific antigen binding molecule of
claim 6, wherein the second antigen binding moiety comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
8. The T cell activating bispecific antigen binding molecule of
claim 6, wherein the second antigen binding moiety is capable of
specific binding to MCSP and comprises a heavy chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence
selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO:
36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence
selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO:
46, SEQ ID NO: 47 and SEQ ID NO: 51.
9. The T cell activating bispecific antigen binding molecule of
claim 8, wherein the second antigen binding moiety comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 13 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 17.
10. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the first antigen binding moiety is a crossover
Fab molecule wherein either the variable or the constant regions of
the Fab light chain and the Fab heavy chain are exchanged.
11. The T cell activating bispecific antigen binding molecule of
claim 10, wherein the first antigen binding moiety is a crossover
Fab molecule wherein the constant regions of the Fab light chain
and the Fab heavy chain are exchanged.
12. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the second antigen binding moiety is a
conventional Fab molecule.
13. The T cell activating bispecific antigen binding molecule of
claim 1, comprising not more than one antigen binding moiety
capable of specific binding to CD3.
14. The T cell activating bispecific antigen binding molecule of
claim 1, comprising a third antigen binding moiety which is a Fab
molecule capable of specific binding to a target cell antigen.
15. The T cell activating bispecific antigen binding molecule of
claim 14, wherein the third antigen binding moiety is a
conventional Fab molecule.
16. The T cell activating bispecific antigen binding molecule of
claim 14, wherein the third antigen binding moiety is identical to
the second antigen binding moiety.
17. The T cell activating bispecific antigen binding molecule of
claim 16, wherein the third antigen binding moiety is an antigen
binding moiety capable of specific binding to CEA and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25
and SEQ ID NO: 26 and at least one light chain CDR selected from
the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
18. The T cell activating bispecific antigen binding molecule of
claim 16, wherein the third antigen binding moiety is an antigen
binding moiety capable of specific binding to MCSP and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ
ID NO: 40 and at least one light chain CDR selected from the group
of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ
ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
19. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the first and the second antigen binding moiety
are fused to each other, optionally via a peptide linker.
20. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first antigen binding moiety.
21. The T cell activating bispecific antigen binding molecule of
claim 1, wherein the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second antigen binding moiety.
22. The T cell activating bispecific antigen binding molecule of
claim 20, wherein the Fab light chain of the first antigen binding
moiety and the Fab light chain of the second antigen binding moiety
are fused to each other, optionally via a peptide linker.
23. The T cell activating bispecific antigen binding molecule of
claim 1, additionally comprising (iii) an Fc domain composed of a
first and a second subunit capable of stable association.
24. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
the second subunit of the Fc domain.
25. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
second subunit of the Fc domain.
26. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the first and the second antigen binding moiety
are each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain.
27. The T cell activating bispecific antigen binding molecule of
claim 23, wherein a third antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
second subunit of the Fc domain.
28. The T cell activating bispecific antigen binding molecule of
claim 27, wherein the second and the third antigen binding moiety
are each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety.
29. The T cell activating bispecific antigen binding molecule of
claim 27, wherein the first and the third antigen binding moiety
are each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain, and the second
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first antigen
binding moiety.
30. The T cell activating bispecific antigen binding molecule of
claim 29, wherein the first and the third antigen binding moiety
and the Fc domain are part of an immunoglobulin molecule,
particularly an IgG class immunoglobulin.
31. A T cell activating bispecific antigen binding molecule
comprising (i) a first antigen binding moiety which is a Fab
molecule capable of specific binding to CD3, comprising the heavy
chain complementarity determining region (CDR) 1 of SEQ ID NO: 4,
the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ
ID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain
CDR 2 of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to CEA comprising the heavy chain CDR 1 of SEQ ID NO: 24,
the heavy chain CDR 2 of SEQ ID NO: 25, the heavy chain CDR 3 of
SEQ ID NO: 26, the light chain CDR 1 of SEQ ID NO: 28, the light
chain CDR 2 of SEQ ID NO: 29 and the light chain CDR3 of SEQ ID NO:
30.
32. The T cell activating bispecific antigen binding molecule of
claim 31, comprising (i) a first antigen binding moiety which is a
Fab molecule capable of specific binding to CD3 comprising a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 3 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 7, wherein the first antigen binding moiety is a crossover
Fab molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to CEA comprising heavy chain variable region comprising an
amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to the amino acid sequence of SEQ ID NO: 23 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 27.
33. A T cell activating bispecific antigen binding molecule
comprising (i) a first antigen binding moiety which is a Fab
molecule capable of specific binding to CD3, comprising the heavy
chain complementarity determining region (CDR) 1 of SEQ ID NO: 4,
the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ
ID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain
CDR 2 of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to MCSP comprising the heavy chain CDR 1 of SEQ ID NO: 14,
the heavy chain CDR 2 of SEQ ID NO: 15, the heavy chain CDR 3 of
SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO: 18, the light
chain CDR 2 of SEQ ID NO: 19 and the light chain CDR3 of SEQ ID NO:
20.
34. The T cell activating bispecific antigen binding molecule of
claim 33, comprising (i) a first antigen binding moiety which is a
Fab molecule capable of specific binding to CD3 comprising a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 3 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 7, wherein the first antigen binding moiety is a crossover
Fab molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to MCSP comprising a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 13
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 17.
35. The T cell activating bispecific antigen binding molecule of
claim 31, further comprising (iii) an Fc domain composed of a first
and a second subunit capable of stable association, wherein the
second antigen binding moiety is fused at the C-terminus of the Fab
heavy chain to the N-terminus of the Fab heavy chain of the first
antigen binding moiety, and the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the first subunit of the Fc domain, and wherein the third antigen
binding moiety is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the second subunit of the Fc domain.
36. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the Fc domain is an IgG, specifically an
IgG.sub.1 or IgG.sub.4, Fc domain.
37. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the Fc domain is a human Fc domain.
38. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the Fc domain comprises a modification promoting
the association of the first and the second subunit of the Fc
domain.
39. The T cell activating bispecific antigen binding molecule of
claim 38, wherein in the CH3 domain of the first subunit of the Fc
domain an amino acid residue is replaced with an amino acid residue
having a larger side chain volume, thereby generating a
protuberance within the CH3 domain of the first subunit which is
positionable in a cavity within the CH3 domain of the second
subunit, and in the CH3 domain of the second subunit of the Fc
domain an amino acid residue is replaced with an amino acid residue
having a smaller side chain volume, thereby generating a cavity
within the CH3 domain of the second subunit within which the
protuberance within the CH3 domain of the first subunit is
positionable.
40. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the Fc domain exhibits reduced binding affinity
to an Fc receptor and/or reduced effector function, as compared to
a native IgG.sub.1 Fc domain.
41. The T cell activating bispecific antigen binding molecule of
claim 23, wherein the Fc domain comprises one or more amino acid
substitution that reduces binding to an Fc receptor and/or effector
function.
42. The T cell activating bispecific antigen binding molecule of
claim 41, wherein said one or more amino acid substitution is at
one or more position selected from the group of L234, L235, and
P329 (Kabat numbering).
43. The T cell activating bispecific antigen binding molecule of
claim 42, wherein each subunit of the Fc domain comprises three
amino acid substitutions that reduce binding to an activating Fc
receptor and/or effector function wherein said amino acid
substitutions are L234A, L235A and P329G.
44. The T cell activating bispecific antigen binding molecule of
claim 40, wherein the Fc receptor is an Fey receptor.
45. The T cell activating bispecific antigen binding molecule of
claim 40, wherein the effector function is antibody-dependent
cell-mediated cytotoxicity (ADCC).
46. An isolated polynucleotide encoding the T cell activating
bispecific antigen binding molecule of claim 1 or a fragment
thereof.
47. An isolated polynucleotide encoding the T cell activating
bispecific antigen binding molecule of claim 31 or a fragment
thereof.
48. An isolated polynucleotide encoding the T cell activating
bispecific antigen binding molecule of claim 33 or a fragment
thereof.
49. A polypeptide encoded by the polynucleotide of claim 46.
50. A vector, particularly an expression vector, comprising the
polynucleotide of claim 46.
51. A host cell comprising the vector of claim 50.
52. A method of producing the T cell activating bispecific antigen
binding molecule capable of specific binding to CD3 and a target
cell antigen, comprising the steps of a) culturing the host cell of
claim 51 under conditions suitable for the expression of the T cell
activating bispecific antigen binding molecule and b) recovering
the T cell activating bispecific antigen binding molecule.
53. A T cell activating bispecific antigen binding molecule
produced by the method of claim 52.
54. A pharmaceutical composition comprising the T cell activating
bispecific antigen binding molecule of claim 1 and a
pharmaceutically acceptable carrier.
55. A pharmaceutical composition comprising the T cell activating
bispecific antigen binding molecule of claim 31 and a
pharmaceutically acceptable carrier.
56. A pharmaceutical composition comprising the T cell activating
bispecific antigen binding molecule of claim 33 and a
pharmaceutically acceptable carrier.
57. A pharmaceutical composition comprising the T cell activating
bispecific antigen binding molecule of claim 53 and a
pharmaceutically acceptable carrier.
58. The T cell activating bispecific antigen binding molecule of
claim 1 for use as a medicament.
59. The T cell activating bispecific antigen binding molecule of
claim 31 for use as a medicament.
60. The T cell activating bispecific antigen binding molecule of
claim 33 for use as a medicament.
61. The T cell activating bispecific antigen binding molecule of
claim 53 for use as a medicament.
62. The T cell activating bispecific antigen binding molecule of
claim 1 for use in the treatment of a disease in an individual in
need thereof.
63. The T cell activating bispecific antigen binding molecule of
claim 62, wherein the disease is cancer.
64. The T cell activating bispecific antigen binding molecule of
claim 31 for use in the treatment of a disease in an individual in
need thereof.
65. The T cell activating bispecific antigen binding molecule of
claim 33 for use in the treatment of a disease in an individual in
need thereof.
66. The T cell activating bispecific antigen binding molecule of
claim 53 for use in the treatment of a disease in an individual in
need thereof.
67. A method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising the T cell activating bispecific
antigen binding molecule of claim 1 in a pharmaceutically
acceptable form.
68. The method of claim 67, wherein said disease is cancer.
69. A method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising the T cell activating bispecific
antigen binding molecule of claim 31 in a pharmaceutically
acceptable form.
70. A method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising the T cell activating bispecific
antigen binding molecule of claim 33 in a pharmaceutically
acceptable form.
71. A method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising the T cell activating bispecific
antigen binding molecule of claim 53 in a pharmaceutically
acceptable form.
72. A method for inducing lysis of a target cell, comprising
contacting a target cell with the T cell activating bispecific
antigen binding molecule of claim 1 in the presence of a T
cell.
73. A method for inducing lysis of a target cell, comprising
contacting a target cell with the T cell activating bispecific
antigen binding molecule of claim 31 in the presence of a T
cell.
74. A method for inducing lysis of a target cell, comprising
contacting a target cell with the T cell activating bispecific
antigen binding molecule of claim 33 in the presence of a T
cell.
75. A method for inducing lysis of a target cell, comprising
contacting a target cell with the T cell activating bispecific
antigen binding molecule of claim 53 in the presence of a T
cell.
76. The T cell activating bispecific antigen binding molecule of
claim 16, wherein the third antigen binding moiety is an antigen
binding moiety capable of specific binding to CEA and comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 23 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 27.
77. The T cell activating bispecific antigen binding molecule of
claim 16, wherein the third antigen binding moiety is an antigen
binding moiety capable of specific binding to MCSP and comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an
amino acid sequence selected from the group of SEQ ID NO: 13, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an
amino acid sequence selected from the group of SEQ ID NO: 17, SEQ
ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
78. The T cell activating bispecific antigen binding molecule of
claim 21, wherein the Fab light chain of the first antigen binding
moiety and the Fab light chain of the second antigen binding moiety
are fused to each other, optionally via a peptide linker.
79. A host cell comprising the polynucleotide of claim 46.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to bispecific
antigen binding molecules for activating T cells. In addition, the
present invention relates to polynucleotides encoding such
bispecific antigen binding molecules, and vectors and host cells
comprising such polynucleotides. The invention further relates to
methods for producing the bispecific antigen binding molecules of
the invention, and to methods of using these bispecific antigen
binding molecules in the treatment of disease.
BACKGROUND
[0002] The selective destruction of an individual cell or a
specific cell type is often desirable in a variety of clinical
settings. For example, it is a primary goal of cancer therapy to
specifically destroy tumor cells, while leaving healthy cells and
tissues intact and undamaged.
[0003] An attractive way of achieving this is by inducing an immune
response against the tumor, to make immune effector cells such as
natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack
and destroy tumor cells. CTLs constitute the most potent effector
cells of the immune system, however they cannot be activated by the
effector mechanism mediated by the Fc domain of conventional
therapeutic antibodies.
[0004] In this regard, bispecific antibodies designed to bind with
one "arm" to a surface antigen on target cells, and with the second
"arm" to an activating, invariant component of the T cell receptor
(TCR) complex, have become of interest in recent years. The
simultaneous binding of such an antibody to both of its targets
will force a temporary interaction between target cell and T cell,
causing activation of any cytotoxic T cell and subsequent lysis of
the target cell. Hence, the immune response is re-directed to the
target cells and is independent of peptide antigen presentation by
the target cell or the specificity of the T cell as would be
relevant for normal MHC-restricted activation of CTLs. In this
context it is crucial that CTLs are only activated when a target
cell is presenting the bispecific antibody to them, i.e. the
immunological synapse is mimicked. Particularly desirable are
bispecific antibodies that do not require lymphocyte
preconditioning or co-stimulation in order to elicit efficient
lysis of target cells.
[0005] Several bispecific antibody formats have been developed and
their suitability for T cell mediated immunotherapy investigated.
Out of these, the so-called BiTE (bispecific T cell engager)
molecules have been very well characterized and already shown some
promise in the clinic (reviewed in Nagorsen and Bauerle, Exp Cell
Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules wherein
two scFv molecules are fused by a flexible linker. Further
bispecific formats being evaluated for T cell engagement include
diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and
derivatives thereof, such as tandem diabodies (Kipriyanov et al., J
Mol Biol 293, 41-66 (1999)). A more recent development are the
so-called DART (dual affinity retargeting) molecules, which are
based on the diabody format but feature a C-terminal disulfide
bridge for additional stabilization (Moore et al., Blood 117,
4542-51 (2011)). The so-called triomabs, which are whole hybrid
mouse/rat IgG molecules and also currently being evaluated in
clinical trials, represent a larger sized format (reviewed in
Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). The variety
of formats that are being developed shows the great potential
attributed to T cell re-direction and activation in immunotherapy.
The task of generating bispecific antibodies suitable therefor is,
however, by no means trivial, but involves a number of challenges
that have to be met related to efficacy, toxicity, applicability
and produceability of the antibodies.
[0006] Small constructs such as, for example, BiTE molecules--while
being able to efficiently crosslink effector and target cells--have
a very short serum half life requiring them to be administered to
patients by continuous infusion. IgG-like formats on the other
hand--while having the great benefit of a long half life--suffer
from toxicity associated with the native effector functions
inherent to IgG molecules. Their immunogenic potential constitutes
another unfavorable feature of IgG-like bispecific antibodies,
especially non-human formats, for successful therapeutic
development. Finally, a major challenge in the general development
of bispecific antibodies has been the production of bispecific
antibody constructs at a clinically sufficient quantity and purity,
due to the mispairing of antibody heavy and light chains of
different specificities upon co-expression, which decreases the
yield of the correctly assembled construct and results in a number
of non-functional side products from which the desired bispecific
antibody may be difficult to separate. Given the difficulties and
disadvantages associated with currently available bispecific
antibodies for T cell mediated immunotherapy, there remains a need
for novel, improved formats of such molecules. The present
invention provides bispecific antigen binding molecules designed
for T cell activation and re-direction that combine good efficacy
and produceability with low toxicity and favorable pharmacokinetic
properties.
SUMMARY OF THE INVENTION
[0007] In a first aspect the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at
least one light chain CDR selected from the group of SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to a target
cell antigen.
[0008] In one embodiment the first antigen binding moiety which is
a Fab molecule capable of specific binding to CD3 comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO:
32 and SEQ ID NO: 33 and a variable light chain comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to an amino acid sequence selected from the group
of: SEQ ID NO: 7 and SEQ ID NO: 31.
[0009] In one embodiment the first antigen binding moiety which is
a Fab molecule capable of specific binding to CD3 comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 3 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 7. In a specific embodiment the second antigen binding
moiety is capable of specific binding to Carcinoembryonic Antigen
(CEA, CEACAM5) and comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at
least one light chain CDR selected from the group of SEQ ID NO: 28,
SEQ ID NO: 29 and SEQ ID NO: 30.
[0010] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to CEA and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 23 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 27.
[0011] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP (CSPG4) and comprises
at least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ
ID NO: 40 and at least one light chain CDR selected from the group
of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ
ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0012] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to Melanoma-associated
Chondroitin Sulfate Proteoglycan (MCSP, CSPG4) and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0013] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO:
34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO:
43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0014] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 13 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 17.
[0015] In a particular embodiment, the first antigen binding moiety
is a crossover Fab molecule wherein either the variable or the
constant regions of the Fab light chain and the Fab heavy chain are
exchanged. In an even more particular embodiment, the first antigen
binding moiety is a crossover Fab molecule wherein the constant
regions of the Fab light chain and the Fab heavy chain are
exchanged.
[0016] In one embodiment, the second antigen binding moiety is a
conventional Fab molecule.
[0017] In a further particular embodiment, not more than one
antigen binding moiety capable of specific binding to CD3 is
present in the T cell activating bispecific antigen binding
molecule (i.e. the T cell activating bispecific antigen binding
molecule provides monovalent binding to CD3).
[0018] In a further embodiment said T cell activating bispecific
antigen binding molecule further comprises a third antigen binding
moiety which is a Fab molecule capable of specific binding to a
target cell antigen. In one embodiment said third antigen binding
molecule is a conventional Fab molecule. In one embodiment said
third antigen binding molecule is identical to the second antigen
binding moiety.
[0019] In a particular embodiment said T cell activating bispecific
antigen binding molecule further comprises a third antigen binding
moiety which is a Fab molecule capable of specific binding to CEA,
and comprises at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 24,
SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR
selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID
NO: 30.
[0020] In a particular embodiment said T cell activating bispecific
antigen binding molecule further comprises a third antigen binding
moiety which is a Fab molecule capable of specific binding to CEA,
and comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 23 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 27.
[0021] In one embodiment said T cell activating bispecific antigen
binding molecule further comprises a third antigen binding moiety
which is a Fab molecule capable of specific binding to MCSP, and
comprises at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 38 and SEQ ID NO: 40 and at least one light chain CDR selected
from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ
ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID
NO: 50.
[0022] In a particular embodiment said T cell activating bispecific
antigen binding molecule further comprises a third antigen binding
moiety which is a Fab molecule capable of specific binding to MCSP,
and comprises at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 14,
SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR
selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID
NO: 20.
[0023] In one embodiment said T cell activating bispecific antigen
binding molecule further comprises a third antigen binding moiety
which is a Fab molecule capable of specific binding to MCSP, and
comprises a heavy chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to an amino acid sequence selected from the group of SEQ
ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID
NO: 41 and a light chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence selected from the group of SEQ
ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID
NO: 51.
[0024] In a particular embodiment said T cell activating bispecific
antigen binding molecule further comprises a third antigen binding
moiety which is a Fab molecule capable of specific binding to MCSP,
and comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 13 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 17.
[0025] In some embodiments, the first and the second antigen
binding moiety of the T cell activating bispecific antigen binding
molecule are fused to each other, optionally via a peptide linker.
In one such embodiment, the second antigen binding moiety is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the first antigen binding moiety. In another
such embodiment, the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second antigen binding moiety. In embodiments
wherein either (i) the second antigen binding moiety is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first antigen binding moiety or (ii) the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety, additionally the Fab light chain of the
first antigen binding moiety and the Fab light chain of the second
antigen binding moiety may be fused to each other, optionally via a
peptide linker.
[0026] In one embodiment said T cell activating bispecific antigen
binding molecule further comprises (iii) an Fc domain composed of a
first and a second subunit capable of stable association.
[0027] In one embodiment, the second antigen binding moiety of the
T cell activating bispecific antigen binding molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the
first or the second subunit of the Fc domain. In another
embodiment, the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
second subunit of the Fc domain. In one embodiment, the first and
the second antigen binding moiety of the T cell activating
bispecific antigen binding molecule are each fused at the
C-terminus of the Fab heavy chain to the N-terminus of one of the
subunits of the Fc domain.
[0028] In one embodiment, the third antigen binding moiety is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
first or second subunit of the Fc domain. In a particular
embodiment, the second and the third antigen binding moiety of the
T cell activating antigen binding molecule are each fused at the
C-terminus of the Fab heavy chain to the N-terminus of one of the
subunits of the Fc domain, and the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the second antigen binding moiety. In
another particular embodiment, the first and the third antigen
binding moiety of the T cell activating antigen binding molecule
are each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain, and the second
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first antigen
binding moiety. The components of the T cell activating bispecific
antigen binding molecule may be fused directly or through suitable
peptide linkers. In one embodiment the first and the third antigen
binding moiety and the Fc domain are part of an immunoglobulin
molecule.
[0029] In a particular embodiment the immunoglobulin molecule is an
IgG class immunoglobulin. In an even more particular embodiment the
immunoglobulin is an IgG.sub.1 subclass immunoglobulin. In another
embodiment, the immunoglobulin is an IgG.sub.4 subclass
immunoglobulin.
[0030] In a particular embodiment, the Fc domain is an IgG Fc
domain. In a specific embodiment, the Fc domain is an IgG.sub.1 Fc
domain. In another specific embodiment, the Fc domain is an
IgG.sub.4 Fc domain. In an even more specific embodiment, the Fc
domain is an IgG.sub.4 Fc domain comprising the amino acid
substitution S228P (Kabat numbering). In particular embodiments the
Fc domain is a human Fc domain.
[0031] In particular embodiments the Fc domain comprises a
modification promoting the association of the first and the second
Fc domain subunit. In a specific such embodiment, an amino acid
residue in the CH3 domain of the first subunit of the Fc domain is
replaced with an amino acid residue having a larger side chain
volume, thereby generating a protuberance within the CH3 domain of
the first subunit which is positionable in a cavity within the CH3
domain of the second subunit, and an amino acid residue in the CH3
domain of the second subunit of the Fc domain is replaced with an
amino acid residue having a smaller side chain volume, thereby
generating a cavity within the CH3 domain of the second subunit
within which the protuberance within the CH3 domain of the first
subunit is positionable.
[0032] In a particular embodiment the Fc domain exhibits reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain. In certain
embodiments the Fc domain is engineered to have reduced binding
affinity to an Fc receptor and/or reduced effector function, as
compared to a non-engineered Fc domain. In one embodiment, the Fc
domain comprises one or more amino acid substitution that reduces
binding to an Fc receptor and/or effector function. In one
embodiment, the one or more amino acid substitution in the Fc
domain that reduces binding to an Fc receptor and/or effector
function is at one or more position selected from the group of
L234, L235, and P329 (Kabat numbering). In particular embodiments,
each subunit of the Fc domain comprises three amino acid
substitutions that reduce binding to an Fc receptor and/or effector
function wherein said amino acid substitutions are L234A, L235A and
P329G. In one such embodiment, the Fc domain is an IgG.sub.1 Fc
domain, particularly a human IgG.sub.1 Fc domain. In other
embodiments, each subunit of the Fc domain comprises two amino acid
substitutions that reduce binding to an Fc receptor and/or effector
function wherein said amino acid substitutions are L235E and P329G.
In one such embodiment, the Fc domain is an IgG.sub.4 Fc domain,
particularly a human IgG.sub.4 Fc domain. In one embodiment, the Fc
domain of the T cell activating bispecific antigen binding molecule
is an IgG.sub.4 Fc domain and comprises the amino acid
substitutions L235E and S228P (SPLE).
[0033] In one embodiment the Fc receptor is an Fc.gamma. receptor.
In one embodiment the Fc receptor is a human Fc receptor. In one
embodiment, the Fc receptor is an activating Fc receptor. In a
specific embodiment, the Fc receptor is human Fc.gamma.RIIa,
Fc.gamma.RI, and/or Fc.gamma.RIIIa. In one embodiment, the effector
function is antibody-dependent cell-mediated cytotoxicity
(ADCC).
[0034] According to another aspect of the invention there is
provided an isolated polynucleotide encoding a T cell activating
bispecific antigen binding molecule of the invention or a fragment
thereof. The invention also encompasses polypeptides encoded by the
polynucleotides of the invention. The invention further provides an
expression vector comprising the isolated polynucleotide of the
invention, and a host cell comprising the isolated polynucleotide
or the expression vector of the invention. In some embodiments the
host cell is a eukaryotic cell, particularly a mammalian cell. In
another aspect is provided a method of producing the T cell
activating bispecific antigen binding molecule of the invention,
comprising the steps of a) culturing the host cell of the invention
under conditions suitable for the expression of the T cell
activating bispecific antigen binding molecule and b) recovering
the T cell activating bispecific antigen binding molecule. The
invention also encompasses a T cell activating bispecific antigen
binding molecule produced by the method of the invention.
[0035] The invention further provides a pharmaceutical composition
comprising the T cell activating bispecific antigen binding
molecule of the invention and a pharmaceutically acceptable
carrier.
[0036] Also encompassed by the invention are methods of using the T
cell activating bispecific antigen binding molecule and
pharmaceutical composition of the invention. In one aspect the
invention provides a T cell activating bispecific antigen binding
molecule or a pharmaceutical composition of the invention for use
as a medicament. In one aspect is provided a T cell activating
bispecific antigen binding molecule or a pharmaceutical composition
according to the invention for use in the treatment of a disease in
an individual in need thereof. In a specific embodiment the disease
is cancer.
[0037] Also provided is the use of a T cell activating bispecific
antigen binding molecule of the invention for the manufacture of a
medicament for the treatment of a disease in an individual in need
thereof; as well as a method of treating a disease in an
individual, comprising administering to said individual a
therapeutically effective amount of a composition comprising the T
cell activating bispecific antigen binding molecule according to
the invention in a pharmaceutically acceptable form. In a specific
embodiment the disease is cancer. In any of the above embodiments
the individual preferably is a mammal, particularly a human.
[0038] The invention also provides a method for inducing lysis of a
target cell, particularly a tumor cell, comprising contacting a
target cell with a T cell activating bispecific antigen binding
molecule of the invention in the presence of a T cell, particularly
a cytotoxic T cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1. Exemplary configurations of the T cell activating
bispecific antigen binding molecules (TCBs) of the invention. (A)
Illustration of the "1+1 IgG Crossfab" molecule. (B) Illustration
of the "2+1 IgG Crossfab" molecule. (C) Illustration of the "2+1
IgG Crossfab" molecule with alternative order of Crossfab and Fab
components ("inverted"). (D) Illustration of the "1+1 CrossMab"
molecule. (E) Illustration of the "2+1 IgG Crossfab, linked light
chain" molecule. (F) Illustration of the "1+1 IgG Crossfab, linked
light chain" molecule. (G) Illustration of the "2+1 IgG Crossfab,
inverted, linked light chain" molecule. (H) Illustration of the
"1+1 IgG Crossfab, inverted, linked light chain" molecule. Black
dot: optional modification in the Fc domain promoting
heterodimerization.
[0040] FIG. 2. Alignment of affinity matured anti-MCSP clones
compared to the non-matured parental clone (M4-3 ML2).
[0041] FIG. 3. Schematic drawing of the MCSP TCB (2+1 Crossfab-IgG
P329G LALA inverted) molecule.
[0042] FIG. 4. CE-SDS analyses of MCSP TCB (2+1 Crossfab-IgG P329G
LALA inverted, SEQ ID NOs: 12, 53, 54 and 55). Electropherogram
shown as SDS-Page of MCSP TCB: A) non reduced, B) reduced.
[0043] FIG. 5. Schematic drawing of CEA TCB (2+1 Crossfab-IgG P329G
LALA inverted) molecule.
[0044] FIG. 6. CE-SDS analyses of CEA TCB (2+1 Crossfab-IgG P329G
LALA inverted, SEQ ID NOs: 22, 56, 57 and 58) molecule.
Electropherogram shown as SDS-Page of CEA TCB: A) non reduced, B)
reduced.
[0045] FIG. 7. Binding of MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55)
to A375 cells (MCSP.sup.+) (A) and Jurkat (CD3.sup.+ cells) (B).
"Untargeted TCB": bispecific antibody engaging CD3 but no second
antigen (SEQ ID NOs:59, 60, 61 and 62).
[0046] FIG. 8. T-cell killing induced by MCSP TCB antibody (SEQ ID
NOs: 12, 53, 54 and 55) of A375 (high MCSP) (A), MV-3 (medium MCSP)
(B), HCT-116 (low MCSP) (C) and LS180 (MCSP negative) (D) target
cells (E:T=10:1, effectors human PBMCs, incubation time 24 h).
"Untargeted TCB": bispecific antibody engaging CD3 but no second
antigen (SEQ ID NOs:59, 60, 61 and 62).
[0047] FIG. 9. Upregulation of CD25 and CD69 on human CD8.sup.+ (A,
B) and CD4.sup.+ (C, D) T cells after T cell-mediated killing of
MV3 melanoma cells (E:T=10:1,24 h incubation) induced by MCSP TCB
antibody (SEQ ID NOs: 12, 53, 54 and 55). "Untargeted TCB":
bispecific antibody engaging CD3 but no second antigen (SEQ ID NOs:
59, 60, 61 and 62).
[0048] FIG. 10. Secretion of IL-2 (A), IFN-.gamma. (B), TNF.alpha.
(C), IL-4 (D), IL-10 (E) and Granzyme B (F) by human PBMCs after T
cell mediated killing of MV3 melanoma cells (E:T=10:1, 24 h
incubation) induced by MCSP TCB antibody (SEQ ID NOs: 12, 53, 54
and 55). "Untargeted TCB": bispecific antibody engaging CD3 but no
second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0049] FIG. 11. Binding of CEA TCB (SEQ ID NOs: 22, 56, 57 and 58)
to CEA-expressing A549 lung adenocarcinoma cells (A) and
CD3-expressing immortalized human and cynomolgus T lymphocyte lines
(Jurkat (B) and HSC-F (C), respectively).
[0050] FIG. 12. T-cell killing induced by CEA TCB (SEQ ID NOs: 22,
56, 57 and 58) of HPAFII (high CEA) (A, E), BxPC-3 (medium CEA) (B,
F), ASPC-1 (low CEA) (C, G) and HCT-116 cells (CEA negative) (D,
H). E:T=10:1, effectors human PBMCs, incubation time 24 h (A-D) or
48 h (E-H). "Untargeted TCB": bispecific antibody engaging CD3 but
no second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0051] FIG. 13. Human CD8.sup.+ and CD4.sup.+ T cell proliferation
(A-D) and upregulation of CD25 on human CD8.sup.+ and CD4 T cells
(E-H) 5 days after T cell-mediated killing of HPAFII (high CEA) (A,
E), BxPC-3 (medium CEA) (B, F), ASPC-1 (low CEA) (C, G) and HCT-116
cells (CEA negative) (D, H) induced by CEA TCB (SEQ ID NOs: 22, 56,
57 and 58). "DP47 TCB": bispecific antibody engaging CD3 but no
second antigen (SEQ ID NOs: 59, 60, 61 and 62).
[0052] FIG. 14. Secretion of IFN-.gamma. (A), TNF.alpha. (B),
Granzyme B (C), IL-2 (D), IL-6 (E) and IL-10 (F) after T cell
mediated killing of MKN45 tumor cells (E:T=10:1, 48 h incubation)
induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58). "Untargeted
TCB": bispecific antibody engaging CD3 but no second antigen (SEQ
ID NOs: 59, 60, 61 and 62).
[0053] FIG. 15. T cell-mediated killing of CEA-expressing LS180
tumor target cells induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and
58) in presence of increasing concentrations of shed CEA (sCEA),
detected 24 h (A) or 48 h (B) after incubation with the CEA TCB and
sCEA.
[0054] FIG. 16. T cell-mediated killing of A549 (lung
adenocarcinoma) cells overexpressing human CEA (A549-hCEA),
assessed 21 h (A, B) and 40 h (C, D) after incubation with CEA TCB
(SEQ ID NOs: 22, 56, 57 and 58) and human PBMCs (A, C) or
cynomolgus PBMCs (B, D) as effector cells.
[0055] FIG. 17. T cell-mediated killing of CEA-expressing human
colorectal cancer cell lines induced by CEA TCB (SEQ ID NOs: 22,
56, 57 and 58) at 0.8 nM (A), 4 nM (B) and 20 nM (C). (D)
correlation between CEA expression and % specific lysis at 20 nM of
CEA TCB, (E) correlation between CEA expression and EC.sub.50 of
CEA TCB.
[0056] FIG. 18. In vivo anti-tumor efficacy of CEA TCB (SEQ ID NOs:
22, 56, 57 and 58) in a LS174T-fluc2 human colon carcinoma
co-grafted with human PBMC (E:T ratio 5:1). Results show average
and SEM from 12 mice of tumor volume measured by caliper (A and C)
and by bioluminescence (Total Flux, B and D) in the different study
groups. (A, B) early treatment starting at day 1, (C, D) delayed
treatment starting at day 7. The MCSP TCB (SEQ ID NOs: 12, 53, 54
and 55) was used as negative control.
[0057] FIG. 19. In vivo anti-tumor efficacy of CEA TCB (SEQ ID NOs:
22, 56, 57 and 58) in a LS174T-fluc2 human colon carcinoma
co-grafted with human PBMC (E:T ratio 1:1). Results show average
and SEM from 10 mice of tumor volume measured by caliper (A) and by
bioluminescence (Total Flux, B) in the different study groups. The
MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55) was used as negative
control.
[0058] FIG. 20. In vivo efficacy of murinized CEA TCB in a
Panco2-huCEA orthotopic tumor model in immunocompetent huCD3c/huCEA
transgenic mice.
[0059] FIG. 21. Thermal stability of CEA TCB. Dynamic Light
Scattering measured in a temperature ramp from 25-75.degree. C. at
0.05.degree. C./min. Duplicate is shown in grey.
[0060] FIG. 22. Thermal stability of MCSP TCB. Dynamic Light
Scattering measured in a temperature ramp from 25-75.degree. C. at
0.05.degree. C./min. Duplicate is shown as grey line.
[0061] FIG. 23. T cell-mediated killing induced by MCSP TCB (SEQ ID
NOs: 12, 53, 54 and 55) and MCSP 1+1 CrossMab TCB antibodies of (A)
A375 (high MCSP), (B) MV-3 (medium MCSP) and (C) HCT-116 (low MCSP)
tumor target cells. (D) LS180 (MCSP negative tumor cell line) was
used as negative control. Tumor cell killing was assessed 24 h
(A-D) and 48 h (E-H) post incubation of target cells with the
antibodies and effector cells (human PBMCs).
[0062] FIG. 24. CD25 and CD69 upregulation on CD8.sup.+ and
CD4.sup.+ T cells after T-cell killing of MCSP-expressing tumor
cells (A375, A-D and MV-3, E-H) mediated by the MCSP TCB (SEQ ID
NOs: 12, 53, 54 and 55) and MCSP 1+1 CrossMab TCB antibodies.
[0063] FIG. 25. CE-SDS analyses of DP47 GS TCB (2+1 Crossfab-IgG
P329G LALA inverted="Untargeted TCB" SEQ ID NOs: 59, 60, 61 and 62)
containing DP47 GS as non binding antibody and humanized CH2527 as
anti CD3 antibody. Electropherogram shown as SDS-PAGE of DP47 GS
TCB: A) non reduced, B) reduced.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0064] Terms are used herein as generally used in the art, unless
otherwise defined in the following.
[0065] As used herein, the term "antigen binding molecule" refers
in its broadest sense to a molecule that specifically binds an
antigenic determinant. Examples of antigen binding molecules are
immunoglobulins and derivatives, e.g. fragments, thereof.
[0066] The term "bispecific" means that the antigen binding
molecule is able to specifically bind to at least two distinct
antigenic determinants. Typically, a bispecific antigen binding
molecule comprises two antigen binding sites, each of which is
specific for a different antigenic determinant. In certain
embodiments the bispecific antigen binding molecule is capable of
simultaneously binding two antigenic determinants, particularly two
antigenic determinants expressed on two distinct cells.
[0067] The term "valent" as used herein denotes the presence of a
specified number of antigen binding sites in an antigen binding
molecule. As such, the term "monovalent binding to an antigen"
denotes the presence of one (and not more than one) antigen binding
site specific for the antigen in the antigen binding molecule.
[0068] An "antigen binding site" refers to the site, i.e. one or
more amino acid residues, of an antigen binding molecule which
provides interaction with the antigen. For example, the antigen
binding site of an antibody comprises amino acid residues from the
complementarity determining regions (CDRs). A native immunoglobulin
molecule typically has two antigen binding sites, a Fab molecule
typically has a single antigen binding site.
[0069] As used herein, the term "antigen binding moiety" refers to
a polypeptide molecule that specifically binds to an antigenic
determinant. In one embodiment, an antigen binding moiety is able
to direct the entity to which it is attached (e.g. a second antigen
binding moiety) to a target site, for example to a specific type of
tumor cell or tumor stroma bearing the antigenic determinant. In
another embodiment an antigen binding moiety is able to activate
signaling through its target antigen, for example a T cell receptor
complex antigen. Antigen binding moieties include antibodies and
fragments thereof as further defined herein. Particular antigen
binding moieties include an antigen binding domain of an antibody,
comprising an antibody heavy chain variable region and an antibody
light chain variable region. In certain embodiments, the antigen
binding moieties may comprise antibody constant regions as further
defined herein and known in the art. Useful heavy chain constant
regions include any of the five isotypes: .alpha., .delta.,
.epsilon., .gamma., or .mu.. Useful light chain constant regions
include any of the two isotypes: .kappa. and .lamda..
[0070] As used herein, the term "antigenic determinant" is
synonymous with "antigen" and "epitope," and refers to a site (e.g.
a contiguous stretch of amino acids or a conformational
configuration made up of different regions of non-contiguous amino
acids) on a polypeptide macromolecule to which an antigen binding
moiety binds, forming an antigen binding moiety-antigen complex.
Useful antigenic determinants can be found, for example, on the
surfaces of tumor cells, on the surfaces of virus-infected cells,
on the surfaces of other diseased cells, on the surface of immune
cells, free in blood serum, and/or in the extracellular matrix
(ECM). The proteins referred to as antigens herein (e.g. MCSP, CEA,
CD3) can be any native form the proteins from any vertebrate
source, including mammals such as primates (e.g. humans) and
rodents (e.g. mice and rats), unless otherwise indicated. In a
particular embodiment the antigen is a human protein. Where
reference is made to a specific protein herein, the term
encompasses the "full-length", unprocessed protein as well as any
form of the protein that results from processing in the cell. The
term also encompasses naturally occurring variants of the protein,
e.g. splice variants or allelic variants. Exemplary human proteins
useful as antigens include, but are not limited to:
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), also
known as Chondroitin Sulfate Proteoglycan 4 (CSPG4, UniProt no.
Q6UVK1 (version 70), NCBI RefSeq no. NP_001888.2); Carcinoembroynic
antigen (CEA), also known as Carcinoembryonic antigen-related cell
adhesion molecule 5 (CEACAM5, UniProt no. P06731 (version 119),
NCBI RefSeq no. NP_004354.2); and CD3, particularly the epsilon
subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq
no. NP_000724.1, SEQ ID NO: 103 for the human sequence; or UniProt
no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO:
104 for the cynomolgus [Macaca fascicularis] sequence). In certain
embodiments the T cell activating bispecific antigen binding
molecule of the invention binds to an epitope of CD3 or a target
cell antigen that is conserved among the CD3 or target antigen from
different species. In certain embodiments the T cell activating
bispecific antigen binding molecule of the invention binds to CD3
and CEACAM5, but does not bind to CEACAM1 or CEACAM6. By "specific
binding" is meant that the binding is selective for the antigen and
can be discriminated from unwanted or non-specific interactions.
The ability of an antigen binding moiety to bind to a specific
antigenic determinant can be measured either through an
enzyme-linked immunosorbent assay (ELISA) or other techniques
familiar to one of skill in the art, e.g. surface plasmon resonance
(SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et
al., Glyco J 17, 323-329 (2000)), and traditional binding assays
(Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the
extent of binding of an antigen binding moiety to an unrelated
protein is less than about 10% of the binding of the antigen
binding moiety to the antigen as measured, e.g., by SPR. In certain
embodiments, an antigen binding moiety that binds to the antigen,
or an antigen binding molecule comprising that antigen binding
moiety, has a dissociation constant (K.sub.D) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g.
from 10.sup.-8M to 10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13
M).
[0071] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., a receptor) and its binding partner (e.g., a
ligand). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., an antigen
binding moiety and an antigen, or a receptor and its ligand). The
affinity of a molecule X for its partner Y can generally be
represented by the dissociation constant (K.sub.D), which is the
ratio of dissociation and association rate constants (k.sub.off and
k.sub.on, respectively). Thus, equivalent affinities may comprise
different rate constants, as long as the ratio of the rate
constants remains the same. Affinity can be measured by well
established methods known in the art, including those described
herein. A particular method for measuring affinity is Surface
Plasmon Resonance (SPR).
[0072] "Reduced binding", for example reduced binding to an Fc
receptor, refers to a decrease in affinity for the respective
interaction, as measured for example by SPR. For clarity the term
includes also reduction of the affinity to zero (or below the
detection limit of the analytic method), i.e. complete abolishment
of the interaction. Conversely, "increased binding" refers to an
increase in binding affinity for the respective interaction.
[0073] "T cell activation" as used herein refers to one or more
cellular response of a T lymphocyte, particularly a cytotoxic T
lymphocyte, selected from: proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. The T cell activating
bispecific antigen binding molecules of the invention are capable
of inducing T cell activation. Suitable assays to measure T cell
activation are known in the art described herein.
[0074] A "target cell antigen" as used herein refers to an
antigenic determinant presented on the surface of a target cell,
for example a cell in a tumor such as a cancer cell or a cell of
the tumor stroma.
[0075] As used herein, the terms "first" and "second" with respect
to antigen binding moieties etc., are used for convenience of
distinguishing when there is more than one of each type of moiety.
Use of these terms is not intended to confer a specific order or
orientation of the T cell activating bispecific antigen binding
molecule unless explicitly so stated.
[0076] A "Fab molecule" refers to a protein consisting of the VH
and CH1 domain of the heavy chain (the "Fab heavy chain") and the
VL and CL domain of the light chain (the "Fab light chain") of an
immunoglobulin.
[0077] By "fused" is meant that the components (e.g. a Fab molecule
and an Fc domain subunit) are linked by peptide bonds, either
directly or via one or more peptide linkers.
[0078] As used herein, the term "single-chain" refers to a molecule
comprising amino acid monomers linearly linked by peptide bonds. In
certain embodiments, one of the antigen binding moieties is a
single-chain Fab molecule, i.e. a Fab molecule wherein the Fab
light chain and the Fab heavy chain are connected by a peptide
linker to form a single peptide chain. In a particular such
embodiment, the C-terminus of the Fab light chain is connected to
the N-terminus of the Fab heavy chain in the single-chain Fab
molecule.
[0079] By a "crossover" Fab molecule (also termed "Crossfab") is
meant a Fab molecule wherein either the variable regions or the
constant regions of the Fab heavy and light chain are exchanged,
i.e. the crossover Fab molecule comprises a peptide chain composed
of the light chain variable region and the heavy chain constant
region, and a peptide chain composed of the heavy chain variable
region and the light chain constant region. For clarity, in a
crossover Fab molecule wherein the variable regions of the Fab
light chain and the Fab heavy chain are exchanged, the peptide
chain comprising the heavy chain constant region is referred to
herein as the "heavy chain" of the crossover Fab molecule.
Conversely, in a crossover Fab molecule wherein the constant
regions of the Fab light chain and the Fab heavy chain are
exchanged, the peptide chain comprising the heavy chain variable
region is referred to herein as the "heavy chain" of the crossover
Fab molecule.
[0080] In contrast thereto, by a "conventional" Fab molecule is
meant a Fab molecule in its natural format, i.e. comprising a heavy
chain composed of the heavy chain variable and constant regions
(VH-CH1), and a light chain composed of the light chain variable
and constant regions (VL-CL).
[0081] The term "immunoglobulin molecule" refers to a protein
having the structure of a naturally occurring antibody. For
example, immunoglobulins of the IgG class are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light
chains and two heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3), also called
a heavy chain constant region. Similarly, from N- to C-terminus,
each light chain has a variable region (VL), also called a variable
light domain or a light chain variable domain, followed by a
constant light (CL) domain, also called a light chain constant
region. The heavy chain of an immunoglobulin may be assigned to one
of five types, called .alpha. (IgA), .delta. (IgD), .epsilon.
(IgE), .gamma. (IgG), or .mu. (IgM), some of which may be further
divided into subtypes, e.g. .gamma..sub.1 (IgG.sub.1),
.gamma..sub.2 (IgG.sub.2), .gamma..sub.3 (IgG.sub.3), .gamma..sub.4
(IgG.sub.4), .alpha..sub.1 (IgA.sub.1) and .alpha..sub.2
(IgA.sub.2). The light chain of an immunoglobulin may be assigned
to one of two types, called kappa (.kappa.) and lambda (.lamda.),
based on the amino acid sequence of its constant domain. An
immunoglobulin essentially consists of two Fab molecules and an Fc
domain, linked via the immunoglobulin hinge region.
[0082] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, and antibody
fragments so long as they exhibit the desired antigen-binding
activity.
[0083] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2, diabodies, linear antibodies, single-chain
antibody molecules (e.g. scFv), and single-domain antibodies. For a
review of certain antibody fragments, see Hudson et al., Nat Med 9,
129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun,
in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody
fragments with two antigen-binding sites that may be bivalent or
bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et
al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl
Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are
also described in Hudson et al., Nat Med 9, 129-134 (2003).
Single-domain antibodies are antibody fragments comprising all or a
portion of the heavy chain variable domain or all or a portion of
the light chain variable domain of an antibody. In certain
embodiments, a single-domain antibody is a human single-domain
antibody (Domantis, Inc., Waltham, Mass.; see e.g. U.S. Pat. No.
6,248,516 B 1). Antibody fragments can be made by various
techniques, including but not limited to proteolytic digestion of
an intact antibody as well as production by recombinant host cells
(e.g. E. coli or phage), as described herein.
[0084] The term "antigen binding domain" refers to the part of an
antibody that comprises the area which specifically binds to and is
complementary to part or all of an antigen. An antigen binding
domain may be provided by, for example, one or more antibody
variable domains (also called antibody variable regions).
Particularly, an antigen binding domain comprises an antibody light
chain variable region (VL) and an antibody heavy chain variable
region (VH).
[0085] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007). A
single VH or VL domain may be sufficient to confer antigen-binding
specificity.
[0086] The term "hypervariable region" or "HVR", as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
complementarity determining regions (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. With the exception of CDR1 in VH, CDRs generally
comprise the amino acid residues that form the hypervariable loops.
Hypervariable regions (HVRs) are also referred to as
"complementarity determining regions" (CDRs), and these terms are
used herein interchangeably in reference to portions of the
variable region that form the antigen binding regions. This
particular region has been described by Kabat et al., U.S. Dept. of
Health and Human Services, Sequences of Proteins of Immunological
Interest (1983) and by Chothia et al., J Mol Biol 196:901-917
(1987), where the definitions include overlapping or subsets of
amino acid residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table A as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00001 TABLE A CDR Definitions.sup.1 CDR Kabat Chothia
AbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58
50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-32
24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97
.sup.1Numbering of all CDR definitions in Table A is according to
the numbering conventions set forth by Kabat et al. (see below).
.sup.2"AbM" with a lowercase "b" as used in Table A refers to the
CDRs as defined by Oxford Molecular's "AbM" antibody modeling
software.
[0087] Kabat et al. also defined a numbering system for variable
region sequences that is applicable to any antibody. One of
ordinary skill in the art can unambiguously assign this system of
"Kabat numbering" to any variable region sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an antibody variable region are according to
the Kabat numbering system.
[0088] The polypeptide sequences of the sequence listing are not
numbered according to the Kabat numbering system. However, it is
well within the ordinary skill of one in the art to convert the
numbering of the sequences of the Sequence Listing to Kabat
numbering.
[0089] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3
(L3)-FR4.
[0090] The "class" of an antibody or immunoglobulin refers to the
type of constant domain or constant region possessed by its heavy
chain. There are five major classes of antibodies: IgA, IgD, IgE,
IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively.
[0091] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an immunoglobulin heavy chain that contains
at least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. Although the boundaries
of the Fc region of an IgG heavy chain might vary slightly, the
human IgG heavy chain Fc region is usually defined to extend from
Cys226, or from Pro230, to the carboxyl-terminus of the heavy
chain. However, the C-terminal lysine (Lys447) of the Fc region may
or may not be present. Unless otherwise specified herein, numbering
of amino acid residues in the Fc region or constant region is
according to the EU numbering system, also called the EU index, as
described in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md., 1991. A "subunit" of an Fc domain as used
herein refers to one of the two polypeptides forming the dimeric Fc
domain, i.e. a polypeptide comprising C-terminal constant regions
of an immunoglobulin heavy chain, capable of stable
self-association. For example, a subunit of an IgG Fc domain
comprises an IgG CH2 and an IgG CH3 constant domain.
[0092] A "modification promoting the association of the first and
the second subunit of the Fc domain" is a manipulation of the
peptide backbone or the post-translational modifications of an Fc
domain subunit that reduces or prevents the association of a
polypeptide comprising the Fc domain subunit with an identical
polypeptide to form a homodimer. A modification promoting
association as used herein particularly includes separate
modifications made to each of the two Fc domain subunits desired to
associate (i.e. the first and the second subunit of the Fc domain),
wherein the modifications are complementary to each other so as to
promote association of the two Fc domain subunits. For example, a
modification promoting association may alter the structure or
charge of one or both of the Fc domain subunits so as to make their
association sterically or electrostatically favorable,
respectively. Thus, (hetero)dimerization occurs between a
polypeptide comprising the first Fc domain subunit and a
polypeptide comprising the second Fc domain subunit, which might be
non-identical in the sense that further components fused to each of
the subunits (e.g. antigen binding moieties) are not the same. In
some embodiments the modification promoting association comprises
an amino acid mutation in the Fc domain, specifically an amino acid
substitution. In a particular embodiment, the modification
promoting association comprises a separate amino acid mutation,
specifically an amino acid substitution, in each of the two
subunits of the Fc domain.
[0093] The term "effector functions" refers to those biological
activities attributable to the Fc region of an antibody, which vary
with the antibody isotype. Examples of antibody effector functions
include: C1q binding and complement dependent cytotoxicity (CDC),
Fc receptor binding, antibody-dependent cell-mediated cytotoxicity
(ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine
secretion, immune complex-mediated antigen uptake by antigen
presenting cells, down regulation of cell surface receptors (e.g. B
cell receptor), and B cell activation.
[0094] As used herein, the terms "engineer, engineered,
engineering", are considered to include any manipulation of the
peptide backbone or the post-translational modifications of a
naturally occurring or recombinant polypeptide or fragment thereof.
Engineering includes modifications of the amino acid sequence, of
the glycosylation pattern, or of the side chain group of individual
amino acids, as well as combinations of these approaches.
[0095] The term "amino acid mutation" as used herein is meant to
encompass amino acid substitutions, deletions, insertions, and
modifications. Any combination of substitution, deletion,
insertion, and modification can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., reduced binding to an Fc receptor, or
increased association with another peptide. Amino acid sequence
deletions and insertions include amino- and/or carboxy-terminal
deletions and insertions of amino acids. Particular amino acid
mutations are amino acid substitutions. For the purpose of altering
e.g. the binding characteristics of an Fc region, non-conservative
amino acid substitutions, i.e. replacing one amino acid with
another amino acid having different structural and/or chemical
properties, are particularly preferred. Amino acid substitutions
include replacement by non-naturally occurring amino acids or by
naturally occurring amino acid derivatives of the twenty standard
amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine,
homoserine, 5-hydroxylysine). Amino acid mutations can be generated
using genetic or chemical methods well known in the art. Genetic
methods may include site-directed mutagenesis, PCR, gene synthesis
and the like. It is contemplated that methods of altering the side
chain group of an amino acid by methods other than genetic
engineering, such as chemical modification, may also be useful.
Various designations may be used herein to indicate the same amino
acid mutation. For example, a substitution from proline at position
329 of the Fc domain to glycine can be indicated as 329G, G329,
G329, P329G, or Pro329Gly.
[0096] As used herein, term "polypeptide" refers to a molecule
composed of monomers (amino acids) linearly linked by amide bonds
(also known as peptide bonds). The term "polypeptide" refers to any
chain of two or more amino acids, and does not refer to a specific
length of the product. Thus, peptides, dipeptides, tripeptides,
oligopeptides, "protein," "amino acid chain," or any other term
used to refer to a chain of two or more amino acids, are included
within the definition of "polypeptide," and the term "polypeptide"
may be used instead of, or interchangeably with any of these terms.
The term "polypeptide" is also intended to refer to the products of
post-expression modifications of the polypeptide, including without
limitation glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino acids. A
polypeptide may be derived from a natural biological source or
produced by recombinant technology, but is not necessarily
translated from a designated nucleic acid sequence. It may be
generated in any manner, including by chemical synthesis. A
polypeptide of the invention may be of a size of about 3 or more, 5
or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or
more, 100 or more, 200 or more, 500 or more, 1,000 or more, or
2,000 or more amino acids. Polypeptides may have a defined
three-dimensional structure, although they do not necessarily have
such structure. Polypeptides with a defined three-dimensional
structure are referred to as folded, and polypeptides which do not
possess a defined three-dimensional structure, but rather can adopt
a large number of different conformations, and are referred to as
unfolded.
[0097] By an "isolated" polypeptide or a variant, or derivative
thereof is intended a polypeptide that is not in its natural
milieu. No particular level of purification is required. For
example, an isolated polypeptide can be removed from its native or
natural environment. Recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated for the
purpose of the invention, as are native or recombinant polypeptides
which have been separated, fractionated, or partially or
substantially purified by any suitable technique.
[0098] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary. In situations where ALIGN-2 is employed
for amino acid sequence comparisons, the % amino acid sequence
identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which can alternatively be phrased as
a given amino acid sequence A that has or comprises a certain %
amino acid sequence identity to, with, or against a given amino
acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0099] The term "polynucleotide" refers to an isolated nucleic acid
molecule or construct, e.g. messenger RNA (mRNA), virally-derived
RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a
conventional phosphodiester bond or a non-conventional bond (e.g.
an amide bond, such as found in peptide nucleic acids (PNA). The
term "nucleic acid molecule" refers to any one or more nucleic acid
segments, e.g. DNA or RNA fragments, present in a
polynucleotide.
[0100] By "isolated" nucleic acid molecule or polynucleotide is
intended a nucleic acid molecule, DNA or RNA, which has been
removed from its native environment. For example, a recombinant
polynucleotide encoding a polypeptide contained in a vector is
considered isolated for the purposes of the present invention.
Further examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. An
isolated polynucleotide includes a polynucleotide molecule
contained in cells that ordinarily contain the polynucleotide
molecule, but the polynucleotide molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of the present
invention, as well as positive and negative strand forms, and
double-stranded forms. Isolated polynucleotides or nucleic acids
according to the present invention further include such molecules
produced synthetically. In addition, a polynucleotide or a nucleic
acid may be or may include a regulatory element such as a promoter,
ribosome binding site, or a transcription terminator.
[0101] By a nucleic acid or polynucleotide having a nucleotide
sequence at least, for example, 95% "identical" to a reference
nucleotide sequence of the present invention, it is intended that
the nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence. As a practical matter, whether any particular
polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence of the present
invention can be determined conventionally using known computer
programs, such as the ones discussed above for polypeptides (e.g.
ALIGN-2).
[0102] The term "expression cassette" refers to a polynucleotide
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a target cell. The recombinant
expression cassette can be incorporated into a plasmid, chromosome,
mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
Typically, the recombinant expression cassette portion of an
expression vector includes, among other sequences, a nucleic acid
sequence to be transcribed and a promoter. In certain embodiments,
the expression cassette of the invention comprises polynucleotide
sequences that encode bispecific antigen binding molecules of the
invention or fragments thereof.
[0103] The term "vector" or "expression vector" is synonymous with
"expression construct" and refers to a DNA molecule that is used to
introduce and direct the expression of a specific gene to which it
is operably associated in a target cell. The term includes the
vector as a self-replicating nucleic acid structure as well as the
vector incorporated into the genome of a host cell into which it
has been introduced. The expression vector of the present invention
comprises an expression cassette. Expression vectors allow
transcription of large amounts of stable mRNA. Once the expression
vector is inside the target cell, the ribonucleic acid molecule or
protein that is encoded by the gene is produced by the cellular
transcription and/or translation machinery. In one embodiment, the
expression vector of the invention comprises an expression cassette
that comprises polynucleotide sequences that encode bispecific
antigen binding molecules of the invention or fragments thereof.
The terms "host cell", "host cell line," and "host cell culture"
are used interchangeably and refer to cells into which exogenous
nucleic acid has been introduced, including the progeny of such
cells. Host cells include "transformants" and "transformed cells,"
which include the primary transformed cell and progeny derived
therefrom without regard to the number of passages. Progeny may not
be completely identical in nucleic acid content to a parent cell,
but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. A host cell is any
type of cellular system that can be used to generate the bispecific
antigen binding molecules of the present invention. Host cells
include cultured cells, e.g. mammalian cultured cells, such as CHO
cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63
mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells,
yeast cells, insect cells, and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue.
[0104] An "activating Fc receptor" is an Fc receptor that following
engagement by an Fc domain of an antibody elicits signaling events
that stimulate the receptor-bearing cell to perform effector
functions. Human activating Fc receptors include Fc.gamma.RIIIa
(CD16a), Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI
(CD89).
[0105] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an
immune mechanism leading to the lysis of antibody-coated target
cells by immune effector cells. The target cells are cells to which
antibodies or derivatives thereof comprising an Fc region
specifically bind, generally via the protein part that is
N-terminal to the Fc region. As used herein, the term "reduced
ADCC" is defined as either a reduction in the number of target
cells that are lysed in a given time, at a given concentration of
antibody in the medium surrounding the target cells, by the
mechanism of ADCC defined above, and/or an increase in the
concentration of antibody in the medium surrounding the target
cells, required to achieve the lysis of a given number of target
cells in a given time, by the mechanism of ADCC. The reduction in
ADCC is relative to the ADCC mediated by the same antibody produced
by the same type of host cells, using the same standard production,
purification, formulation and storage methods (which are known to
those skilled in the art), but that has not been engineered. For
example the reduction in ADCC mediated by an antibody comprising in
its Fc domain an amino acid substitution that reduces ADCC, is
relative to the ADCC mediated by the same antibody without this
amino acid substitution in the Fc domain. Suitable assays to
measure ADCC are well known in the art (see e.g. PCT publication
no. WO 2006/082515 or PCT publication no. WO 2012/130831).
[0106] An "effective amount" of an agent refers to the amount that
is necessary to result in a physiological change in the cell or
tissue to which it is administered.
[0107] A "therapeutically effective amount" of an agent, e.g. a
pharmaceutical composition, refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result. A therapeutically effective
amount of an agent for example eliminates, decreases, delays,
minimizes or prevents adverse effects of a disease.
[0108] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g. humans and non-human
primates such as monkeys), rabbits, and rodents (e.g. mice and
rats). Particularly, the individual or subject is a human.
[0109] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0110] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical composition, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0111] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of a disease
in the individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, T cell
activating bispecific antigen binding molecules of the invention
are used to delay development of a disease or to slow the
progression of a disease.
[0112] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0113] In a first aspect the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, and which comprises at least one heavy
chain complementarity determining region (CDR) selected from the
group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and
at least one light chain CDR selected from the group of SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10; (ii) a second antigen binding
moiety which is a Fab molecule capable of specific binding to a
target cell antigen.
[0114] In one embodiment the first antigen binding moiety comprises
a heavy chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
an amino acid sequence selected from the group of: SEQ ID NO: 3,
SEQ ID NO: 32 and SEQ ID NO: 33 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to an amino acid sequence selected
from the group of: SEQ ID NO: 7 and SEQ ID NO: 31.
[0115] In one embodiment the first antigen binding moiety comprises
a heavy chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 3 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 7.
[0116] In a specific embodiment the second antigen binding moiety
is capable of specific binding to CEA and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO:
26 and at least one light chain CDR selected from the group of SEQ
ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0117] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to CEA and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 23 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 27.
[0118] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ
ID NO: 40 and at least one light chain CDR selected from the group
of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ
ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0119] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
[0120] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO:
34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino
acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO:
43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0121] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to MCSP and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 13 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 17.
[0122] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at
least one light chain CDR selected from the group of SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to CEA
comprising at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 24,
SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR
selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID
NO: 30.
[0123] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 3 and a light chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 7, (ii) a second
antigen binding moiety which is a Fab molecule capable of specific
binding to CEA comprising heavy chain variable region comprising an
amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to the amino acid sequence of SEQ ID NO: 23 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 27.
[0124] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at
least one light chain CDR selected from the group of SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to MCSP
comprising at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 14,
SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR
selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID
NO: 20.
[0125] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 3 and a light chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 7. (ii) a second
antigen binding moiety which is a Fab molecule capable of specific
binding to MCSP comprising a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 13
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 17.
[0126] In a particular embodiment, the first antigen binding moiety
is a crossover Fab molecule wherein either the variable or the
constant regions of the Fab light chain and the Fab heavy chain are
exchanged.
[0127] In one embodiment, the second antigen binding moiety is a
conventional Fab molecule.
[0128] In a particular embodiment, the first antigen binding moiety
is a crossover Fab molecule wherein the constant regions of the Fab
light chain and the Fab heavy chain are exchanged, and the second
antigen binding moiety is a conventional Fab molecule. In a further
particular embodiment, the first and the second antigen binding
moiety are fused to each other, optionally through a peptide
linker.
[0129] In particular embodiments, the T cell activating bispecific
antigen binding molecule further comprises an Fc domain composed of
a first and a second subunit capable of stable association.
[0130] In a further particular embodiment, not more than one
antigen binding moiety capable of specific binding to CD3 is
present in the T cell activating bispecific antigen binding
molecule (i.e. the T cell activating bispecific antigen binding
molecule provides monovalent binding to CD3).
T Cell Activating Bispecific Antigen Binding Molecule Formats
[0131] The components of the T cell activating bispecific antigen
binding molecule can be fused to each other in a variety of
configurations. Exemplary configurations are depicted in FIGS. 1, 3
and 5.
[0132] In particular embodiments, the T cell activating bispecific
antigen binding molecule comprises an Fc domain composed of a first
and a second subunit capable of stable association. In some
embodiments, the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
the second subunit of the Fc domain.
[0133] In one such embodiment, the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the second antigen binding moiety. In a
specific such embodiment, the T cell activating bispecific antigen
binding molecule essentially consists of a first and a second
antigen binding moiety, an Fc domain composed of a first and a
second subunit, and optionally one or more peptide linkers, wherein
the first antigen binding moiety is fused at the C-terminus of the
Fab heavy chain to the N-terminus of the Fab heavy chain of the
second antigen binding moiety, and the second antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the first or the second subunit of the Fc domain.
Optionally, the Fab light chain of the first antigen binding moiety
and the Fab light chain of the second antigen binding moiety may
additionally be fused to each other.
[0134] In another such embodiment, the first antigen binding moiety
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the first or second subunit of the Fc domain. In a specific such
embodiment, the T cell activating bispecific antigen binding
molecule essentially consists of a first and a second antigen
binding moiety, an Fc domain composed of a first and a second
subunit, and optionally one or more peptide linkers, wherein the
first and the second antigen binding moiety are each fused at the
C-terminus of the Fab heavy chain to the N-terminus of one of the
subunits of the Fc domain.
[0135] In other embodiments, the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the first or second subunit of the Fc domain.
[0136] In a particular such embodiment, the second antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first antigen binding
moiety. In a specific such embodiment, the T cell activating
bispecific antigen binding molecule essentially consists of a first
and a second antigen binding moiety, an Fc domain composed of a
first and a second subunit, and optionally one or more peptide
linkers, wherein the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first antigen binding moiety, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the first or the second subunit of the
Fc domain. Optionally, the Fab light chain of the first antigen
binding moiety and the Fab light chain of the second antigen
binding moiety may additionally be fused to each other.
[0137] The antigen binding moieties may be fused to the Fc domain
or to each other directly or through a peptide linker, comprising
one or more amino acids, typically about 2-20 amino acids. Peptide
linkers are known in the art and are described herein. Suitable,
non-immunogenic peptide linkers include, for example,
(G.sub.4S).sub.n, (SG.sub.4).sub.n, (G.sub.4S).sub.n or
G.sub.4(SG.sub.4).sub.n peptide linkers. "n" is generally a number
between 1 and 10, typically between 2 and 4. A particularly
suitable peptide linker for fusing the Fab light chains of the
first and the second antigen binding moiety to each other is
(G.sub.4S).sub.2. An exemplary peptide linker suitable for
connecting the Fab heavy chains of the first and the second antigen
binding moiety is EPKSC(D)-(G.sub.4S).sub.2 (SEQ ID NOs 105 and
106). Additionally, linkers may comprise (a portion of) an
immunoglobulin hinge region. Particularly where an antigen binding
moiety is fused to the N-terminus of an Fc domain subunit, it may
be fused via an immunoglobulin hinge region or a portion thereof,
with or without an additional peptide linker.
[0138] A T cell activating bispecific antigen binding molecule with
a single antigen binding moiety capable of specific binding to a
target cell antigen (for example as shown in FIG. 1A, 1D, 1F or 1H)
is useful, particularly in cases where internalization of the
target cell antigen is to be expected following binding of a high
affinity antigen binding moiety. In such cases, the presence of
more than one antigen binding moiety specific for the target cell
antigen may enhance internalization of the target cell antigen,
thereby reducing its availability.
[0139] In many other cases, however, it will be advantageous to
have a T cell activating bispecific antigen binding molecule
comprising two or more antigen binding moieties specific for a
target cell antigen (see examples in shown in FIG. 1B, 1C, 1E or
1G), for example to optimize targeting to the target site or to
allow crosslinking of target cell antigens.
[0140] Accordingly, in certain embodiments, the T cell activating
bispecific antigen binding molecule of the invention further
comprises a third antigen binding moiety which is a Fab molecule
capable of specific binding to a target cell antigen. In one
embodiment, the third antigen binding moiety is a conventional Fab
molecule. In one embodiment, the third antigen binding moiety is
capable of specific binding to the same target cell antigen as the
second antigen binding moiety. In a particular embodiment, the
first antigen binding moiety is capable of specific binding to CD3,
and the second and third antigen binding moieties are capable of
specific binding to a target cell antigen. In a particular
embodiment, the second and the third antigen binding moiety are
identical (i.e. they comprise the same amino acid sequences).
[0141] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to CEA,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25
and SEQ ID NO: 26 and at least one light chain CDR selected from
the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0142] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6
and at least one light chain CDR selected from the group of SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third
antigen binding moieties are capable of specific binding to CEA,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25
and SEQ ID NO: 26 and at least one light chain CDR selected from
the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0143] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6
and at least one light chain CDR selected from the group of SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third
antigen binding moieties are capable of specific binding to CEA,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25
and SEQ ID NO: 26 and at least one light chain CDR selected from
the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
[0144] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid
sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32
and SEQ ID NO: 33 and a light chain variable region comprising an
amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to an amino acid sequence selected from the group
of: SEQ ID NO: 7 and SEQ ID NO: 31, and the second and third
antigen binding moieties are capable of specific binding to CEA,
wherein the second and third antigen binding moieties comprise a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 23 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 27.
[0145] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 3, and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 7, and the second and third antigen binding moieties are
capable of specific binding to CEA, wherein the second and third
antigen binding moieties comprise a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 23 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 27.
[0146] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to MCSP,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ
ID NO: 40 and at least one light chain CDR selected from the group
of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ
ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50. In a
particular embodiment, the first antigen binding moiety is capable
of specific binding to CD3, and comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at
least one light chain CDR selected from the group of SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10; and the second and third antigen
binding moieties are capable of specific binding to MCSP, wherein
the second and third antigen binding moieties comprise at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40
and at least one light chain CDR selected from the group of SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45,
SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50. In one embodiment,
the first antigen binding moiety is capable of specific binding to
CD3, and comprises at least one heavy chain complementarity
determining region (CDR) selected from the group consisting of SEQ
ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one light
chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10; and the second and third antigen binding moieties
are capable of specific binding to MCSP, wherein the second and
third antigen binding moieties comprise at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at
least one light chain CDR selected from the group of SEQ ID NO: 18,
SEQ ID NO: 19 and SEQ ID NO: 20.
[0147] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid
sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32
and SEQ ID NO: 33 and a light chain variable region comprising an
amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to an amino acid sequence selected from the group
of: SEQ ID NO: 7 and SEQ ID NO: 31, and the second and third
antigen binding moieties are capable of specific binding to MCSP,
wherein the second and third antigen binding moieties comprise a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an
amino acid sequence selected from the group of SEQ ID NO: 13, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an
amino acid sequence selected from the group of SEQ ID NO: 17, SEQ
ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0148] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 3 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 7, and the second and third antigen binding moieties are
capable of specific binding to MCSP, wherein the second and third
antigen binding moieties comprise a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 13 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 17.
[0149] In one embodiment, the third antigen binding moiety is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
first or second subunit of the Fc domain. In a more specific
embodiment, the second and the third antigen binding moiety are
each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety. Optionally, the Fab light chain of the
first antigen binding moiety and the Fab light chain of the second
antigen binding moiety may additionally be fused to each other.
[0150] The second and the third antigen binding moiety may be fused
to the Fc domain directly or through a peptide linker. In a
particular embodiment the second and the third antigen binding
moiety are each fused to the Fc domain through an immunoglobulin
hinge region. In a specific embodiment, the immunoglobulin hinge
region is a human IgG.sub.1 hinge region. In one embodiment the
second and the third antigen binding moiety and the Fc domain are
part of an immunoglobulin molecule. In a particular embodiment the
immunoglobulin molecule is an IgG class immunoglobulin. In an even
more particular embodiment the immunoglobulin is an IgG.sub.1
subclass immunoglobulin. In another embodiment the immunoglobulin
is an IgG.sub.4 subclass immunoglobulin. In a further particular
embodiment the immunoglobulin is a human immunoglobulin. In other
embodiments the immunoglobulin is a chimeric immunoglobulin or a
humanized immunoglobulin. In one embodiment, the T cell activating
bispecific antigen binding molecule essentially consists of an
immunoglobulin molecule capable of specific binding to a target
cell antigen, and an antigen binding moiety capable of specific
binding to CD3 wherein the antigen binding moiety is a Fab
molecule, particularly a crossover Fab molecule, fused to the
N-terminus of one of the immunoglobulin heavy chains, optionally
via a peptide linker.
[0151] In a particular embodiment, the first and the third antigen
binding moiety are each fused at the C-terminus of the Fab heavy
chain to the N-terminus of one of the subunits of the Fc domain,
and the second antigen binding moiety is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the Fab heavy chain of the
first antigen binding moiety. In a specific such embodiment, the T
cell activating bispecific antigen binding molecule essentially
consists of a first, a second and a third antigen binding moiety,
an Fc domain composed of a first and a second subunit, and
optionally one or more peptide linkers, wherein the second antigen
binding moiety is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the Fab heavy chain of the first antigen binding
moiety, and the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first
subunit of the Fc domain, and wherein the third antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the second subunit of the Fc domain. Optionally, the
Fab light chain of the first antigen binding moiety and the Fab
light chain of the second antigen binding moiety may additionally
be fused to each other.
[0152] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 4, the
heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID
NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2
of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to CEA comprising the heavy chain CDR 1 of SEQ ID NO: 24, the heavy
chain CDR 2 of SEQ ID NO: 25, the heavy chain CDR 3 of SEQ ID NO:
26, the light chain CDR 1 of SEQ ID NO: 28, the light chain CDR 2
of SEQ ID NO: 29 and the light chain CDR3 of SEQ ID NO: 30.
[0153] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 3 and a light chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 7, wherein the
first antigen binding moiety is a crossover Fab molecule wherein
either the variable or the constant regions, particularly the
constant regions, of the Fab light chain and the Fab heavy chain
are exchanged; (ii) a second and a third antigen binding moiety
each of which is a Fab molecule capable of specific binding to CEA
comprising heavy chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 23 and a light
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 27.
[0154] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 4, the
heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID
NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2
of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to MCSP comprising the heavy chain CDR 1 of SEQ ID NO: 14, the
heavy chain CDR 2 of SEQ ID NO: 15, the heavy chain CDR 3 of SEQ ID
NO: 16, the light chain CDR 1 of SEQ ID NO: 18, the light chain CDR
2 of SEQ ID NO: 19 and the light chain CDR3 of SEQ ID NO: 20.
[0155] In one embodiment the present invention provides a T cell
activating bispecific antigen binding molecule comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 3 and a light chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 7, wherein the
first antigen binding moiety is a crossover Fab molecule wherein
either the variable or the constant regions, particularly the
constant regions, of the Fab light chain and the Fab heavy chain
are exchanged; (ii) a second and a third antigen binding moiety
each of which is a Fab molecule capable of specific binding to MCSP
comprising a heavy chain variable region comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 13 and a light
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 17.
[0156] The T cell activating bispecific antigen binding molecule
according to any of the four above embodiments may further comprise
(iii) an Fc domain composed of a first and a second subunit capable
of stable association, wherein the second antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the first antigen binding moiety, and the
first antigen binding moiety is fused at the C-terminus of the Fab
heavy chain to the N-terminus of the first subunit of the Fc
domain, and wherein the third antigen binding moiety is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the
second subunit of the Fc domain.
[0157] In some of the T cell activating bispecific antigen binding
molecule of the invention, the Fab light chain of the first antigen
binding moiety and the Fab light chain of the second antigen
binding moiety are fused to each other, optionally via a linker
peptide. Depending on the configuration of the first and the second
antigen binding moiety, the Fab light chain of the first antigen
binding moiety may be fused at its C-terminus to the N-terminus of
the Fab light chain of the second antigen binding moiety, or the
Fab light chain of the second antigen binding moiety may be fused
at its C-terminus to the N-terminus of the Fab light chain of the
first antigen binding moiety. Fusion of the Fab light chains of the
first and the second antigen binding moiety further reduces
mispairing of unmatched Fab heavy and light chains, and also
reduces the number of plasmids needed for expression of some of the
T cell activating bispecific antigen binding molecules of the
invention.
[0158] In certain embodiments the T cell activating bispecific
antigen binding molecule comprises a polypeptide wherein the Fab
light chain variable region of the first antigen binding moiety
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of the first antigen binding moiety (i.e. a the
first antigen binding moiety comprises a crossover Fab heavy chain,
wherein the heavy chain variable region is replaced by a light
chain variable region), which in turn shares a carboxy-terminal
peptide bond with an Fc domain subunit
(VL.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)), and a polypeptide wherein a
the Fab heavy chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In some embodiments the T
cell activating bispecific antigen binding molecule further
comprises a polypeptide wherein the Fab heavy chain variable region
of the first antigen binding moiety shares a carboxy-terminal
peptide bond with the Fab light chain constant region of the first
antigen binding moiety (VH.sub.(1)-CL.sub.(1)) and the Fab light
chain polypeptide of the second antigen binding moiety
(VL.sub.(2)-CL.sub.(2)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0159] In alternative embodiments the T cell activating bispecific
antigen binding molecule comprises a polypeptide wherein the Fab
heavy chain variable region of the first antigen binding moiety
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of the first antigen binding moiety (i.e. the first
antigen binding moiety comprises a crossover Fab heavy chain,
wherein the heavy chain constant region is replaced by a light
chain constant region), which in turn shares a carboxy-terminal
peptide bond with an Fc domain subunit
(VH.sub.(1)-CL.sub.(1)-CH2-CH3(-CH4)), and a polypeptide wherein
the Fab heavy chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In some embodiments the T
cell activating bispecific antigen binding molecule further
comprises a polypeptide wherein the Fab light chain variable region
of the first antigen binding moiety shares a carboxy-terminal
peptide bond with the Fab heavy chain constant region of the first
antigen binding moiety (VL.sub.(1)-CH1.sub.(1)) and the Fab light
chain polypeptide of the second antigen binding moiety
(VL.sub.(2)-CL.sub.(2)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0160] In some embodiments, the T cell activating bispecific
antigen binding molecule comprises a polypeptide wherein the Fab
light chain variable region of the first antigen binding moiety
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of the first antigen binding moiety (i.e. the first
antigen binding moiety comprises a crossover Fab heavy chain,
wherein the heavy chain variable region is replaced by a light
chain variable region), which in turn shares a carboxy-terminal
peptide bond with the Fab heavy chain of the second antigen binding
moiety, which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit
(VL.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In
other embodiments, the T cell activating bispecific antigen binding
molecule comprises a polypeptide wherein the Fab heavy chain
variable region of the first antigen binding moiety shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the first antigen binding moiety (i.e. the first antigen
binding moiety comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the second antigen binding moiety, which in
turn shares a carboxy-terminal peptide bond with an Fc domain
subunit
(VH.sub.(1)-CL.sub.(1)-VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In
still other embodiments, the T cell activating bispecific antigen
binding molecule comprises a polypeptide wherein the Fab heavy
chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with the Fab light chain variable
region of the first antigen binding moiety which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the first antigen binding moiety (i.e. the first antigen
binding moiety comprises a crossover Fab heavy chain, wherein the
heavy chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-VL.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In
other embodiments, the T cell activating bispecific antigen binding
molecule comprises a polypeptide wherein the Fab heavy chain of the
second antigen binding moiety shares a carboxy-terminal peptide
bond with the Fab heavy chain variable region of the first antigen
binding moiety which in turn shares a carboxy-terminal peptide bond
with the Fab light chain constant region of the first antigen
binding moiety (i.e. the first antigen binding moiety comprises a
crossover Fab heavy chain, wherein the heavy chain constant region
is replaced by a light chain constant region), which in turn shares
a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CL.sub.(1)-CH2-CH3(-CH4)).
[0161] In some of these embodiments the T cell activating
bispecific antigen binding molecule further comprises a crossover
Fab light chain polypeptide of the first antigen binding moiety,
wherein the Fab heavy chain variable region of the first antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the first antigen binding moiety
(VH.sub.(1)-CL.sub.(1)), and the Fab light chain polypeptide of the
second antigen binding moiety (VL.sub.(2)-CL.sub.(2)). In others of
these embodiments the T cell activating bispecific antigen binding
molecule further comprises a crossover Fab light chain polypeptide,
wherein the Fab light chain variable region of the first antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of the first antigen binding moiety
(VL.sub.(1)-CH1.sub.(1)), and the Fab light chain polypeptide of
the second antigen binding moiety (VL.sub.(2)-CL.sub.(2)). In still
others of these embodiments the T cell activating bispecific
antigen binding molecule further comprises a polypeptide wherein
the Fab light chain variable region of the first antigen binding
moiety shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the first antigen binding moiety which in
turn shares a carboxy-terminal peptide bond with the Fab light
chain polypeptide of the second antigen binding moiety
(VL.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CL.sub.(2)), a polypeptide
wherein the Fab heavy chain variable region of the first antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the first antigen binding moiety
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain polypeptide of the second antigen binding moiety
(VH.sub.(1)-CL.sub.(1)-VL.sub.(2)-CL.sub.(2)), a polypeptide
wherein the Fab light chain polypeptide of the second antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
light chain variable region of the first antigen binding moiety
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of the first antigen binding moiety
(VL.sub.(2)-CL.sub.(2)-VL.sub.(1)-CH1.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the second antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
heavy chain variable region of the first antigen binding moiety
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the first antigen binding moiety
(VL.sub.(2)-CL.sub.(2)-VH.sub.(1)-CL.sub.(1)).
[0162] The T cell activating bispecific antigen binding molecule
according to these embodiments may further comprise (i) an Fc
domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide
wherein the Fab heavy chain of a third antigen binding moiety
shares a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(3)-CH1.sub.(3)-CH2-CH3(-CH4)) and the Fab light chain
polypeptide of a third antigen binding moiety
(VL.sub.(3)-CL.sub.(3)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0163] According to any of the above embodiments, components of the
T cell activating bispecific antigen binding molecule (e.g. antigen
binding moiety, Fc domain) may be fused directly or through various
linkers, particularly peptide linkers comprising one or more amino
acids, typically about 2-20 amino acids, that are described herein
or are known in the art. Suitable, non-immunogenic peptide linkers
include, for example, (G.sub.4S).sub.n, (SG.sub.4).sub.n,
(G.sub.4S).sub.n or G.sub.4(SG.sub.4).sub.n peptide linkers,
wherein n is generally a number between 1 and 10, typically between
2 and 4.
Fc Domain
[0164] The Fc domain of the T cell activating bispecific antigen
binding molecule consists of a pair of polypeptide chains
comprising heavy chain domains of an immunoglobulin molecule. For
example, the Fc domain of an immunoglobulin G (IgG) molecule is a
dimer, each subunit of which comprises the CH2 and CH3 IgG heavy
chain constant domains. The two subunits of the Fc domain are
capable of stable association with each other. In one embodiment
the T cell activating bispecific antigen binding molecule of the
invention comprises not more than one Fc domain. In one embodiment
according the invention the Fc domain of the T cell activating
bispecific antigen binding molecule is an IgG Fc domain. In a
particular embodiment the Fc domain is an IgG.sub.1 Fc domain. In
another embodiment the Fc domain is an IgG.sub.4 Fc domain. In a
more specific embodiment, the Fc domain is an IgG.sub.4 Fc domain
comprising an amino acid substitution at position 5228 (Kabat
numbering), particularly the amino acid substitution S228P. This
amino acid substitution reduces in vivo Fab arm exchange of
IgG.sub.4 antibodies (see Stubenrauch et al., Drug Metabolism and
Disposition 38, 84-91 (2010)). In a further particular embodiment
the Fc domain is human. An exemplary sequence of a human IgG.sub.1
Fc region is given in SEQ ID NO: 107.
Fc Domain Modifications Promoting Heterodimerization
[0165] T cell activating bispecific antigen binding molecules
according to the invention comprise different antigen binding
moieties, fused to one or the other of the two subunits of the Fc
domain, thus the two subunits of the Fc domain are typically
comprised in two non-identical polypeptide chains. Recombinant
co-expression of these polypeptides and subsequent dimerization
leads to several possible combinations of the two polypeptides. To
improve the yield and purity of T cell activating bispecific
antigen binding molecules in recombinant production, it will thus
be advantageous to introduce in the Fc domain of the T cell
activating bispecific antigen binding molecule a modification
promoting the association of the desired polypeptides.
[0166] Accordingly, in particular embodiments the Fc domain of the
T cell activating bispecific antigen binding molecule according to
the invention comprises a modification promoting the association of
the first and the second subunit of the Fc domain. The site of most
extensive protein-protein interaction between the two subunits of a
human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in
one embodiment said modification is in the CH3 domain of the Fc
domain.
[0167] In a specific embodiment said modification is a so-called
"knob-into-hole" modification, comprising a "knob" modification in
one of the two subunits of the Fc domain and a "hole" modification
in the other one of the two subunits of the Fc domain.
[0168] The knob-into-hole technology is described e.g. in U.S. Pat.
Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621
(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the
method involves introducing a protuberance ("knob") at the
interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine).
[0169] Accordingly, in a particular embodiment, in the CH3 domain
of the first subunit of the Fc domain of the T cell activating
bispecific antigen binding molecule an amino acid residue is
replaced with an amino acid residue having a larger side chain
volume, thereby generating a protuberance within the CH3 domain of
the first subunit which is positionable in a cavity within the CH3
domain of the second subunit, and in the CH3 domain of the second
subunit of the Fc domain an amino acid residue is replaced with an
amino acid residue having a smaller side chain volume, thereby
generating a cavity within the CH3 domain of the second subunit
within which the protuberance within the CH3 domain of the first
subunit is positionable.
[0170] The protuberance and cavity can be made by altering the
nucleic acid encoding the polypeptides, e.g. by site-specific
mutagenesis, or by peptide synthesis.
[0171] In a specific embodiment, in the CH3 domain of the first
subunit of the Fc domain the threonine residue at position 366 is
replaced with a tryptophan residue (T366W), and in the CH3 domain
of the second subunit of the Fc domain the tyrosine residue at
position 407 is replaced with a valine residue (Y407V). In one
embodiment, in the second subunit of the Fc domain additionally the
threonine residue at position 366 is replaced with a serine residue
(T366S) and the leucine residue at position 368 is replaced with an
alanine residue (L368A).
[0172] In yet a further embodiment, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C), and in the second subunit of the
Fc domain additionally the tyrosine residue at position 349 is
replaced by a cysteine residue (Y349C). Introduction of these two
cysteine residues results in formation of a disulfide bridge
between the two subunits of the Fc domain, further stabilizing the
dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0173] In a particular embodiment the antigen binding moiety
capable of binding to CD3 is fused (optionally via the antigen
binding moiety capable of binding to a target cell antigen) to the
first subunit of the Fc domain (comprising the "knob"
modification). Without wishing to be bound by theory, fusion of the
antigen binding moiety capable of binding to CD3 to the
knob-containing subunit of the Fc domain will (further) minimize
the generation of antigen binding molecules comprising two antigen
binding moieties capable of binding to CD3 (steric clash of two
knob-containing polypeptides).
[0174] In an alternative embodiment a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g. as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable.
Fc Domain Modifications Reducing Fc Receptor Binding and/or
Effector Function
[0175] The Fc domain confers to the T cell activating bispecific
antigen binding molecule favorable pharmacokinetic properties,
including a long serum half-life which contributes to good
accumulation in the target tissue and a favorable tissue-blood
distribution ratio. At the same time it may, however, lead to
undesirable targeting of the T cell activating bispecific antigen
binding molecule to cells expressing Fc receptors rather than to
the preferred antigen-bearing cells. Moreover, the co-activation of
Fc receptor signaling pathways may lead to cytokine release which,
in combination with the T cell activating properties and the long
half-life of the antigen binding molecule, results in excessive
activation of cytokine receptors and severe side effects upon
systemic administration. Activation of (Fc receptor-bearing) immune
cells other than T cells may even reduce efficacy of the T cell
activating bispecific antigen binding molecule due to the potential
destruction of T cells e.g. by NK cells.
[0176] Accordingly, in particular embodiments the Fc domain of the
T cell activating bispecific antigen binding molecules according to
the invention exhibits reduced binding affinity to an Fc receptor
and/or reduced effector function, as compared to a native IgG.sub.1
Fc domain. In one such embodiment the Fc domain (or the T cell
activating bispecific antigen binding molecule comprising said Fc
domain) exhibits less than 50%, preferably less than 20%, more
preferably less than 10% and most preferably less than 5% of the
binding affinity to an Fc receptor, as compared to a native
IgG.sub.1 Fc domain (or a T cell activating bispecific antigen
binding molecule comprising a native IgG.sub.1 Fc domain), and/or
less than 50%, preferably less than 20%, more preferably less than
10% and most preferably less than 5% of the effector function, as
compared to a native IgG.sub.1 Fc domain domain (or a T cell
activating bispecific antigen binding molecule comprising a native
IgG.sub.1 Fc domain). In one embodiment, the Fc domain domain (or
the T cell activating bispecific antigen binding molecule
comprising said Fc domain) does not substantially bind to an Fc
receptor and/or induce effector function. In a particular
embodiment the Fc receptor is an Fc.gamma. receptor. In one
embodiment the Fc receptor is a human Fc receptor. In one
embodiment the Fc receptor is an activating Fc receptor. In a
specific embodiment the Fc receptor is an activating human Fey
receptor, more specifically human Fc.gamma.RIIIa, Fc.gamma.RI or
Fc.gamma.RIIa, most specifically human Fc.gamma.RIIIa. In one
embodiment the effector function is one or more selected from the
group of CDC, ADCC, ADCP, and cytokine secretion. In a particular
embodiment the effector function is ADCC. In one embodiment the Fc
domain domain exhibits substantially similar binding affinity to
neonatal Fc receptor (FcRn), as compared to a native IgG.sub.1 Fc
domain domain. Substantially similar binding to FcRn is achieved
when the Fc domain (or the T cell activating bispecific antigen
binding molecule comprising said Fc domain) exhibits greater than
about 70%, particularly greater than about 80%, more particularly
greater than about 90% of the binding affinity of a native
IgG.sub.1 Fc domain (or the T cell activating bispecific antigen
binding molecule comprising a native IgG.sub.1 Fc domain) to
FcRn.
[0177] In certain embodiments the Fc domain is engineered to have
reduced binding affinity to an Fc receptor and/or reduced effector
function, as compared to a non-engineered Fc domain. In particular
embodiments, the Fc domain of the T cell activating bispecific
antigen binding molecule comprises one or more amino acid mutation
that reduces the binding affinity of the Fc domain to an Fc
receptor and/or effector function. Typically, the same one or more
amino acid mutation is present in each of the two subunits of the
Fc domain. In one embodiment the amino acid mutation reduces the
binding affinity of the Fc domain to an Fc receptor. In one
embodiment the amino acid mutation reduces the binding affinity of
the Fc domain to an Fc receptor by at least 2-fold, at least
5-fold, or at least 10-fold. In embodiments where there is more
than one amino acid mutation that reduces the binding affinity of
the Fc domain to the Fc receptor, the combination of these amino
acid mutations may reduce the binding affinity of the Fc domain to
an Fc receptor by at least 10-fold, at least 20-fold, or even at
least 50-fold. In one embodiment the T cell activating bispecific
antigen binding molecule comprising an engineered Fc domain
exhibits less than 20%, particularly less than 10%, more
particularly less than 5% of the binding affinity to an Fc receptor
as compared to a T cell activating bispecific antigen binding
molecule comprising a non-engineered Fc domain. In a particular
embodiment the Fc receptor is an Fc.gamma. receptor. In some
embodiments the Fc receptor is a human Fc receptor. In some
embodiments the Fc receptor is an activating Fc receptor. In a
specific embodiment the Fc receptor is an activating human Fey
receptor, more specifically human Fc.gamma.RIIIa, Fc.gamma.RI or
Fc.gamma.RIIa, most specifically human Fc.gamma.RIIIa. Preferably,
binding to each of these receptors is reduced. In some embodiments
binding affinity to a complement component, specifically binding
affinity to C1q, is also reduced. In one embodiment binding
affinity to neonatal Fc receptor (FcRn) is not reduced.
Substantially similar binding to FcRn, i.e. preservation of the
binding affinity of the Fc domain to said receptor, is achieved
when the Fc domain (or the T cell activating bispecific antigen
binding molecule comprising said Fc domain) exhibits greater than
about 70% of the binding affinity of a non-engineered form of the
Fc domain (or the T cell activating bispecific antigen binding
molecule comprising said non-engineered form of the Fc domain) to
FcRn. The Fc domain, or T cell activating bispecific antigen
binding molecules of the invention comprising said Fc domain, may
exhibit greater than about 80% and even greater than about 90% of
such affinity. In certain embodiments the Fc domain of the T cell
activating bispecific antigen binding molecule is engineered to
have reduced effector function, as compared to a non-engineered Fc
domain. The reduced effector function can include, but is not
limited to, one or more of the following: reduced complement
dependent cytotoxicity (CDC), reduced antibody-dependent
cell-mediated cytotoxicity (ADCC), reduced antibody-dependent
cellular phagocytosis (ADCP), reduced cytokine secretion, reduced
immune complex-mediated antigen uptake by antigen-presenting cells,
reduced binding to NK cells, reduced binding to macrophages,
reduced binding to monocytes, reduced binding to polymorphonuclear
cells, reduced direct signaling inducing apoptosis, reduced
crosslinking of target-bound antibodies, reduced dendritic cell
maturation, or reduced T cell priming. In one embodiment the
reduced effector function is one or more selected from the group of
reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine
secretion. In a particular embodiment the reduced effector function
is reduced ADCC. In one embodiment the reduced ADCC is less than
20% of the ADCC induced by a non-engineered Fc domain (or a T cell
activating bispecific antigen binding molecule comprising a
non-engineered Fc domain).
[0178] In one embodiment the amino acid mutation that reduces the
binding affinity of the Fc domain to an Fc receptor and/or effector
function is an amino acid substitution. In one embodiment the Fc
domain comprises an amino acid substitution at a position selected
from the group of E233, L234, L235, N297, P331 and P329. In a more
specific embodiment the Fc domain comprises an amino acid
substitution at a position selected from the group of L234, L235
and P329. In some embodiments the Fc domain comprises the amino
acid substitutions L234A and L235A. In one such embodiment, the Fc
domain is an IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc
domain. In one embodiment the Fc domain comprises an amino acid
substitution at position P329. In a more specific embodiment the
amino acid substitution is P329A or P329G, particularly P329G. In
one embodiment the Fc domain comprises an amino acid substitution
at position P329 and a further amino acid substitution at a
position selected from E233, L234, L235, N297 and P331. In a more
specific embodiment the further amino acid substitution is E233P,
L234A, L235A, L235E, N297A, N297D or P331S. In particular
embodiments the Fc domain comprises amino acid substitutions at
positions P329, L234 and L235. In more particular embodiments the
Fc domain comprises the amino acid mutations L234A, L235A and P329G
("P329G LALA"). In one such embodiment, the Fc domain is an
IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc domain. The
"P329G LALA" combination of amino acid substitutions almost
completely abolishes Fc.gamma. receptor (as well as complement)
binding of a human IgG.sub.1 Fc domain, as described in PCT
publication no. WO 2012/130831, incorporated herein by reference in
its entirety. WO 2012/130831 also describes methods of preparing
such mutant Fc domains and methods for determining its properties
such as Fc receptor binding or effector functions.
[0179] IgG.sub.4 antibodies exhibit reduced binding affinity to Fc
receptors and reduced effector functions as compared to IgG.sub.1
antibodies. Hence, in some embodiments the Fc domain of the T cell
activating bispecific antigen binding molecules of the invention is
an IgG.sub.4 Fc domain, particularly a human IgG.sub.4 Fc domain.
In one embodiment the IgG.sub.4 Fc domain comprises amino acid
substitutions at position 5228, specifically the amino acid
substitution S228P. To further reduce its binding affinity to an Fc
receptor and/or its effector function, in one embodiment the
IgG.sub.4 Fc domain comprises an amino acid substitution at
position L235, specifically the amino acid substitution L235E. In
another embodiment, the IgG.sub.4 Fc domain comprises an amino acid
substitution at position P329, specifically the amino acid
substitution P329G. In a particular embodiment, the IgG.sub.4 Fc
domain comprises amino acid substitutions at positions S228, L235
and P329, specifically amino acid substitutions S228P, L235E and
P329G. Such IgG.sub.4 Fc domain mutants and their Fc.gamma.
receptor binding properties are described in PCT publication no. WO
2012/130831, incorporated herein by reference in its entirety.
[0180] In a particular embodiment the Fc domain exhibiting reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain, is a human
IgG.sub.1 Fc domain comprising the amino acid substitutions L234A,
L235A and optionally P329G, or a human IgG.sub.4 Fc domain
comprising the amino acid substitutions S228P, L235E and optionally
P329G.
[0181] In certain embodiments N-glycosylation of the Fc domain has
been eliminated. In one such embodiment the Fc domain comprises an
amino acid mutation at position N297, particularly an amino acid
substitution replacing asparagine by alanine (N297A) or aspartic
acid (N297D).
[0182] In addition to the Fc domains described hereinabove and in
PCT publication no. WO 2012/130831, Fc domains with reduced Fc
receptor binding and/or effector function also include those with
substitution of one or more of Fc domain residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0183] Mutant Fc domains can be prepared by amino acid deletion,
substitution, insertion or modification using genetic or chemical
methods well known in the art. Genetic methods may include
site-specific mutagenesis of the encoding DNA sequence, PCR, gene
synthesis, and the like. The correct nucleotide changes can be
verified for example by sequencing.
[0184] Binding to Fc receptors can be easily determined e.g. by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BIAcore instrument (GE Healthcare), and
Fc receptors such as may be obtained by recombinant expression. A
suitable such binding assay is described herein. Alternatively,
binding affinity of Fc domains or cell activating bispecific
antigen binding molecules comprising an Fc domain for Fc receptors
may be evaluated using cell lines known to express particular Fc
receptors, such as human NK cells expressing Fc.gamma.IIIa
receptor.
[0185] Effector function of an Fc domain, or a T cell activating
bispecific antigen binding molecule comprising an Fc domain, can be
measured by methods known in the art. A suitable assay for
measuring ADCC is described herein. Other examples of in vitro
assays to assess ADCC activity of a molecule of interest are
described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl
Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl
Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;
Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,
non-radioactive assays methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.)).
Useful effector cells for such assays include peripheral blood
mononuclear cells (PB MC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g. in a animal model such as
that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656
(1998).
[0186] In some embodiments, binding of the Fc domain to a
complement component, specifically to C1q, is reduced. Accordingly,
in some embodiments wherein the Fc domain is engineered to have
reduced effector function, said reduced effector function includes
reduced CDC. C1q binding assays may be carried out to determine
whether the T cell activating bispecific antigen binding molecule
is able to bind C1q and hence has CDC activity. See e.g., C1q and
C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess
complement activation, a CDC assay may be performed (see, for
example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996);
Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie,
Blood 103, 2738-2743 (2004)).
Antigen Binding Moieties
[0187] The antigen binding molecule of the invention is bispecific,
i.e. it comprises at least two antigen binding moieties capable of
specific binding to two distinct antigenic determinants. According
to the invention, the antigen binding moieties are Fab molecules
(i.e. antigen binding domains composed of a heavy and a light
chain, each comprising a variable and a constant region). In one
embodiment said Fab molecules are human. In another embodiment said
Fab molecules are humanized. In yet another embodiment said Fab
molecules comprise human heavy and light chain constant
regions.
[0188] At least one of the antigen binding moieties is a crossover
Fab molecule. Such modification prevent mispairing of heavy and
light chains from different Fab molecules, thereby improving the
yield and purity of the T cell activating bispecific antigen
binding molecule of the invention in recombinant production. In a
particular crossover Fab molecule useful for the T cell activating
bispecific antigen binding molecule of the invention, the constant
regions of the Fab light chain and the Fab heavy chain are
exchanged. In another crossover Fab molecule useful for the T cell
activating bispecific antigen binding molecule of the invention,
the variable regions of the Fab light chain and the Fab heavy chain
are exchanged.
[0189] In a particular embodiment according to the invention, the T
cell activating bispecific antigen binding molecule is capable of
simultaneous binding to a target cell antigen, particularly a tumor
cell antigen, and CD3. In one embodiment, the T cell activating
bispecific antigen binding molecule is capable of crosslinking a T
cell and a target cell by simultaneous binding to a target cell
antigen and CD3. In an even more particular embodiment, such
simultaneous binding results in lysis of the target cell,
particularly a tumor cell. In one embodiment, such simultaneous
binding results in activation of the T cell. In other embodiments,
such simultaneous binding results in a cellular response of a T
lymphocyte, particularly a cytotoxic T lymphocyte, selected from
the group of: proliferation, differentiation, cytokine secretion,
cytotoxic effector molecule release, cytotoxic activity, and
expression of activation markers. In one embodiment, binding of the
T cell activating bispecific antigen binding molecule to CD3
without simultaneous binding to the target cell antigen does not
result in T cell activation.
[0190] In one embodiment, the T cell activating bispecific antigen
binding molecule is capable of re-directing cytotoxic activity of a
T cell to a target cell. In a particular embodiment, said
re-direction is independent of MHC-mediated peptide antigen
presentation by the target cell and and/or specificity of the T
cell.
[0191] Particularly, a T cell according to any of the embodiments
of the invention is a cytotoxic T cell. In some embodiments the T
cell is a CD4.sup.+ or a CD8.sup.+ T cell, particularly a CD8.sup.+
T cell.
CD3 Binding Moiety
[0192] The T cell activating bispecific antigen binding molecule of
the invention comprises at least one antigen binding moiety capable
of binding to CD3 (also referred to herein as an "CD3 antigen
binding moiety" or "first antigen binding moiety"). In a particular
embodiment, the T cell activating bispecific antigen binding
molecule comprises not more than one antigen binding moiety capable
of specific binding to CD3. In one embodiment the T cell activating
bispecific antigen binding molecule provides monovalent binding to
CD3. The CD3 antigen binding is a crossover Fab molecule, i.e. a
Fab molecule wherein either the variable or the constant regions of
the Fab heavy and light chains are exchanged. In embodiments where
there is more than one antigen binding moiety capable of specific
binding to a target cell antigen comprised in the T cell activating
bispecific antigen binding molecule, the antigen binding moiety
capable of specific binding to CD3 preferably is a crossover Fab
molecule and the antigen binding moieties capable of specific
binding to a target cell antigen are conventional Fab
molecules.
[0193] In a particular embodiment CD3 is human CD3 (SEQ ID NO: 103)
or cynomolgus CD3 (SEQ ID NO: 104), most particularly human CD3. In
a particular embodiment the CD3 antigen binding moiety is
cross-reactive for (i.e. specifically binds to) human and
cynomolgus CD3. In some embodiments, the first antigen binding
moiety is capable of specific binding to the epsilon subunit of
CD3.
[0194] The CD3 antigen binding moiety comprises at least one heavy
chain complementarity determining region (CDR) selected from the
group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and
at least one light chain CDR selected from the group of SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10.
[0195] In one embodiment the CD3 antigen binding moiety comprises
the heavy chain CDR1 of SEQ ID NO: 4, the heavy chain CDR2 of SEQ
ID NO: 5, the heavy chain CDR3 of SEQ ID NO: 6, the light chain
CDR1 of SEQ ID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the
light chain CDR3 of SEQ ID NO: 10.
[0196] In one embodiment the CD3 antigen binding moiety comprises a
heavy chain variable region sequence that is at least about 95%,
96%, 97%, 98%, 99% or 100% identical to an amino acid sequence
selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID
NO: 33, and a light chain variable region sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid
sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO:
31.
[0197] In one embodiment the CD3 antigen binding moiety comprises a
heavy chain variable region comprising an amino acid sequence
selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID
NO: 33 and a light chain variable region comprising an amino acid
sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO:
31.
[0198] In one embodiment the CD3 antigen binding moiety comprises a
heavy chain variable region sequence that is at least about 95%,
96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 and a light
chain variable region sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 7.
[0199] In one embodiment the CD3 antigen binding moiety comprises a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 3 and a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 7.
[0200] In one embodiment the CD3 antigen binding moiety comprises
the heavy chain variable region sequence of SEQ ID NO: 3 and the
light chain variable region sequence of SEQ ID NO: 7.
Target Cell Antigen Binding Moiety
[0201] The T cell activating bispecific antigen binding molecule of
the invention comprises at least one antigen binding moiety capable
of binding to a target cell antigen (also referred to herein as an
"target cell antigen binding moiety" or "second" or "third" antigen
binding moiety). In certain embodiments, the T cell activating
bispecific antigen binding molecule comprises two antigen binding
moieties capable of binding to a target cell antigen. In a
particular such embodiment, each of these antigen binding moieties
specifically binds to the same antigenic determinant. In an even
more particular embodiment, all of these antigen binding moieties
are identical. In one embodiment, the T cell activating bispecific
antigen binding molecule comprises an immunoglobulin molecule
capable of specific binding to a target cell antigen. In one
embodiment the T cell activating bispecific antigen binding
molecule comprises not more than two antigen binding moieties
capable of binding to a target cell antigen.
[0202] The target cell antigen binding moiety is generally a Fab
molecule, particularly a conventional Fab molecule that binds to a
specific antigenic determinant and is able to direct the T cell
activating bispecific antigen binding molecule to a target site,
for example to a specific type of tumor cell that bears the
antigenic determinant.
[0203] In certain embodiments the target cell antigen binding
moiety specifically binds to a cell surface antigen. In a
particular embodiment the target cell antigen binding moiety
specifically binds to a membrane-proximal region of a cell surface
antigen. In a specific such embodiment the cell surface antigen is
Carcinoembryonic Antigen (CEA) and the membrane-proximal region is
the B3 domain of CEA (residues 208-286 of SEQ ID NO: 119). In
another specific such embodiment the cell surface antigen is
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP) and the
membrane-proximal region is the D3 domain of MSCP (SEQ ID NO:
118).
[0204] In certain embodiments the target cell antigen binding
moiety is directed to an antigen associated with a pathological
condition, such as an antigen presented on a tumor cell or on a
virus-infected cell. Suitable antigens are cell surface antigens,
for example, but not limited to, cell surface receptors. In
particular embodiments the antigen is a human antigen. In a
specific embodiment the target cell antigen is selected from
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP, CSPG4)
and Carcinoembryonic Antigen (CEA, CEACAM5).
[0205] In some embodiments the T cell activating bispecific antigen
binding molecule comprises at least one antigen binding moiety that
is specific for Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP). In one embodiment, the antigen binding moiety
that is specific for MCSP comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least
one light chain CDR selected from the group of SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:
48, SEQ ID NO: 49 and SEQ ID NO: 50.
[0206] In one embodiment, the antigen binding moiety that is
specific for MCSP comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at
least one light chain CDR selected from the group of SEQ ID NO: 18,
SEQ ID NO: 19 and SEQ ID NO: 20.
[0207] In one embodiment, the antigen binding moiety that is
specific for MCSP comprises the heavy chain CDR1 of SEQ ID NO: 14,
the heavy chain CDR2 of SEQ ID NO: 15, the heavy chain CDR3 of SEQ
ID NO: 16, the light chain CDR1 of SEQ ID NO: 18, the light chain
CDR2 of SEQ ID NO: 19, and the light chain CDR3 of SEQ ID NO:
20.
[0208] In a further embodiment, the antigen binding moiety that is
specific for MCSP comprises a heavy chain variable region sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
an amino acid sequence selected from the group of SEQ ID NO: 13,
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a
light chain variable region sequence that is at least about 95%,
96%, 97%, 98%, 99% or 100% identical to an amino acid sequence
selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO:
46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0209] In a further embodiment, the antigen binding moiety that is
specific for MCSP comprises a heavy chain variable region
comprising an amino acid sequence selected from the group of SEQ ID
NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO:
41 and a light chain variable region comprising an amino acid
sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43,
SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.
[0210] In a further embodiment, the antigen binding moiety that is
specific for MCSP comprises a heavy chain variable region sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO:13 and a light chain variable region sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
17 or variants thereof that retain functionality.
[0211] In one embodiment, the antigen binding moiety that is
specific for MCSP comprises a heavy chain variable region
comprising an amino acid sequence of SEQ ID NO: 13 and a light
chain variable region comprising an amino acid sequence of SEQ ID
NO: 17.
[0212] In one embodiment, the antigen binding moiety that is
specific for MCSP comprises the heavy chain variable region
sequence of SEQ ID NO: 13 and the light chain variable region
sequence of SEQ ID NO: 17.
[0213] In one embodiment the T cell activating bispecific antigen
binding molecule comprises a polypeptide sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12, a
polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to SEQ ID NO: 53, a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 54, and a polypeptide sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 55.
[0214] In particular embodiments the T cell activating bispecific
antigen binding molecule comprises at least one antigen binding
moiety that is specific for Carcinoembryonic Antigen (CEA). In one
embodiment, the antigen binding moiety that is specific for CEA
comprises at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 24,
SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR
selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID
NO: 30.
[0215] In one embodiment, the antigen binding moiety that is
specific for CEA comprises the heavy chain CDR1 of SEQ ID NO: 24,
the heavy chain CDR2 of SEQ ID NO: 25, the heavy chain CDR3 of SEQ
ID NO: 26, the light chain CDR1 of SEQ ID NO: 28, the light chain
CDR2 of SEQ ID NO: 29, and the light chain CDR3 of SEQ ID NO:
30.
[0216] In a further embodiment, the antigen binding moiety that is
specific for CEA comprises a heavy chain variable region sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 23 and a light chain variable region sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
27, or variants thereof that retain functionality.
[0217] In one embodiment, the antigen binding moiety that is
specific for CEA comprises a heavy chain variable region comprising
an amino acid sequence of SEQ ID NO: 23 and a light chain variable
region comprising an amino acid sequence of SEQ ID NO: 27.
[0218] In one embodiment, the antigen binding moiety that is
specific for CEA comprises the heavy chain variable region sequence
of SEQ ID NO: 23 and the light chain variable region sequence of
SEQ ID NO: 27.
[0219] In one embodiment the T cell activating bispecific antigen
binding molecule comprises a polypeptide sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22, a
polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to SEQ ID NO: 56, a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 57, and a polypeptide sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 58.
Polynucleotides
[0220] The invention further provides isolated polynucleotides
encoding a T cell activating bispecific antigen binding molecule as
described herein or a fragment thereof. In some embodiments, said
fragment is an antigen binding fragment.
[0221] Polynucleotides of the invention include those that are at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequences set forth in SEQ ID NOs 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 and 98
including functional fragments or variants thereof.
[0222] The polynucleotides encoding T cell activating bispecific
antigen binding molecules of the invention may be expressed as a
single polynucleotide that encodes the entire T cell activating
bispecific antigen binding molecule or as multiple (e.g., two or
more) polynucleotides that are co-expressed. Polypeptides encoded
by polynucleotides that are co-expressed may associate through,
e.g., disulfide bonds or other means to form a functional T cell
activating bispecific antigen binding molecule. For example, the
light chain portion of an antigen binding moiety may be encoded by
a separate polynucleotide from the portion of the T cell activating
bispecific antigen binding molecule comprising the heavy chain
portion of the antigen binding moiety, an Fc domain subunit and
optionally (part of) another antigen binding moiety. When
co-expressed, the heavy chain polypeptides will associate with the
light chain polypeptides to form the antigen binding moiety. In
another example, the portion of the T cell activating bispecific
antigen binding molecule comprising one of the two Fc domain
subunits and optionally (part of) one or more antigen binding
moieties could be encoded by a separate polynucleotide from the
portion of the T cell activating bispecific antigen binding
molecule comprising the other of the two Fc domain subunits and
optionally (part of) an antigen binding moiety. When co-expressed,
the Fc domain subunits will associate to form the Fc domain.
[0223] In some embodiments, the isolated polynucleotide encodes the
entire T cell activating bispecific antigen binding molecule
according to the invention as described herein. In other
embodiments, the isolated polynucleotide encodes a polypeptides
comprised in the T cell activating bispecific antigen binding
molecule according to the invention as described herein.
[0224] In another embodiment, the present invention is directed to
an isolated polynucleotide encoding a T cell activating bispecific
antigen binding molecule of the invention or a fragment thereof,
wherein the polynucleotide comprises a sequence that encodes a
variable region sequence as shown in SEQ ID NOs 3, 7, 13, 17, 23,
27, 31, 32, 33, 34, 36, 39, 41, 43, 46, 47 or 51 In another
embodiment, the present invention is directed to an isolated
polynucleotide encoding a T cell activating bispecific antigen
binding molecule or fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a polypeptide sequence as shown
in SEQ ID NOs 22, 56, 57, 58, 12, 53, 54 and 55 In another
embodiment, the invention is further directed to an isolated
polynucleotide encoding a T cell activating bispecific antigen
binding molecule of the invention or a fragment thereof, wherein
the polynucleotide comprises a sequence that is at least about 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide
sequence shown in SEQ ID NOs 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97 or 98. In another embodiment,
the invention is directed to an isolated polynucleotide encoding a
T cell activating bispecific antigen binding molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises the nucleic acid sequence shown in SEQ ID NOs 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98.
In another embodiment, the invention is directed to an isolated
polynucleotide encoding a T cell activating bispecific antigen
binding molecule of the invention or a fragment thereof, wherein
the polynucleotide comprises a sequence that encodes a variable
region sequence that is at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to the amino acid sequence in SEQ ID NOs
3, 7, 13, 17, 23, 27, 31, 32, 33, 34, 36, 39, 41, 43, 46, 47 or 51.
In another embodiment, the invention is directed to an isolated
polynucleotide encoding a T cell activating bispecific antigen
binding molecule or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes a polypeptide sequence that is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the
amino acid sequence in SEQ ID NOs 22, 56, 57, 58, 12, 53, 54 or 55.
The invention encompasses an isolated polynucleotide encoding a T
cell activating bispecific antigen binding molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes the variable region sequence of
SEQ ID NOs SEQ ID NOs 3, 7, 13, 17, 23, 27, 31, 32, 33, 34, 36, 39,
41, 43, 46, 47 or 51 with conservative amino acid substitutions.
The invention also encompasses an isolated polynucleotide encoding
a T cell activating bispecific antigen binding molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes the polypeptide sequence of SEQ
ID NOs 22, 56, 57, 58, 12, 53, 54 or 55 with conservative amino
acid substitutions.
[0225] In certain embodiments the polynucleotide or nucleic acid is
DNA. In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA (mRNA).
RNA of the present invention may be single stranded or double
stranded.
Recombinant Methods
[0226] T cell activating bispecific antigen binding molecules of
the invention may be obtained, for example, by solid-state peptide
synthesis (e.g. Merrifield solid phase synthesis) or recombinant
production. For recombinant production one or more polynucleotide
encoding the T cell activating bispecific antigen binding molecule
(fragment), e.g., as described above, is isolated and inserted into
one or more vectors for further cloning and/or expression in a host
cell. Such polynucleotide may be readily isolated and sequenced
using conventional procedures. In one embodiment a vector,
preferably an expression vector, comprising one or more of the
polynucleotides of the invention is provided. Methods which are
well known to those skilled in the art can be used to construct
expression vectors containing the coding sequence of a T cell
activating bispecific antigen binding molecule (fragment) along
with appropriate transcriptional/translational control signals.
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold
Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and
Wiley Interscience, N.Y (1989). The expression vector can be part
of a plasmid, virus, or may be a nucleic acid fragment. The
expression vector includes an expression cassette into which the
polynucleotide encoding the T cell activating bispecific antigen
binding molecule (fragment) (i.e. the coding region) is cloned in
operable association with a promoter and/or other transcription or
translation control elements. As used herein, a "coding region" is
a portion of nucleic acid which consists of codons translated into
amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not
translated into an amino acid, it may be considered to be part of a
coding region, if present, but any flanking sequences, for example
promoters, ribosome binding sites, transcriptional terminators,
introns, 5' and 3' untranslated regions, and the like, are not part
of a coding region. Two or more coding regions can be present in a
single polynucleotide construct, e.g. on a single vector, or in
separate polynucleotide constructs, e.g. on separate (different)
vectors. Furthermore, any vector may contain a single coding
region, or may comprise two or more coding regions, e.g. a vector
of the present invention may encode one or more polypeptides, which
are post- or co-translationally separated into the final proteins
via proteolytic cleavage. In addition, a vector, polynucleotide, or
nucleic acid of the invention may encode heterologous coding
regions, either fused or unfused to a polynucleotide encoding the T
cell activating bispecific antigen binding molecule (fragment) of
the invention, or variant or derivative thereof. Heterologous
coding regions include without limitation specialized elements or
motifs, such as a secretory signal peptide or a heterologous
functional domain. An operable association is when a coding region
for a gene product, e.g. a polypeptide, is associated with one or
more regulatory sequences in such a way as to place expression of
the gene product under the influence or control of the regulatory
sequence(s). Two DNA fragments (such as a polypeptide coding region
and a promoter associated therewith) are "operably associated" if
induction of promoter function results in the transcription of mRNA
encoding the desired gene product and if the nature of the linkage
between the two DNA fragments does not interfere with the ability
of the expression regulatory sequences to direct the expression of
the gene product or interfere with the ability of the DNA template
to be transcribed. Thus, a promoter region would be operably
associated with a nucleic acid encoding a polypeptide if the
promoter was capable of effecting transcription of that nucleic
acid. The promoter may be a cell-specific promoter that directs
substantial transcription of the DNA only in predetermined cells.
Other transcription control elements, besides a promoter, for
example enhancers, operators, repressors, and transcription
termination signals, can be operably associated with the
polynucleotide to direct cell-specific transcription. Suitable
promoters and other transcription control regions are disclosed
herein. A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions, which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (e.g. the immediate early promoter, in
conjunction with intron-A), simian virus 40 (e.g. the early
promoter), and retroviruses (such as, e.g. Rous sarcoma virus).
Other transcription control regions include those derived from
vertebrate genes such as actin, heat shock protein, bovine growth
hormone and rabbit a-globin, as well as other sequences capable of
controlling gene expression in eukaryotic cells. Additional
suitable transcription control regions include tissue-specific
promoters and enhancers as well as inducible promoters (e.g.
promoters inducible tetracyclins). Similarly, a variety of
translation control elements are known to those of ordinary skill
in the art. These include, but are not limited to ribosome binding
sites, translation initiation and termination codons, and elements
derived from viral systems (particularly an internal ribosome entry
site, or IRES, also referred to as a CITE sequence). The expression
cassette may also include other features such as an origin of
replication, and/or chromosome integration elements such as
retroviral long terminal repeats (LTRs), or adeno-associated viral
(AAV) inverted terminal repeats (ITRs).
[0227] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. For example, if secretion of the T cell
activating bispecific antigen binding molecule is desired, DNA
encoding a signal sequence may be placed upstream of the nucleic
acid encoding a T cell activating bispecific antigen binding
molecule of the invention or a fragment thereof. According to the
signal hypothesis, proteins secreted by mammalian cells have a
signal peptide or secretory leader sequence which is cleaved from
the mature protein once export of the growing protein chain across
the rough endoplasmic reticulum has been initiated. Those of
ordinary skill in the art are aware that polypeptides secreted by
vertebrate cells generally have a signal peptide fused to the
N-terminus of the polypeptide, which is cleaved from the translated
polypeptide to produce a secreted or "mature" form of the
polypeptide. In certain embodiments, the native signal peptide,
e.g. an immunoglobulin heavy chain or light chain signal peptide is
used, or a functional derivative of that sequence that retains the
ability to direct the secretion of the polypeptide that is operably
associated with it. Alternatively, a heterologous mammalian signal
peptide, or a functional derivative thereof, may be used. For
example, the wild-type leader sequence may be substituted with the
leader sequence of human tissue plasminogen activator (TPA) or
mouse .beta.-glucuronidase. Exemplary amino acid and polynucleotide
sequences of secretory signal peptides are given in SEQ ID NOs
108-116.
[0228] DNA encoding a short protein sequence that could be used to
facilitate later purification (e.g. a histidine tag) or assist in
labeling the T cell activating bispecific antigen binding molecule
may be included within or at the ends of the T cell activating
bispecific antigen binding molecule (fragment) encoding
polynucleotide.
[0229] In a further embodiment, a host cell comprising one or more
polynucleotides of the invention is provided. In certain
embodiments a host cell comprising one or more vectors of the
invention is provided. The polynucleotides and vectors may
incorporate any of the features, singly or in combination,
described herein in relation to polynucleotides and vectors,
respectively. In one such embodiment a host cell comprises (e.g.
has been transformed or transfected with) a vector comprising a
polynucleotide that encodes (part of) a T cell activating
bispecific antigen binding molecule of the invention. As used
herein, the term "host cell" refers to any kind of cellular system
which can be engineered to generate the T cell activating
bispecific antigen binding molecules of the invention or fragments
thereof. Host cells suitable for replicating and for supporting
expression of T cell activating bispecific antigen binding
molecules are well known in the art. Such cells may be transfected
or transduced as appropriate with the particular expression vector
and large quantities of vector containing cells can be grown for
seeding large scale fermenters to obtain sufficient quantities of
the T cell activating bispecific antigen binding molecule for
clinical applications. Suitable host cells include prokaryotic
microorganisms, such as E. coli, or various eukaryotic cells, such
as Chinese hamster ovary cells (CHO), insect cells, or the like.
For example, polypeptides may be produced in bacteria in particular
when glycosylation is not needed. After expression, the polypeptide
may be isolated from the bacterial cell paste in a soluble fraction
and can be further purified. In addition to prokaryotes, eukaryotic
microbes such as filamentous fungi or yeast are suitable cloning or
expression hosts for polypeptide-encoding vectors, including fungi
and yeast strains whose glycosylation pathways have been
"humanized", resulting in the production of a polypeptide with a
partially or fully human glycosylation pattern. See Gerngross, Nat
Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24,
210-215 (2006). Suitable host cells for the expression of
(glycosylated) polypeptides are also derived from multicellular
organisms (invertebrates and vertebrates). Examples of invertebrate
cells include plant and insect cells. Numerous baculoviral strains
have been identified which may be used in conjunction with insect
cells, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures can also be utilized as hosts. See e.g.
U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and
6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants). Vertebrate cells may also be used
as hosts. For example, mammalian cell lines that are adapted to
grow in suspension may be useful. Other examples of useful
mammalian host cell lines are monkey kidney CV1 line transformed by
SV40 (COS-7); human embryonic kidney line (293 or 293T cells as
described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby
hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as
described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey
kidney cells (CV1), African green monkey kidney cells (VERO-76),
human cervical carcinoma cells (HELA), canine kidney cells (MDCK),
buffalo rat liver cells (BRL 3A), human lung cells (W138), human
liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI
cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci
383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including dhfr.sup.- CHO cells (Urlaub et al., Proc Natl
Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO,
NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell
lines suitable for protein production, see, e.g., Yazaki and Wu,
Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana
Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include
cultured cells, e.g., mammalian cultured cells, yeast cells, insect
cells, bacterial cells and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue. In one embodiment, the host
cell is a eukaryotic cell, preferably a mammalian cell, such as a
Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK)
cell or a lymphoid cell (e.g., YO, NS0, Sp20 cell).
[0230] Standard technologies are known in the art to express
foreign genes in these systems. Cells expressing a polypeptide
comprising either the heavy or the light chain of an antigen
binding domain such as an antibody, may be engineered so as to also
express the other of the antibody chains such that the expressed
product is an antibody that has both a heavy and a light chain.
[0231] In one embodiment, a method of producing a T cell activating
bispecific antigen binding molecule according to the invention is
provided, wherein the method comprises culturing a host cell
comprising a polynucleotide encoding the T cell activating
bispecific antigen binding molecule, as provided herein, under
conditions suitable for expression of the T cell activating
bispecific antigen binding molecule, and recovering the T cell
activating bispecific antigen binding molecule from the host cell
(or host cell culture medium).
[0232] The components of the T cell activating bispecific antigen
binding molecule are genetically fused to each other. T cell
activating bispecific antigen binding molecule can be designed such
that its components are fused directly to each other or indirectly
through a linker sequence. The composition and length of the linker
may be determined in accordance with methods well known in the art
and may be tested for efficacy. Examples of linker sequences
between different components of T cell activating bispecific
antigen binding molecules are found in the sequences provided
herein. Additional sequences may also be included to incorporate a
cleavage site to separate the individual components of the fusion
if desired, for example an endopeptidase recognition sequence.
[0233] In certain embodiments the one or more antigen binding
moieties of the T cell activating bispecific antigen binding
molecules comprise at least an antibody variable region capable of
binding an antigenic determinant. Variable regions can form part of
and be derived from naturally or non-naturally occurring antibodies
and fragments thereof. Methods to produce polyclonal antibodies and
monoclonal antibodies are well known in the art (see e.g. Harlow
and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor
Laboratory, 1988). Non-naturally occurring antibodies can be
constructed using solid phase-peptide synthesis, can be produced
recombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can
be obtained, for example, by screening combinatorial libraries
comprising variable heavy chains and variable light chains (see
e.g. U.S. Pat. No. 5,969,108 to McCafferty).
[0234] Any animal species of antibody, antibody fragment, antigen
binding domain or variable region can be used in the T cell
activating bispecific antigen binding molecules of the invention.
Non-limiting antibodies, antibody fragments, antigen binding
domains or variable regions useful in the present invention can be
of murine, primate, or human origin. If the T cell activating
bispecific antigen binding molecule is intended for human use, a
chimeric form of antibody may be used wherein the constant regions
of the antibody are from a human. A humanized or fully human form
of the antibody can also be prepared in accordance with methods
well known in the art (see e. g. U.S. Pat. No. 5,565,332 to
Winter). Humanization may be achieved by various methods including,
but not limited to (a) grafting the non-human (e.g., donor
antibody) CDRs onto human (e.g. recipient antibody) framework and
constant regions with or without retention of critical framework
residues (e.g. those that are important for retaining good antigen
binding affinity or antibody functions), (b) grafting only the
non-human specificity-determining regions (SDRs or a-CDRs; the
residues critical for the antibody-antigen interaction) onto human
framework and constant regions, or (c) transplanting the entire
non-human variable domains, but "cloaking" them with a human-like
section by replacement of surface residues. Humanized antibodies
and methods of making them are reviewed, e.g., in Almagro and
Fransson, Front Biosci 13, 1619-1633 (2008), and are further
described, e.g., in Riechmann et al., Nature 332, 323-329 (1988);
Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S.
Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et
al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad
Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92
(1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan,
Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36,
25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol
28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al.,
Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn
et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83,
252-260 (2000) (describing the "guided selection" approach to FR
shuffling). Human antibodies and human variable regions can be
produced using various techniques known in the art. Human
antibodies are described generally in van Dijk and van de Winkel,
Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol
20, 450-459 (2008). Human variable regions can form part of and be
derived from human monoclonal antibodies made by the hybridoma
method (see e.g. Monoclonal Antibody Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
Human antibodies and human variable regions may also be prepared by
administering an immunogen to a transgenic animal that has been
modified to produce intact human antibodies or intact antibodies
with human variable regions in response to antigenic challenge (see
e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies
and human variable regions may also be generated by isolating Fv
clone variable region sequences selected from human-derived phage
display libraries (see e.g., Hoogenboom et al. in Methods in
Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001); and McCafferty et al., Nature 348, 552-554;
Clackson et al., Nature 352, 624-628 (1991)). Phage typically
display antibody fragments, either as single-chain Fv (scFv)
fragments or as Fab fragments.
[0235] In certain embodiments, the antigen binding moieties useful
in the present invention are engineered to have enhanced binding
affinity according to, for example, the methods disclosed in U.S.
Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are
hereby incorporated by reference. The ability of the T cell
activating bispecific antigen binding molecule of the invention to
bind to a specific antigenic determinant can be measured either
through an enzyme-linked immunosorbent assay (ELISA) or other
techniques familiar to one of skill in the art, e.g. surface
plasmon resonance technique (analyzed on a BIACORE T100 system)
(Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional
binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition
assays may be used to identify an antibody, antibody fragment,
antigen binding domain or variable domain that competes with a
reference antibody for binding to a particular antigen, e.g. an
antibody that competes with the V9 antibody for binding to CD3. In
certain embodiments, such a competing antibody binds to the same
epitope (e.g. a linear or a conformational epitope) that is bound
by the reference antibody. Detailed exemplary methods for mapping
an epitope to which an antibody binds are provided in Morris (1996)
"Epitope Mapping Protocols," in Methods in Molecular Biology vol.
66 (Humana Press, Totowa, N.J.). In an exemplary competition assay,
immobilized antigen (e.g. CD3) is incubated in a solution
comprising a first labeled antibody that binds to the antigen (e.g.
V9 antibody, described in U.S. Pat. No. 6,054,297) and a second
unlabeled antibody that is being tested for its ability to compete
with the first antibody for binding to the antigen. The second
antibody may be present in a hybridoma supernatant. As a control,
immobilized antigen is incubated in a solution comprising the first
labeled antibody but not the second unlabeled antibody. After
incubation under conditions permissive for binding of the first
antibody to the antigen, excess unbound antibody is removed, and
the amount of label associated with immobilized antigen is
measured. If the amount of label associated with immobilized
antigen is substantially reduced in the test sample relative to the
control sample, then that indicates that the second antibody is
competing with the first antibody for binding to the antigen. See
Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0236] T cell activating bispecific antigen binding molecules
prepared as described herein may be purified by art-known
techniques such as high performance liquid chromatography, ion
exchange chromatography, gel electrophoresis, affinity
chromatography, size exclusion chromatography, and the like. The
actual conditions used to purify a particular protein will depend,
in part, on factors such as net charge, hydrophobicity,
hydrophilicity etc., and will be apparent to those having skill in
the art. For affinity chromatography purification an antibody,
ligand, receptor or antigen can be used to which the T cell
activating bispecific antigen binding molecule binds. For example,
for affinity chromatography purification of T cell activating
bispecific antigen binding molecules of the invention, a matrix
with protein A or protein G may be used. Sequential Protein A or G
affinity chromatography and size exclusion chromatography can be
used to isolate a T cell activating bispecific antigen binding
molecule essentially as described in the Examples. The purity of
the T cell activating bispecific antigen binding molecule can be
determined by any of a variety of well known analytical methods
including gel electrophoresis, high pressure liquid chromatography,
and the like. For example, the heavy chain fusion proteins
expressed as described in the Examples were shown to be intact and
properly assembled as demonstrated by reducing SDS-PAGE (see e.g.
FIG. 4). Three bands were resolved at approximately Mr 25,000, Mr
50,000 and Mr 75,000, corresponding to the predicted molecular
weights of the T cell activating bispecific antigen binding
molecule light chain, heavy chain and heavy chain/light chain
fusion protein.
Assays
[0237] T cell activating bispecific antigen binding molecules
provided herein may be identified, screened for, or characterized
for their physical/chemical properties and/or biological activities
by various assays known in the art.
Affinity Assays
[0238] The affinity of the T cell activating bispecific antigen
binding molecule for an Fc receptor or a target antigen can be
determined in accordance with the methods set forth in the Examples
by surface plasmon resonance (SPR), using standard instrumentation
such as a BIAcore instrument (GE Healthcare), and receptors or
target proteins such as may be obtained by recombinant expression.
Alternatively, binding of T cell activating bispecific antigen
binding molecules for different receptors or target antigens may be
evaluated using cell lines expressing the particular receptor or
target antigen, for example by flow cytometry (FACS). A specific
illustrative and exemplary embodiment for measuring binding
affinity is described in the following and in the Examples
below.
[0239] According to one embodiment, K.sub.D is measured by surface
plasmon resonance using a BIACORE.RTM. T100 machine (GE Healthcare)
at 25.degree. C.
[0240] To analyze the interaction between the Fc-portion and Fc
receptors, His-tagged recombinant Fc-receptor is captured by an
anti-Penta His antibody (Qiagen) immobilized on CM5 chips and the
bispecific constructs are used as analytes. Briefly,
carboxymethylated dextran biosensor chips (CM5, GE Healthcare) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Anti Penta-His antibody is diluted with 10
mM sodium acetate, pH 5.0, to 40 .mu.g/ml before injection at a
flow rate of 5 .mu.l/min to achieve approximately 6500 response
units (RU) of coupled protein. Following the injection of the
ligand, 1 M ethanolamine is injected to block unreacted groups.
Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.
For kinetic measurements, four-fold serial dilutions of the
bispecific construct (range between 500 nM and 4000 nM) are
injected in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM
EDTA, 0.05% Surfactant P20, pH 7.4) at 25.degree. C. at a flow rate
of 30 .mu.l/min for 120 s.
[0241] To determine the affinity to the target antigen, bispecific
constructs are captured by an anti human Fab specific antibody (GE
Healthcare) that is immobilized on an activated CM5-sensor chip
surface as described for the anti Penta-His antibody. The final
amount of coupled protein is approximately 12000 RU. The bispecific
constructs are captured for 90 s at 300 nM. The target antigens are
passed through the flow cells for 180 s at a concentration range
from 250 to 1000 nM with a flowrate of 30 .mu.l/min. The
dissociation is monitored for 180 s.
[0242] Bulk refractive index differences are corrected for by
subtracting the response obtained on reference flow cell. The
steady state response was used to derive the dissociation constant
K.sub.D by non-linear curve fitting of the Langmuir binding
isotherm. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. T100 Evaluation Software version 1.1.1)
by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (K.sub.D) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J Mol Biol 293, 865-881 (1999).
Activity Assays
[0243] Biological activity of the T cell activating bispecific
antigen binding molecules of the invention can be measured by
various assays as described in the Examples. Biological activities
may for example include the induction of proliferation of T cells,
the induction of signaling in T cells, the induction of expression
of activation markers in T cells, the induction of cytokine
secretion by T cells, the induction of lysis of target cells such
as tumor cells, and the induction of tumor regression and/or the
improvement of survival.
Compositions, Formulations, and Routes of Administration
[0244] In a further aspect, the invention provides pharmaceutical
compositions comprising any of the T cell activating bispecific
antigen binding molecules provided herein, e.g., for use in any of
the below therapeutic methods. In one embodiment, a pharmaceutical
composition comprises any of the T cell activating bispecific
antigen binding molecules provided herein and a pharmaceutically
acceptable carrier. In another embodiment, a pharmaceutical
composition comprises any of the T cell activating bispecific
antigen binding molecules provided herein and at least one
additional therapeutic agent, e.g., as described below.
[0245] Further provided is a method of producing a T cell
activating bispecific antigen binding molecule of the invention in
a form suitable for administration in vivo, the method comprising
(a) obtaining a T cell activating bispecific antigen binding
molecule according to the invention, and (b) formulating the T cell
activating bispecific antigen binding molecule with at least one
pharmaceutically acceptable carrier, whereby a preparation of T
cell activating bispecific antigen binding molecule is formulated
for administration in vivo.
[0246] Pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of one or more T cell
activating bispecific antigen binding molecule dissolved or
dispersed in a pharmaceutically acceptable carrier. The phrases
"pharmaceutical or pharmacologically acceptable" refers to
molecular entities and compositions that are generally non-toxic to
recipients at the dosages and concentrations employed, i.e. do not
produce an adverse, allergic or other untoward reaction when
administered to an animal, such as, for example, a human, as
appropriate. The preparation of a pharmaceutical composition that
contains at least one T cell activating bispecific antigen binding
molecule and optionally an additional active ingredient will be
known to those of skill in the art in light of the present
disclosure, as exemplified by Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, incorporated herein by
reference. Moreover, for animal (e.g., human) administration, it
will be understood that preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards or corresponding authorities in
other countries. Preferred compositions are lyophilized
formulations or aqueous solutions. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, buffers, dispersion media, coatings, surfactants,
antioxidants, preservatives (e.g. antibacterial agents, antifungal
agents), isotonic agents, absorption delaying agents, salts,
preservatives, antioxidants, proteins, drugs, drug stabilizers,
polymers, gels, binders, excipients, disintegration agents,
lubricants, sweetening agents, flavoring agents, dyes, such like
materials and combinations thereof, as would be known to one of
ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.
1289-1329, incorporated herein by reference). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated.
[0247] The composition may comprise different types of carriers
depending on whether it is to be administered in solid, liquid or
aerosol form, and whether it need to be sterile for such routes of
administration as injection. T cell activating bispecific antigen
binding molecules of the present invention (and any additional
therapeutic agent) can be administered intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostatically,
intrasplenically, intrarenally, intrapleurally, intratracheally,
intranasally, intravitreally, intravaginally, intrarectally,
intratumorally, intramuscularly, intraperitoneally, subcutaneously,
subconjunctivally, intravesicularlly, mucosally,
intrapericardially, intraumbilically, intraocularally, orally,
topically, locally, by inhalation (e.g. aerosol inhalation),
injection, infusion, continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g. liposomes), or by other method
or any combination of the forgoing as would be known to one of
ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference). Parenteral administration, in
particular intravenous injection, is most commonly used for
administering polypeptide molecules such as the T cell activating
bispecific antigen binding molecules of the invention.
[0248] Parenteral compositions include those designed for
administration by injection, e.g. subcutaneous, intradermal,
intralesional, intravenous, intraarterial intramuscular,
intrathecal or intraperitoneal injection. For injection, the T cell
activating bispecific antigen binding molecules of the invention
may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. The solution may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the T cell activating bispecific
antigen binding molecules may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before
use. Sterile injectable solutions are prepared by incorporating the
T cell activating bispecific antigen binding molecules of the
invention in the required amount in the appropriate solvent with
various of the other ingredients enumerated below, as required.
Sterility may be readily accomplished, e.g., by filtration through
sterile filtration membranes. Generally, dispersions are prepared
by incorporating the various sterilized active ingredients into a
sterile vehicle which contains the basic dispersion medium and/or
the other ingredients. In the case of sterile powders for the
preparation of sterile injectable solutions, suspensions or
emulsion, the preferred methods of preparation are vacuum-drying or
freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered liquid medium thereof. The liquid medium should be
suitably buffered if necessary and the liquid diluent first
rendered isotonic prior to injection with sufficient saline or
glucose. The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. It will be
appreciated that endotoxin contamination should be kept minimally
at a safe level, for example, less that 0.5 ng/mg protein. Suitable
pharmaceutically acceptable carriers include, but are not limited
to: buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Aqueous injection suspensions may
contain compounds which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, dextran, or the
like. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl cleats or
triglycerides, or liposomes.
[0249] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing
Company, 1990). Sustained-release preparations may be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
e.g. films, or microcapsules. In particular embodiments, prolonged
absorption of an injectable composition can be brought about by the
use in the compositions of agents delaying absorption, such as, for
example, aluminum monostearate, gelatin or combinations
thereof.
[0250] In addition to the compositions described previously, the T
cell activating bispecific antigen binding molecules may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the T cell activating bispecific antigen binding molecules may be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0251] Pharmaceutical compositions comprising the T cell activating
bispecific antigen binding molecules of the invention may be
manufactured by means of conventional mixing, dissolving,
emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions may be formulated in conventional
manner using one or more physiologically acceptable carriers,
diluents, excipients or auxiliaries which facilitate processing of
the proteins into preparations that can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen.
[0252] The T cell activating bispecific antigen binding molecules
may be formulated into a composition in a free acid or base,
neutral or salt form. Pharmaceutically acceptable salts are salts
that substantially retain the biological activity of the free acid
or base. These include the acid addition salts, e.g., those formed
with the free amino groups of a proteinaceous composition, or which
are formed with inorganic acids such as for example, hydrochloric
or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric or mandelic acid. Salts formed with the free carboxyl
groups can also be derived from inorganic bases such as for
example, sodium, potassium, ammonium, calcium or ferric hydroxides;
or such organic bases as isopropylamine, trimethylamine, histidine
or procaine. Pharmaceutical salts tend to be more soluble in
aqueous and other protic solvents than are the corresponding free
base forms.
Therapeutic Methods and Compositions
[0253] Any of the T cell activating bispecific antigen binding
molecules provided herein may be used in therapeutic methods. T
cell activating bispecific antigen binding molecules of the
invention can be used as immunotherapeutic agents, for example in
the treatment of cancers.
[0254] For use in therapeutic methods, T cell activating bispecific
antigen binding molecules of the invention would be formulated,
dosed, and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners.
[0255] In one aspect, T cell activating bispecific antigen binding
molecules of the invention for use as a medicament are provided. In
further aspects, T cell activating bispecific antigen binding
molecules of the invention for use in treating a disease are
provided. In certain embodiments, T cell activating bispecific
antigen binding molecules of the invention for use in a method of
treatment are provided. In one embodiment, the invention provides a
T cell activating bispecific antigen binding molecule as described
herein for use in the treatment of a disease in an individual in
need thereof. In certain embodiments, the invention provides a T
cell activating bispecific antigen binding molecule for use in a
method of treating an individual having a disease comprising
administering to the individual a therapeutically effective amount
of the T cell activating bispecific antigen binding molecule. In
certain embodiments the disease to be treated is a proliferative
disorder. In a particular embodiment the disease is cancer. In
certain embodiments the method further comprises administering to
the individual a therapeutically effective amount of at least one
additional therapeutic agent, e.g., an anti-cancer agent if the
disease to be treated is cancer. In further embodiments, the
invention provides a T cell activating bispecific antigen binding
molecule as described herein for use in inducing lysis of a target
cell, particularly a tumor cell. In certain embodiments, the
invention provides a T cell activating bispecific antigen binding
molecule for use in a method of inducing lysis of a target cell,
particularly a tumor cell, in an individual comprising
administering to the individual an effective amount of the T cell
activating bispecific antigen binding molecule to induce lysis of a
target cell. An "individual" according to any of the above
embodiments is a mammal, preferably a human.
[0256] In a further aspect, the invention provides for the use of a
T cell activating bispecific antigen binding molecule of the
invention in the manufacture or preparation of a medicament. In one
embodiment the medicament is for the treatment of a disease in an
individual in need thereof. In a further embodiment, the medicament
is for use in a method of treating a disease comprising
administering to an individual having the disease a therapeutically
effective amount of the medicament. In certain embodiments the
disease to be treated is a proliferative disorder. In a particular
embodiment the disease is cancer. In one embodiment, the method
further comprises administering to the individual a therapeutically
effective amount of at least one additional therapeutic agent,
e.g., an anti-cancer agent if the disease to be treated is cancer.
In a further embodiment, the medicament is for inducing lysis of a
target cell, particularly a tumor cell. In still a further
embodiment, the medicament is for use in a method of inducing lysis
of a target cell, particularly a tumor cell, in an individual
comprising administering to the individual an effective amount of
the medicament to induce lysis of a target cell. An "individual"
according to any of the above embodiments may be a mammal,
preferably a human.
[0257] In a further aspect, the invention provides a method for
treating a disease. In one embodiment, the method comprises
administering to an individual having such disease a
therapeutically effective amount of a T cell activating bispecific
antigen binding molecule of the invention. In one embodiment a
composition is administered to said individual, comprising the T
cell activating bispecific antigen binding molecule of the
invention in a pharmaceutically acceptable form. In certain
embodiments the disease to be treated is a proliferative disorder.
In a particular embodiment the disease is cancer. In certain
embodiments the method further comprises administering to the
individual a therapeutically effective amount of at least one
additional therapeutic agent, e.g., an anti-cancer agent if the
disease to be treated is cancer. An "individual" according to any
of the above embodiments may be a mammal, preferably a human.
[0258] In a further aspect, the invention provides a method for
inducing lysis of a target cell, particularly a tumor cell. In one
embodiment the method comprises contacting a target cell with a T
cell activating bispecific antigen binding molecule of the
invention in the presence of a T cell, particularly a cytotoxic T
cell. In a further aspect, a method for inducing lysis of a target
cell, particularly a tumor cell, in an individual is provided. In
one such embodiment, the method comprises administering to the
individual an effective amount of a T cell activating bispecific
antigen binding molecule to induce lysis of a target cell. In one
embodiment, an "individual" is a human.
[0259] In certain embodiments the disease to be treated is a
proliferative disorder, particularly cancer. Non-limiting examples
of cancers include bladder cancer, brain cancer, head and neck
cancer, pancreatic cancer, lung cancer, breast cancer, ovarian
cancer, uterine cancer, cervical cancer, endometrial cancer,
esophageal cancer, colon cancer, colorectal cancer, rectal cancer,
gastric cancer, prostate cancer, blood cancer, skin cancer,
squamous cell carcinoma, bone cancer, and kidney cancer. Other cell
proliferation disorders that can be treated using a T cell
activating bispecific antigen binding molecule of the present
invention include, but are not limited to neoplasms located in the:
abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
system (central and peripheral), lymphatic system, pelvic, skin,
soft tissue, spleen, thoracic region, and urogenital system. Also
included are pre-cancerous conditions or lesions and cancer
metastases. In certain embodiments the cancer is chosen from the
group consisting of renal cell cancer, skin cancer, lung cancer,
colorectal cancer, breast cancer, brain cancer, head and neck
cancer. A skilled artisan readily recognizes that in many cases the
T cell activating bispecific antigen binding molecule may not
provide a cure but may only provide partial benefit. In some
embodiments, a physiological change having some benefit is also
considered therapeutically beneficial. Thus, in some embodiments,
an amount of T cell activating bispecific antigen binding molecule
that provides a physiological change is considered an "effective
amount" or a "therapeutically effective amount". The subject,
patient, or individual in need of treatment is typically a mammal,
more specifically a human.
[0260] In some embodiments, an effective amount of a T cell
activating bispecific antigen binding molecule of the invention is
administered to a cell. In other embodiments, a therapeutically
effective amount of a T cell activating bispecific antigen binding
molecule of the invention is administered to an individual for the
treatment of disease.
[0261] For the prevention or treatment of disease, the appropriate
dosage of a T cell activating bispecific antigen binding molecule
of the invention (when used alone or in combination with one or
more other additional therapeutic agents) will depend on the type
of disease to be treated, the route of administration, the body
weight of the patient, the type of T cell activating bispecific
antigen binding molecule, the severity and course of the disease,
whether the T cell activating bispecific antigen binding molecule
is administered for preventive or therapeutic purposes, previous or
concurrent therapeutic interventions, the patient's clinical
history and response to the T cell activating bispecific antigen
binding molecule, and the discretion of the attending physician.
The practitioner responsible for administration will, in any event,
determine the concentration of active ingredient(s) in a
composition and appropriate dose(s) for the individual subject.
Various dosing schedules including but not limited to single or
multiple administrations over various time-points, bolus
administration, and pulse infusion are contemplated herein.
[0262] The T cell activating bispecific antigen binding molecule is
suitably administered to the patient at one time or over a series
of treatments. Depending on the type and severity of the disease,
about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell
activating bispecific antigen binding molecule can be an initial
candidate dosage for administration to the patient, whether, for
example, by one or more separate administrations, or by continuous
infusion. One typical daily dosage might range from about 1
.mu.g/kg to 100 mg/kg or more, depending on the factors mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment would generally be
sustained until a desired suppression of disease symptoms occurs.
One exemplary dosage of the T cell activating bispecific antigen
binding molecule would be in the range from about 0.005 mg/kg to
about 10 mg/kg. In other non-limiting examples, a dose may also
comprise from about 1 microgram/kg body weight, about 5
microgram/kg body weight, about 10 microgram/kg body weight, about
50 microgram/kg body weight, about 100 microgram/kg body weight,
about 200 microgram/kg body weight, about 350 microgram/kg body
weight, about 500 microgram/kg body weight, about 1 milligram/kg
body weight, about 5 milligram/kg body weight, about 10
milligram/kg body weight, about 50 milligram/kg body weight, about
100 milligram/kg body weight, about 200 milligram/kg body weight,
about 350 milligram/kg body weight, about 500 milligram/kg body
weight, to about 1000 mg/kg body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 5
mg/kg body weight to about 100 mg/kg body weight, about 5
microgram/kg body weight to about 500 milligram/kg body weight,
etc., can be administered, based on the numbers described above.
Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or
10 mg/kg (or any combination thereof) may be administered to the
patient. Such doses may be administered intermittently, e.g. every
week or every three weeks (e.g. such that the patient receives from
about two to about twenty, or e.g. about six doses of the T cell
activating bispecific antigen binding molecule). An initial higher
loading dose, followed by one or more lower doses may be
administered. However, other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional
techniques and assays.
[0263] The T cell activating bispecific antigen binding molecules
of the invention will generally be used in an amount effective to
achieve the intended purpose. For use to treat or prevent a disease
condition, the T cell activating bispecific antigen binding
molecules of the invention, or pharmaceutical compositions thereof,
are administered or applied in a therapeutically effective amount.
Determination of a therapeutically effective amount is well within
the capabilities of those skilled in the art, especially in light
of the detailed disclosure provided herein.
[0264] For systemic administration, a therapeutically effective
dose can be estimated initially from in vitro assays, such as cell
culture assays. A dose can then be formulated in animal models to
achieve a circulating concentration range that includes the
IC.sub.50 as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans.
[0265] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One having ordinary skill in the art could readily optimize
administration to humans based on animal data.
[0266] Dosage amount and interval may be adjusted individually to
provide plasma levels of the T cell activating bispecific antigen
binding molecules which are sufficient to maintain therapeutic
effect. Usual patient dosages for administration by injection range
from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1
mg/kg/day. Therapeutically effective plasma levels may be achieved
by administering multiple doses each day. Levels in plasma may be
measured, for example, by HPLC.
[0267] In cases of local administration or selective uptake, the
effective local concentration of the T cell activating bispecific
antigen binding molecules may not be related to plasma
concentration. One having skill in the art will be able to optimize
therapeutically effective local dosages without undue
experimentation.
[0268] A therapeutically effective dose of the T cell activating
bispecific antigen binding molecules described herein will
generally provide therapeutic benefit without causing substantial
toxicity. Toxicity and therapeutic efficacy of a T cell activating
bispecific antigen binding molecule can be determined by standard
pharmaceutical procedures in cell culture or experimental animals.
Cell culture assays and animal studies can be used to determine the
LD.sub.50 (the dose lethal to 50% of a population) and the
ED.sub.50 (the dose therapeutically effective in 50% of a
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index, which can be expressed as the ratio
LD.sub.50/ED.sub.50. T cell activating bispecific antigen binding
molecules that exhibit large therapeutic indices are preferred. In
one embodiment, the T cell activating bispecific antigen binding
molecule according to the present invention exhibits a high
therapeutic index. The data obtained from cell culture assays and
animal studies can be used in formulating a range of dosages
suitable for use in humans. The dosage lies preferably within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon a variety of factors, e.g., the dosage form
employed, the route of administration utilized, the condition of
the subject, and the like. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition (see, e.g., Fingl et al., 1975,
in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1,
incorporated herein by reference in its entirety).
[0269] The attending physician for patients treated with T cell
activating bispecific antigen binding molecules of the invention
would know how and when to terminate, interrupt, or adjust
administration due to toxicity, organ dysfunction, and the like.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administered
dose in the management of the disorder of interest will vary with
the severity of the condition to be treated, with the route of
administration, and the like. The severity of the condition may,
for example, be evaluated, in part, by standard prognostic
evaluation methods. Further, the dose and perhaps dose frequency
will also vary according to the age, body weight, and response of
the individual patient.
Other Agents and Treatments
[0270] The T cell activating bispecific antigen binding molecules
of the invention may be administered in combination with one or
more other agents in therapy. For instance, a T cell activating
bispecific antigen binding molecule of the invention may be
co-administered with at least one additional therapeutic agent. The
term "therapeutic agent" encompasses any agent administered to
treat a symptom or disease in an individual in need of such
treatment. Such additional therapeutic agent may comprise any
active ingredients suitable for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. In certain embodiments, an additional
therapeutic agent is an immunomodulatory agent, a cytostatic agent,
an inhibitor of cell adhesion, a cytotoxic agent, an activator of
cell apoptosis, or an agent that increases the sensitivity of cells
to apoptotic inducers. In a particular embodiment, the additional
therapeutic agent is an anti-cancer agent, for example a
microtubule disruptor, an antimetabolite, a topoisomerase
inhibitor, a DNA intercalator, an alkylating agent, a hormonal
therapy, a kinase inhibitor, a receptor antagonist, an activator of
tumor cell apoptosis, or an antiangiogenic agent. Such other agents
are suitably present in combination in amounts that are effective
for the purpose intended. The effective amount of such other agents
depends on the amount of T cell activating bispecific antigen
binding molecule used, the type of disorder or treatment, and other
factors discussed above. The T cell activating bispecific antigen
binding molecules are generally used in the same dosages and with
administration routes as described herein, or about from 1 to 99%
of the dosages described herein, or in any dosage and by any route
that is empirically/clinically determined to be appropriate.
[0271] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate compositions), and separate administration,
in which case, administration of the T cell activating bispecific
antigen binding molecule of the invention can occur prior to,
simultaneously, and/or following, administration of the additional
therapeutic agent and/or adjuvant. T cell activating bispecific
antigen binding molecules of the invention can also be used in
combination with radiation therapy.
Articles of Manufacture
[0272] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a T cell activating bispecific antigen
binding molecule of the invention. The label or package insert
indicates that the composition is used for treating the condition
of choice. Moreover, the article of manufacture may comprise (a) a
first container with a composition contained therein, wherein the
composition comprises a T cell activating bispecific antigen
binding molecule of the invention; and (b) a second container with
a composition contained therein, wherein the composition comprises
a further cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
EXAMPLES
[0273] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
General Methods
Recombinant DNA Techniques
[0274] Standard methods were used to manipulate DNA as described in
Sambrook et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturers' instructions. General information regarding the
nucleotide sequences of human immunoglobulins light and heavy
chains is given in: Kabat, E. A. et al., (1991) Sequences of
Proteins of Immunological Interest, 5.sup.th ed., NIH Publication
No. 91-3242.
DNA Sequencing
[0275] DNA sequences were determined by double strand
sequencing.
Gene Synthesis
[0276] Desired gene segments where required were either generated
by PCR using appropriate templates or were synthesized by Geneart
AG (Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. In cases where no exact gene
sequence was available, oligonucleotide primers were designed based
on sequences from closest homologues and the genes were isolated by
RT-PCR from RNA originating from the appropriate tissue. The gene
segments flanked by singular restriction endonuclease cleavage
sites were cloned into standard cloning/sequencing vectors. The
plasmid DNA was purified from transformed bacteria and
concentration determined by UV spectroscopy. The DNA sequence of
the subcloned gene fragments was confirmed by DNA sequencing. Gene
segments were designed with suitable restriction sites to allow
sub-cloning into the respective expression vectors. All constructs
were designed with a 5'-end DNA sequence coding for a leader
peptide which targets proteins for secretion in eukaryotic cells.
Exemplary leader peptides and polynucleotide sequences encoding
them are depicted SEQ ID NOs 108-116.
Isolation of Primary Human Pan T Cells from PBMCs
[0277] Peripheral blood mononuclear cells (PBMCs) were prepared by
Histopaque density centrifugation from enriched lymphocyte
preparations (buffy coats) obtained from local blood banks or from
fresh blood from healthy human donors. Briefly, blood was diluted
with sterile PBS and carefully layered over a Histopaque gradient
(Sigma, H8889). After centrifugation for 30 minutes at 450.times.g
at room temperature (brake switched off), part of the plasma above
the PBMC containing interphase was discarded. The PBMCs were
transferred into new 50 ml Falcon tubes and tubes were filled up
with PBS to a total volume of 50 ml. The mixture was centrifuged at
room temperature for 10 minutes at 400.times.g (brake switched on).
The supernatant was discarded and the PBMC pellet washed twice with
sterile PBS (centrifugation steps at 4.degree. C. for 10 minutes at
350.times.g). The resulting PBMC population was counted
automatically (ViCell) and stored in RPMI1640 medium, containing
10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37.degree.
C., 5% CO.sub.2 in the incubator until assay start.
[0278] T cell enrichment from PBMCs was performed using the Pan T
Cell Isolation Kit II (Miltenyi Biotec #130-091-156), according to
the manufacturer's instructions. Briefly, the cell pellets were
diluted in 40 .mu.l cold buffer per 10 million cells (PBS with 0.5%
BSA, 2 mM EDTA, sterile filtered) and incubated with 10 .mu.l
Biotin-Antibody Cocktail per 10 million cells for 10 min at
4.degree. C. 30 .mu.l cold buffer and 20 .mu.l Anti-Biotin magnetic
beads per 10 million cells were added, and the mixture incubated
for another 15 min at 4.degree. C. Cells were washed by adding
10-20.times. the current volume of buffer and a subsequent
centrifugation step at 300.times.g for 10 min. Up to 100 million
cells were resuspended in 500 .mu.l buffer. Magnetic separation of
unlabeled human pan T cells was performed using LS columns
(Miltenyi Biotec #130-042-401) according to the manufacturer's
instructions. The resulting T cell population was counted
automatically (ViCell) and stored in AIM-V medium at 37.degree. C.,
5% CO.sub.2 in the incubator until assay start (not longer than 24
h).
Isolation of Primary Human Naive T Cells from PBMCs
[0279] Peripheral blood mononuclar cells (PBMCs) were prepared by
Histopaque density centrifugation from enriched lymphocyte
preparations (buffy coats) obtained from local blood banks or from
fresh blood from healthy human donors. T-cell enrichment from PBMCs
was performed using the Naive CD8.sup.+ T cell isolation Kit from
Miltenyi Biotec (#130-093-244), according to the manufacturer's
instructions, but skipping the last isolation step of CD8.sup.+ T
cells (also see description for the isolation of primary human pan
T cells).
Isolation of Murine Pan T Cells from Splenocytes
[0280] Spleens were isolated from C57BL/6 mice, transferred into a
GentleMACS C-tube (Miltenyi Biotech #130-093-237) containing MACS
buffer (PBS+0.5% BSA+2 mM EDTA) and dissociated with the GentleMACS
Dissociator to obtain single-cell suspensions according to the
manufacturer's instructions. The cell suspension was passed through
a pre-separation filter to remove remaining undissociated tissue
particles. After centrifugation at 400.times.g for 4 min at
4.degree. C., ACK Lysis Buffer was added to lyse red blood cells
(incubation for 5 min at room temperature). The remaining cells
were washed with MACS buffer twice, counted and used for the
isolation of murine pan T cells. The negative (magnetic) selection
was performed using the Pan T Cell Isolation Kit from Miltenyi
Biotec (#130-090-861), following the manufacturer's instructions.
The resulting T cell population was automatically counted (ViCell)
and immediately used for further assays.
Isolation of Primary Cynomolgus PBMCs from Heparinized Blood
[0281] Peripheral blood mononuclar cells (PBMCs) were prepared by
density centrifugation from fresh blood from healthy cynomolgus
donors, as follows: Heparinized blood was diluted 1:3 with sterile
PBS, and Lymphoprep medium (Axon Lab #1114545) was diluted to 90%
with sterile PBS. Two volumes of the diluted blood were layered
over one volume of the diluted density gradient and the PBMC
fraction was separated by centrifugation for 30 min at 520.times.g,
without brake, at room temperature. The PBMC band was transferred
into a fresh 50 ml Falcon tube and washed with sterile PBS by
centrifugation for 10 min at 400.times.g at 4.degree. C. One
low-speed centrifugation was performed to remove the platelets (15
min at 150.times.g, 4.degree. C.), and the resulting PBMC
population was automatically counted (ViCell) and immediately used
for further assays.
Target Cells
[0282] For the assessment of MCSP-targeting bispecific antigen
binding molecules, the following tumor cell lines were used: the
human melanoma cell line WM266-4 (ATCC #CRL-1676), derived from a
metastatic site of a malignant melanoma and expressing high levels
of human MCSP; the human melanoma cell line MV-3 (a kind gift from
The Radboud University Nijmegen Medical Centre), expressing medium
levels of human MCSP; the human malignant melanoma (primary tumour)
cell line A375 (ECACC #88113005) expressing high levels of MCSP;
the human colon carcinoma cell line HCT-116 (ATCC #CCL-247) that
does not express MCSP; and the human Caucasian colon adenocarcinoma
cell line LS180 (ECACC #87021202) that does not express MCSP. For
the assessment of CEA-targeting bispecific antigen binding
molecules, the following tumor cell lines were used: the human
gastric cancer cell line MKN45 (DSMZ #ACC 409), expressing very
high levels of human CEA; the human pancreas adenocarcinoma cell
line HPAF-II (kind gift of Roche Nutley), expressing high levels of
human CEA; the human primary pancreatic adenocarcinoma cell line
BxPC-3 (ECACC #93120816) expressing medium levels of human CEA; the
human female Caucasian colon adenocarcinoma cell line LS-174T
(ECACC #87060401), expressing medium levels of human CEA; the human
pancreas adenocarcinoma cell line ASPC-1 (ECACC #96020930)
expressing low levels of human CEA; the human epithelioid
pancreatic carcinoma cell line Panc-1 (ATCC #CRL-1469), expressing
(very) low levels of human CEA; the human colon carcinoma cell line
HCT-116 (ATCC #CCL-247) that does not express CEA; a human
adenocarcinomic alveolar basal epithelial cell line A549-huCEA that
was stably transfected in-house to express human CEA; and a murine
colon carcinoma cell line MC38-huCEA, that was engineered in-house
to stably express human CEA.
[0283] In addition, a human T cell leukaemia cell line, Jurkat
(ATCC #TIB-152), was used to assess binding of different bispecific
constructs to human CD3 on cells.
Example 1
Affinity Maturation of Anti-MCSP Antibody M4-3/ML2
[0284] Affinity maturation was performed via the
oligonucleotide-directed mutagenesis procedure. For this the heavy
chain variant M4-3, and the light chain variant ML2 were cloned
into a phagemid vector, similar to those described by Hoogenboom,
(Hoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-4137).
Residues to be randomized were identified by first generating a 3D
model of that antibody via classical homology modeling and then
identifying the solvent accessible residues of the complementary
determining regions (CDRs) of heavy and light chain.
Oligonucleotides with randomization based on trinucleotide
synthesis as shown in Table 1 were purchased from Ella Biotech
(Munich, Germany). Three independent sublibraries were generated
via classical PCR, and comprised randomization in CDR-H1 together
with CDR-H2, or CDR-L1 together with CDR-L2. CDR-L3 was randomized
in a separate approach. The DNA fragments of those libraries were
cloned into the phagemid via restriction digest and ligation, and
subsequently electroporated into TG1 bacteria.
Library Selection
[0285] The antibody variants thus generated were displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants were then screened for their biological
activities (here: binding affinity) and candidates that have one or
more improved activities were used for further development. Methods
for making phage display libraries can be found in Lee et al., J.
Mol. Biol. (2004) 340, 1073-1093. Selections with all affinity
maturation libraries were carried out in solution according to the
following procedure: 1. binding of .about.1012 phagemid particles
of each affinity maturation libraries to 100 nM biotinylated
hu-MCSP(D3 domain)-avi-his (SEQ ID NO: 118) for 0.5 h in a total
volume of 1 ml, 2. capture of biotinylated hu-MCSP(D3
domain)-avi-his and specifically bound phage particles by addition
of 5.4.times.10.sup.7 streptavidin-coated magnetic beads for 10
min, 3. washing of beads using 5-10.times.1 ml PBS/Tween-20 and
5-10.times.1 ml PBS, 4. elution of phage particles by addition of 1
ml 100 mM TEA (triethylamine) for 10 min and neutralization by
adding 500 .mu.l 1M Tris/HCl pH 7.4 and 5. re-infection of
exponentially growing E. coli TG1 bacteria, infection with helper
phage VCSM13 and subsequent PEG/NaCl precipitation of phagemid
particles to be used in subsequent selection rounds. Selections
were carried out over 3-5 rounds using either constant or
decreasing (from 10.sup.-7 M to 2.times.10.sup.-9 M) antigen
concentrations. In round 2, capture of antigen-phage complexes was
performed using neutravidin plates instead of streptavidin beads.
Specific binders were identified by ELISA as follows: 100 .mu.l of
10 nM biotinylated hu-MCSP(D3 domain)-avi-his per well were coated
on neutravidin plates. Fab-containing bacterial supernatants were
added and binding Fabs were detected via their Flag-tags by using
an anti-Flag/HRP secondary antibody. ELISA-positive clones were
bacterially expressed as soluble Fab fragments in 96-well format
and supernatants were subjected to a kinetic screening experiment
by SPR-analysis using ProteOn XPR36 (BioRad). Clones expressing
Fabs with the highest affinity constants were identified and the
corresponding phagemids were sequenced.
TABLE-US-00002 TABLE 1 (excluded were always Cys and Met. Lys was
excluded on top in those cases where the oligonucleotide was a
reverse primer) Position Randomization Heavy chain CDR1 Ser31 S
(40%), rest (60%, 4% each) Gly32 G (40%), rest (60%, 4% each).
Tyr33 Y (40%), rest (60%, 4% each) Tyr34 Y (40%), rest (60%, 4%
each) CDR2 Tyr50 Y 40%, (F, W, L, A, I, 30%, 6% each), rest (30%,
2.5% each) Thr52 T (60%), rest (40%, 2.5% each) Tyr53 Y (40%), rest
(60%, 3.8% each) Asp54 D (40%), rest (60%, 3.8% each) Ser56 S
(40%), rest (60%, 3.8% each) Light chain CDR1 Gln27 Q (40%), (E, D,
N, S, T, R, 40%, 6.7% each), rest (total 20%, 2.2% each) Gly28 G
(40%), (N, T, S, Q, Y, D, E, 40%, 5.7% each), rest (20%, 2.5% each)
Asn31 N (40%), (S, T, G, Q, Y, D, E, R, 50%, 6.3% each), rest (10%,
1.4% each) Tyr32 Y (40%), (W, S, R, 30%, 10% each), rest (30%, 2.3%
each) CDR2 Tyr50 Y (70%), (E, R, K, A, Q, T, S, D, G, W, F, 30%,
2.7% each) Thr51 T (50%), (S, A, G, N, Q, V, 30%, 5% each), rest
(20%, 2% each) Ser52 S (50%), rest (50%, 3.1% each) Ser53 S (40%),
(N, T, Q, Y, D, E, I, 40%, 5.7% each), rest (20%, 2.2% each) CDR3
Tyr91 Y (50%), rest (50%, 3.1% each) Ser92 S (50%), (N, Q, T, A, G
25%, 5% each), rest (25%, 2.3% each) Lys93 K (50%), S (5%), T (5%),
N (5%), rest (35%, 2.7% each) Leu94 L (50%), (Y, F, S, I, A, V,
30%, 5% each), rest (20%, 2% each) Pro95 P (50%), (S, A, 20%, 10%
each), rest (30%, 2.1% each) Trp96 W 50%, (Y, R, L, 15%, 5% each),
rest (35%, 2.5% each)
[0286] FIG. 2 shows an alignment of affinity matured anti-MCSP
clones compared to the non-matured parental clone (M4-3 ML2). Heavy
chain randomization was performed only in the CDR1 and 2. Light
chain randomization was performed in CDR1 and 2, and independently
in CDR3.
[0287] During selection, a few mutations in the frameworks occured
like F71Y in clone G3 or Y87H in clone E10.
Production and Purification of Human IgG.sub.1
[0288] The variable region of heavy and light chain DNA sequences
of the affinity matured variants were subcloned in frame with
either the constant heavy chain or the constant light chain
pre-inserted into the respective recipient mammalian expression
vector. The antibody expression was driven by an MPSV promoter and
carries a synthetic polyA signal sequence at the 3' end of the CDS.
In addition each vector contained an EBV OriP sequence.
[0289] The molecule was produced by co-transfecting HEK293-EBNA
cells with the mammalian expression vectors using polyethylenimine
(PEI). The cells were transfected with the corresponding expression
vectors in a 1:1 ratio. For transfection HEK293 EBNA cells were
cultivated in suspension serum-free in CD CHO culture medium. For
the production in 500 ml shake flask, 400 million HEK293 EBNA cells
were seeded 24 hours before transfection. For transfection cells
were centrifuged for 5 min at 210.times.g, supernatant was replaced
by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in
20 ml CD CHO medium to a final amount of 200 .mu.g DNA. After
addition of 540 .mu.l PEI solution, the mixture was vortexed for 15
s and subsequently incubated for 10 min at room temperature.
Afterwards cells were mixed with the DNA/PEI solution, transferred
to a 500 ml shake flask and incubated for 3 hours at 37.degree. C.
in an incubator with a 5% CO.sub.2 atmosphere. After incubation
time 160 ml F17 medium was added and cells were cultivated for 24
hours. One day after transfection 1 mM valproic acid and 7% Feed 1
(Lonza) was added. After 7 days cultivation supernatant was
collected for purification by centrifugation for 15 min at
210.times.g, the solution was sterile filtered (0.22 .mu.m filter)
and sodium azide in a final concentration of 0.01% w/v was added,
and kept at 4.degree. C.
[0290] The secreted protein was purified from cell culture
supernatants by affinity chromatography using Protein A.
Supernatant was loaded on a HiTrap Protein A HP column (CV=5 mL, GE
Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM
sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was
removed by washing with at least 10 column volumes 20 mM sodium
phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5.
Target protein was eluted during a gradient over 20 column volumes
from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM
sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was
neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target
protein was concentrated and filtrated prior loading on a HiLoad
Superdex 200 column (GE Healthcare) equilibrated with 20 mM
histidine, 140 mM sodium chloride solution of pH 6.0.
[0291] The protein concentration of purified protein samples was
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence. Purity and molecular weight of molecules were
analyzed by CE-SDS analyses in the presence and absence of a
reducing agent. The Caliper LabChip GXII system (Caliper Life
Sciences) was used according to the manufacturer's instruction. 2
.mu.g sample was used for analyses. The aggregate content of
antibody samples was analyzed using a TSKgel G3000 SW XL analytical
size-exclusion column (Tosoh) in 25 mM K.sub.2HPO.sub.4, 125 mM
NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN.sub.3,
pH 6.7 running buffer at 25.degree. C.
TABLE-US-00003 TABLE 2 Production and purification of affinity
matured anti-MCSP IgGs Yield HMW LMW Monomer Construct [mg/l] [%]
[%] [%] M4-3(C1) ML2(G3) 43.9 0 0 100 M4-3(C1) ML2(E10) 59.5 0 0
100 M4-3(C1) ML2(C5) 68.9 0 0.8 99.2
Affinity Determination
ProteOn Analysis
[0292] K.sub.D was measured by surface plasmon resonance using a
ProteOn XPR36 machine (BioRad) at 25.degree. C. with anti-human
F(ab')2 fragment specific capture antibody (Jackson ImmunoResearch
#109-005-006) immobilized by amine coupling on CM5 chips and
subsequent capture of Fabs from bacterial supernatant or from
purified Fab preparations. Briefly, carboxymethylated dextran
biosensor chips (CM5, GE Healthcare) were activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Anti-human F(ab')2 fragment specific capture antibody
was diluted with 10 mM sodium acetate, pH 5.0 at 50 .mu.g/ml before
injection at a flow rate of 10 .mu.l/minute to achieve
approximately up to 10.000 response units (RU) of coupled capture
antibody. Following the injection of the capture antibody, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, Fabs from bacterial supernatant or purified Fabs were
injected at a flow rate of 10 .mu.l/minute for 300 s and a
dissociation of 300 s for capture baseline stabilization. Capture
levels were in the range of 100-500 RU. In a subsequent step, human
MCSP(D3 domain)-avi-his analyte was injected either as a single
concentration or as a concentration series (depending of clone
affinity in a range between 100 nM and 250 pM) diluted into HBS-EP+
(GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%
Surfactant P20, pH 7.4) at 25.degree. C. at a flow rate of 50
.mu.l/min. The surface of the sensorchip was regenerated by
injection of glycine pH 1.5 for 30 s at 90 .mu.l/min followed by
injection of NaOH for 20 s at the same flow rate. Association rates
(k.sub.on) and dissociation rates (k.sub.off) were calculated using
a simple one-to-one Langmuir binding model (ProteOn XPR36
Evaluation Software or Scrubber software (BioLogic)) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (K.sub.D) was
calculated as the ratio k.sub.off/k.sub.on. This data was used to
determine the comparative binding affinity of the affinity matured
variants with the parental antibody. Table 3a shows the data
generated from these assays.
[0293] G3, E10, C5 for the light chain, and D6, A7, B7, B8, C1 for
the heavy chain were chosen for conversion into human IgG.sub.1
format. Since CDR1 and 2 of the light chain were randomized
independent from CDR3, the obtained CDRs were combined during IgG
conversion.
[0294] In the IgG format affinities were measured again to the
human MCSP antigen (SEQ ID NO: 118), in addition also to the
cynomolgus homologue (SEQ ID NO: 117).
[0295] The method used was exactly as described for the Fab
fragments, just using purified IgG from mammalian production.
TABLE-US-00004 TABLE 3a MCSP affinity matured clones: Proteon data.
Human Human Cyno Human Cyno MCSP MCSP MCSP MCSP MCSP Fab K.sub.D
IgG K.sub.D IgG K.sub.D IgG K.sub.D IgG K.sub.D Proteon Comparative
binding generated affinity--Fold Variant affinity data increase
over parent Parental M4-3/ML2 5 * 10.sup.-9 2 * 10.sup.-9 2 *
10.sup.-9 M4-3/ML2(G3) 4 * 10.sup.-10 3 * 10.sup.-10 6 * 10.sup.-10
6.7 3.3 M4-3/ML2(E10) 7 * 10.sup.-10 1 * 10.sup.-9 2 * 10.sup.-9
2.0 1.0 M4- 4 * 10.sup.-10 9 * 10.sup.-10 5.0 2.2 3/ML2(E10/G3)
M4-3/ML2(C5) 7 * 10.sup.-10 4 * 10.sup.-10 1 * 10.sup.-9 5.0 2.0
M4- 7 * 10.sup.-10 1 * 10.sup.-9 2.9 2.0 3/ML2(C5/G3) M4-3(D6)/ML2
2 * 10.sup.-9 4 * 10.sup.-10 1 * 10.sup.-9 5.0 2.0 M4-3(A7)/ML2 2 *
10.sup.-11 8 * 10.sup.-10 1 * 10.sup.-9 2.5 2.0 M4-3(B7)/ML2 5 *
10.sup.-10 7 * 10.sup.-10 4.0 2.9 M4-3(B8)/ML2 3 * 10.sup.-10 9 *
10.sup.-10 1 * 10.sup.-9 2.2 2.0 M4-3(C1)/ML2 6 * 10.sup.-10 9 *
10.sup.-10 8 * 10.sup.-10 2.2 2.5 M4- 7 * 10.sup.-11 2 * 10.sup.-10
28.6 10.0 3(C1)/ML2(G3) M4- 5 * 10.sup.-10 6 * 10.sup.-10 4.0 3.3
3(C1)/ML2(E10) M4- 7 * 10.sup.-11 2 * 10.sup.-10 28.6 10.0
3(A7)/ML2(G3) M4- 3 * 10.sup.-10 7 * 10.sup.-10 6.7 2.9
3(A7)/ML2(E10) M4- 2 * 10.sup.-10 3 * 10.sup.-10 10.0 6.7
3(C1)/ML2(C5) M4- 7 * 10.sup.-11 2 * 10.sup.-10 28.6 10.0
3(A7)/ML2(C5)
Affinity Determination by Surface Plasmon Resonance (SPR) Using
Biacore T200
[0296] Surface plasmon resonance (SPR) experiments to determine the
affinity and the avidity of the affinity matured IgGs were
performed on a Biacore T200 at 25.degree. C. with HBS-EP as running
buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%
Surfactant P20, Biacore, Freiburg/Germany).
[0297] For analyzing the avidity of the interaction of different
anti-MCSP IgGs to human and cynomolgus MCSP D3 direct coupling of
around 9,500 resonance units (RU) of the anti-Penta His antibody
(Qiagen) was performed on a CM5 chip at pH 5.0 using the standard
amine coupling kit (Biacore, Freiburg/Germany). Antigens were
captured for 60 s at 30 nM with 10 .mu.l/min respectively. IgGs
were passed at a concentration of 0.0064-100 nM with a flowrate of
30 .mu.l/min through the flow cells over 280 s. The dissociation
was monitored for 180 s. Bulk refractive index differences were
corrected for by subtracting the response obtained on reference
flow cell. Here, the IgGs were flown over a surface with
immobilized anti-Penta His antibody but on which HBS-EP has been
injected rather than human MCSP D3 or cynomolgus MCSP D3.
[0298] For affinity measurements IgGs were captured on a CM5
sensorchip surface with immobilized anti human Fc. Capture IgG was
coupled to the sensorchip surface by direct immobilization of
around 9,500 resonance units (RU) at pH 5.0 using the standard
amine coupling kit (Biacore, Freiburg/Germany). IgGs are captured
for 25 s at 10 nM with 30 .mu.l/min. Human and cynomolgus MCSP D3
were passed at a concentration of 2-500 nM with a flowrate of 30
.mu.l/min through the flow cells over 120 s. The dissociation was
monitored for 60 s. Association and dissociation for concentration
166 and 500 nM was monitored for 1200 and 600 s, respectively. Bulk
refractive index differences were corrected for by subtracting the
response obtained on reference flow cell. Here, the antigens were
flown over a surface with immobilized anti-human Fc antibody but on
which HBS-EP has been injected rather than anti-MCSP IgGs.
[0299] Kinetic constants were derived using the Biacore T200
Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rate
equations for 1:1 Langmuir binding by numerical integration.
[0300] Higher affinity to human and cynomolgus MCSP D3 were
confirmed by surface plasmon resonance measurements using Biacore
T200. In addition, avidity measurements showed an up to 3-fold
increase in bivalent binding (Table 3b).
TABLE-US-00005 TABLE 3b Affinity and avidity of anti MCSP IgGs to
human MCSP-D3 and cynomolgus MCSP-D3. KD in nM Human MCSP D3
Cynomolgus MCSP D3 T = 25.degree. C. Affinity Avidity Affinity
Avidity M4-3(C1) ML2(G3) 1.8 0.0045 1.4 0.0038 M4-3(C1) ML2(E10)
4.6 0.0063 3.8 0.0044 M4-3(C1) ML2(C5) 1.8 0.0046 1.3 0.0044 M4-3
ML2 (parental) 8.6 0.0090 11.4 0.0123
Example 2
Preparation of MCSP TCB (2+1 Crossfab-IgG P329G LALA Inverted)
Containing M4-3(C1) ML2(G3) as Anti MCSP Antibody and Humanized
CH2527 as Anti CD3 Antibody
[0301] The variable region of heavy and light chain DNA sequences
were subcloned in frame with either the constant heavy chain or the
constant light chain pre-inserted into the respective recipient
mammalian expression vector. The antibody expression was driven by
an MPSV promoter and carries a synthetic polyA signal sequence at
the 3' end of the CDS. In addition each vector contains an EBV OriP
sequence.
[0302] The molecule was produced by co-transfecting HEK293-EBNA
cells with the mammalian expression vectors using polyethylenimine
(PEI). The cells were transfected with the corresponding expression
vectors in a 1:2:1:1 ratio ("vector heavy chain Fc(hole)":"vector
light chain":"vector light chain Crossfab": "vector heavy chain
Fc(knob)-FabCrossfab").
[0303] For transfection HEK293 EBNA cells were cultivated in
suspension serum-free in CD CHO culture medium. For the production
in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24
hours before transfection. For transfection cells were centrifuged
for 5 min at 210.times.g, supernatant was replaced by pre-warmed 20
ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO
medium to a final amount of 200 .mu.g DNA. After addition of 540
.mu.l PEI solution the mixture was vortexed for 15 s and
subsequently incubated for 10 min at room temperature. Afterwards
cells were mixed with the DNA/PEI solution, transferred to a 500 ml
shake flask and incubated for 3 hours at 37.degree. C. in an
incubator with a 5% CO.sub.2 atmosphere. After incubation time 160
ml F17 medium was added and cell were cultivated for 24 hours. One
day after transfection 1 mM valproic acid and 7% Feed 1 (Lonza) was
added. After 7 days cultivation supernatant was collected for
purification by centrifugation for 15 min at 210.times.g, the
solution was sterile filtered (0.22 .mu.m filter) and sodium azide
in a final concentration of 0.01% w/v was added, and kept at
4.degree. C.
[0304] The secreted protein was purified from cell culture
supernatants by affinity chromatography using Protein A.
Supernatant was loaded on a HiTrap Protein A HP column (CV=5 mL, GE
Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM
sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was
removed by washing with at least 10 column volumes 20 mM sodium
phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5.
Target protein was eluted during a gradient over 20 column volumes
from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM
sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was
neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target
protein was concentrated and filtrated prior loading on a HiLoad
Superdex 200 column (GE Healthcare) equilibrated with 20 mM
histidine, 140 mM sodium chloride solution of pH 6.0.
[0305] The protein concentration of purified protein samples was
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence.
[0306] Purity and molecular weight of molecules were analyzed by
CE-SDS analyses in the presence and absence of a reducing agent.
The Caliper LabChip GXII system (Caliper lifescience) was used
according to the manufacturer's instruction. 2 .mu.g sample was
used for analyses.
[0307] The aggregate content of antibody samples was analyzed using
a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine
monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C.
TABLE-US-00006 TABLE 4a Summary production and purification of MCSP
TCB. Aggregate after 1.sup.st Titer Yield purification HMW LMW
Monomer Construct [mg/l] [mg/l] step [%] [%] [%] [%] MCSP TCB 157
0.32 32 3.3 0 96.7
[0308] FIG. 3 shows a schematic drawing of the MCSP TCB (2+1
Crossfab-IgG P329G LALA inverted) molecule.
[0309] FIG. 4 and Table 4b show CE-SDS analyses of a MCSP TCB (2+1
Crossfab-IgG P329G LALA inverted) molecule (SEQ ID NOs: 12, 53, 54
and 55).
TABLE-US-00007 TABLE 4b CE-SDS analyses of MCSP TCB. Peak kDa
Corresponding Chain MCSP TCB non reduced (A) 1 206.47 MCSP TCB
reduced (B) 1 29.15 Light chain ML2 (C1) 2 37.39 Light chain
huCH2527 3 66.07 Fc(hole) 4 94.52 Fc(knob)
Example 3
Preparation of CEA TCB (2+1 Crossfab-IgG P329G LALA Inverted)
Containing CH1A1A 98/99 2F1 as Anti CEA Antibody and Humanized
CH2527 as Anti CD3 Antibody
[0310] The variable region of heavy and light chain DNA sequences
were subcloned in frame with either the constant heavy chain or the
constant light chain pre-inserted into the respective recipient
mammalian expression vector. The antibody expression was driven by
an MPSV promoter and carries a synthetic polyA signal sequence at
the 3' end of the CDS. In addition each vector contains an EBV OriP
sequence.
[0311] The molecule was produced by co-transfecting HEK293 EBNA
cells with the mammalian expression vectors using polyethylenimine
(PEI). The cells were transfected with the corresponding expression
vectors in a 1:2:1:1 ratio ("vector heavy chain Fc(hole)":"vector
light chain":"vector light chain Crossfab": "vector heavy chain
Fc(knob)-FabCrossfab").
[0312] For transfection HEK293 EBNA cells were cultivated in
suspension serum-free in CD CHO culture medium. For the production
in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24
hours before transfection. For transfection cells were centrifuged
for 5 min at 210.times.g, supernatant was replaced by pre-warmed 20
ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO
medium to a final amount of 200 .mu.g DNA. After addition of 540
.mu.l PEI solution the mixture was vortexed for 15 s and
subsequently incubated for 10 min at room temperature. Afterwards
cells were mixed with the DNA/PEI solution, transferred to a 500 ml
shake flask and incubated for 3 hours by 37.degree. C. in an
incubator with a 5% CO.sub.2 atmosphere. After incubation time 160
ml F17 medium was added and cell were cultivated for 24 hours. One
day after transfection 1 mM valproic acid and 7% Feed 1 (Lonza) was
added. After 7 days cultivation supernatant was collected for
purification by centrifugation for 15 min at 210.times.g, the
solution was sterile filtered (0.22 .mu.m filter) and sodium azide
in a final concentration of 0.01% w/v was added, and kept at
4.degree. C.
[0313] The secreted protein was purified from cell culture
supernatants by affinity chromatography using Protein A.
Supernatant was loaded on a HiTrap Protein A HP column (CV=5 mL, GE
Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM
sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was
removed by washing with at least 10 column volumes 20 mM sodium
phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5.
Target protein was eluted during a gradient over 20 column volumes
from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM
sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was
neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target
protein was concentrated and filtrated prior loading on a HiLoad
Superdex 200 column (GE Healthcare) equilibrated with 20 mM
histidine, 140 mM sodium chloride solution of pH 6.0.
[0314] The protein concentration of purified protein samples was
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence.
[0315] Purity and molecular weight of molecules were analyzed by
CE-SDS analyses in the presence and absence of a reducing agent.
The Caliper LabChip GXII system (Caliper lifescience) was used
according to the manufacturer's instructions. 2 .mu.g sample was
used for analyses.
[0316] The aggregate content of antibody samples was analyzed using
a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine
monohydrocloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C.
TABLE-US-00008 TABLE 5 Summary production and purification of CEA
TCB. Aggregate after 1.sup.st Titer Yield purification HMW LMW
Monomer Construct [mg/l] [mg/l] step [%] [%] [%] [%] CEA TCB 66
0.31 21.5 8.1 4.4 87.5
[0317] FIG. 5 shows a schematic drawing of CEA TCB (2+1
Crossfab-IgG P329G LALA inverted) molecule.
[0318] FIG. 6 and Table 6 show CE-SDS analyses of a CEA TCB (2+1
Crossfab-IgG P329G LALA inverted) molecule (SEQ ID NOs: 22, 56, 57
and 58).
TABLE-US-00009 TABLE 6 CE-SDS analyses of CEA TCB. Peak kDa
Corresponding Chain CEA TCB non reduced (A) 1 205.67 Correct
molecule CEA TCB reduced (B) 1 28.23 Light chain CH1A1A 98/99
.times. 2F1 2 36.31 Light chain CH2527 3 63.48 Fc(hole) 4 90.9
Fc(knob)
[0319] In an alternative purification method, the CEA TCB was
captured from harvested and clarified fermentation supernatant by
Protein A affinity chromatography (MabSelect SuRe). The Protein A
eluate was then submitted to cation exchange chromatography (Poros
50 HS) and subsequently fractionated and analyzed by means of
SE-HPLC. and capillary electrophoresis. The product containing
fractions were pooled and subjected to hydrophobic interaction
chromatography (Butyl-Sepharose 4FF) at room temperature in a
bind-elute mode. The eluate therefrom was then fractionated and
analyzed by means of SE-HPLC and capillary electrophoresis. The
product containing fractions were pooled and subsequently anion
exchange chromatography (Q-Sepharose FF) in flow-through mode was
performed. The material obtained using this purification method had
a monomer content of >98%.
Example 4
Binding of MCSP TCB to MCSP- and CD3-Expressing Cells
[0320] The binding of MCSP TCB was tested on a MCSP-expressing
human malignant melanoma cell line (A375) and a CD3-expressing
immortalized T lymphocyte line (Jurkat). Briefly, cells were
harvested, counted, checked for viability and resuspended at
2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l PBS 0.1% BSA).
100 .mu.l cell suspension (containing 0.2.times.10.sup.6 cells)
were incubated in round-bottom 96-well plate for 30 min at
4.degree. C. with increasing concentrations of the MCSP TCB (2.6
pM-200 nM), washed twice with cold PBS 0.1% BSA, re-incubated for
further 30 min at 4.degree. C. with the PE-conjugated AffiniPure
F(ab')2 Fragment goat anti-human IgG Fc.gamma. Fragment Specific
secondary antibody (Jackson Immuno Research Lab PE #109-116-170),
washed twice with cold PBS 0.1% BSA and immediately analyzed by
FACS using a FACS CantoII (Software FACS Diva) by gating live,
DAPI-negative, cells. Binding curves were obtained using
GraphPadPrism5 (FIG. 7A, binding to A375 cells, EC.sub.50=3381 pM;
FIG. 7B, binding to Jurkat cells).
Example 5
T-Cell Killing Induced by MCSP TCB Antibody
[0321] T-cell killing mediated by MCSP TCB antibody was assessed
using a panel of tumor cell lines expressing different levels of
MCSP (A375=MCSP high, MV-3=MSCP medium, HCT-116=MCSP low,
LS180=MCSP negative). Briefly, target cells were harvested with
Trypsin/EDTA, washed, and plated at density of 25 000 cells/well
using flat-bottom 96-well plates. Cells were left to adhere
overnight. Peripheral blood mononuclear cells (PBMCs) were prepared
by Histopaque density centrifugation of enriched lymphocyte
preparations (buffy coats) obtained from healthy human donors.
Fresh blood was diluted with sterile PBS and layered over
Histopaque gradient (Sigma, #H8889). After centrifugation
(450.times.g, 30 minutes, room temperature), the plasma above the
PBMC-containing interphase was discarded and PBMCs transferred in a
new falcon tube subsequently filled with 50 ml of PBS. The mixture
was centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant discarded and the PBMC pellet washed twice with sterile
PBS (centrifugation steps 350.times.g, 10 minutes). The resulting
PBMC population was counted automatically (ViCell) and stored in
RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine
(Biochrom, K0302) at 37.degree. C., 5% CO.sub.2 in cell incubator
until further use (no longer than 24 h). For the killing assay, the
antibody was added at the indicated concentrations (range of 1
pM-10 nM in triplicates). PBMCs were added to target cells at final
effector to target (E:T) ratio of 10:1. Target cell killing was
assessed after 24 h of incubation at 37.degree. C., 5% CO.sub.2 by
quantification of LDH released into cell supernatants by
apoptotic/necrotic cells (LDH detection kit, Roche Applied Science,
#11 644 793 001). Maximal lysis of the target cells (=100%) was
achieved by incubation of target cells with 1% Triton X-100.
Minimal lysis (=0%) refers to target cells co-incubated with
effector cells without bispecific construct. The results show that
MCSP TCB induced a strong and target-specific killing of
MCSP-positive target cell lines with no killing of MCSP-negative
cell lines (FIG. 8, A-D). The EC.sub.50 values related to the
killing assays, calculated using GraphPadPrism5 are given in Table
7.
TABLE-US-00010 TABLE 7 EC.sub.50 values (pM) for T-cell mediated
killing of MCSP- expressing tumor cells induced by MCSP TCB
antibody. Cell line MCSP receptor copy number EC.sub.50 [pM] A375
387 058 12.3 MV-3 260 000 9.4 HCT-116 36770 3.7 LS180 Negative
n.d.
Example 6
[0322] CD25 and CD69 Upregulation on CD8.sup.+ and CD4.sup.+
Effector Cells after T Cell Killing of MCSP-Expressing Tumor Cells
Induced by MCSP TCB Antibody
[0323] Activation of CD8.sup.+ and CD4.sup.+ T cells after T-cell
killing of MCSP-expressing MV-3 tumor cells mediated by the MCSP
TCB antibody was assessed by FACS analysis using antibodies
recognizing the T cell activation markers CD25 (late activation
marker) and CD69 (early activation marker). The antibody and the
killing assay conditions were essentially as described above
(Example 5), using the same antibody concentration range (1 pM-10
nM in triplicates), E:T ratio 10:1 and an incubation time 24 h.
[0324] After the incubation, PBMCs were transferred to a
round-bottom 96-well plate, centrifuged at 350.times.g for 5 min
and washed twice with PBS containing 0.1% BSA. Surface staining for
CD8 (FITC anti-human CD8, BD #555634), CD4 (PECy7 anti-human CD4,
BD #557852), CD69 (PE anti-human CD69, Biolegend #310906) and CD25
(APC anti-human CD25, BD #555434) was performed according to the
suppliers' indications. Cells were washed twice with 150 .mu.l/well
PBS containing 0.1% BSA and fixed for 15 min at 4.degree. C. using
100 .mu.l/well fixation buffer (BD #554655). After centrifugation,
the samples were resuspended in 200 .mu.l/well PBS 0.1% BSA
containing DAPI to exclude dead cells for the FACS measurement.
Samples were analyzed at BD FACS Fortessa. The results show that
MCSP TCB induced a strong and target-specific upregulation of
activation markers (CD25, CD69) on CD8.sup.+ T cells (FIGS. 9A, B)
and CD4.sup.+ T cells (FIGS. 9C, D) after killing.
Example 7
[0325] Cytokine Secretion by Human Effector Cells after T
Cell-Killing of MCSP-Expressing Tumor Cells Induced by MCSP TCB
Antibody
[0326] Cytokine secretion by human PBMCs after T-cell killing of
MCSP-expressing MV-3 tumor cells induced by the MCSP TCB antibody
was assessed by FACS analysis of cell supernatants after the
killing assay.
[0327] The same antibody was used and the killing assay was
performed essentially as described above (Example 5 and 6), using
an E:T ratio of 10:1 and an incubation time of 24 h.
[0328] At the end of the incubation time, the plate was centrifuged
for 5 min at 350.times.g, the supernatant transferred in a new
96-well plate and stored at -20.degree. C. until subsequent
analysis. Granzyme B, TNF.alpha., IFN-.gamma., IL-2, IL-4 and IL-10
secreted into in cell supernatants were detected using the BD CBA
Human Soluble Protein Flex Set, according to manufacturer's
instructions on a FACS CantoII. The following kits were used: BD
CBA human Granzyme B BD CBA human Granzyme B Flex Set #BD 560304;
BD CBA human TNF Flex Set #BD 558273; BD CBA human IFN-.gamma. Flex
Set #BD 558269; BD CBA human IL-2 Flex Set #BD 558270; BD CBA human
IL-4 Flex Set #BD 558272; BD CBA human IL-10 Flex Set #BD
558274.
[0329] The results show that MCSP TCB induced secretion of IL-2,
IFN-.gamma., TNF.alpha., Granzyme B and IL-10 (but no IL-4) upon
killing (FIG. 10, A-F).
[0330] Taken together, these examples show that the MCSP CD3
bispecific antibody [0331] Showed a good binding to MCSP-positive
A375 cells [0332] Induced a strong and target-specific killing of
MCSP-positive target cell lines, and no killing of MCSP-negative
cell lines [0333] Induced a strong and target-specific upregulation
of activation markers (CD25, CD69) on CD8.sup.+ and CD4.sup.+ T
cells after killing [0334] Induced secretion of IL-2, IFN-.gamma.,
TNF.alpha., Granzyme B and IL-10 (no IL-4) upon killing.
Example 8
Binding of CEA TCB to CEA- and CD3-Expressing Cells
[0335] The binding of CEA TCB was tested on transfected
CEA-expressing lung adenocarcinoma cells (A549-huCEA) and
CD3-expressing immortalized human and cynomolgus T lymphocyte lines
(Jurkat and HSC-F, respectively). An untargeted TCB (SEQ ID NOs:
59, 60, 61 and 62; see example 24) was used as control. Briefly,
cells were harvested, counted, checked for viability and
resuspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l
PBS 0.1% BSA). 100 .mu.l cell suspension (containing
0.2.times.10.sup.6 cells) were incubated in round-bottom 96-well
plate for 30 min at 4.degree. C. with increasing concentrations of
the CEA TCB (61 pM-1000 nM), washed twice with cold PBS 0.1% BSA,
re-incubated for further 30 min at 4.degree. C. with the
FITC-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG
F(ab')2 Fragment Specific secondary antibody (Jackson Immuno
Research Lab FITC #109-096-097), washed twice with cold PBS 0.1%
BSA and immediately analyzed by FACS using a FACS CantoII or
Fortessa (Software FACS Diva) by gating live, PI-negative, cells.
Binding curves were obtained using GraphPadPrism5 (FIG. 11A,
binding to A549 cells (EC.sub.50 6.6 nM); FIG. 11B, binding to
Jurkat cells; FIG. 11C, binding to HSC-F cells).
Example 9
T Cell-Mediated Killing of CEA-Expressing Tumor Target Cells
Induced by CEA TCB Antibody
[0336] T cell-mediated killing of target cells induced by CEA TCB
antibody was assessed on HPAFII (high CEA), BxPC-3 (medium CEA) and
ASPC-1 (low CEA) human tumor cells. HCT-116 (CEA negative tumor
cell line) and the untargeted TCB were used as negative controls.
Human PBMCs were used as effectors and killing detected 24 h and 48
h after incubation with the bispecific antibody. Briefly, target
cells were harvested with Trypsin/EDTA, washed, and plated at
density of 25 000 cells/well using flat-bottom 96-well plates.
Cells were left to adhere overnight. Peripheral blood mononuclear
cells (PBMCs) were prepared by Histopaque density centrifugation of
enriched lymphocyte preparations (buffy coats) obtained from
healthy human donors. Fresh blood was diluted with sterile PBS and
layered over Histopaque gradient (Sigma, #H8889). After
centrifugation (450.times.g, 30 minutes, room temperature), the
plasma above the PBMC-containing interphase was discarded and PBMCs
transferred in a new falcon tube subsequently filled with 50 ml of
PBS. The mixture was centrifuged (400.times.g, 10 minutes, room
temperature), the supernatant discarded and the PBMC pellet washed
twice with sterile PBS (centrifugation steps 350.times.g, 10
minutes). The resulting PBMC population was counted automatically
(ViCell) and kept in RPMI1640 medium containing 10% FCS and 1%
L-alanyl-L-glutamine (Biochrom, K0302) in cell incubator
(37.degree. C., 5% CO.sub.2) until further use (no longer than 24
h). For the killing assay, the antibodies were added at indicated
concentrations (range of 6 pM-100 nM in triplicates). PBMCs were
added to target cells at the final E:T ratio of 10:1. Target cell
killing was assessed after 24 h and 48 h of incubation by
quantification of LDH (lactate dehydrogenase) released into cell
supernatants by apoptotic/necrotic cells (LDH detection kit, Roche
Applied Science, #11 644 793 001). Maximal lysis of the target
cells (=100%) was achieved by incubation of target cells with 1%
Triton X-100. Minimal lysis (=0%) refers to target cells
co-incubated with effector cells without bispecific antibody. The
results show that CEA TCB induced a strong and target-specific
killing of CEA-positive target cells (FIG. 12, A-H). The EC.sub.50
values related to the killing assays, calculated using
GraphPadPrism5 are given in Table 8.
TABLE-US-00011 TABLE 8 CEA receptor copy number and EC.sub.50
values (pM) for T-cell mediated killing of CEA-expressing tumor
cells induced by CEA TCB antibody. CEA receptor EC50 [pM] Cell line
copy number 48 h HPAFII 120 000-205 000 667 BxPC-3 41 000 3785
ASPC1 3500-8000 846
Example 10
[0337] T Cell Proliferation and Activation 5 Days after CEA
TCB-Mediated Killing of CEA-Expressing Tumor Target Cells
[0338] T cell proliferation and activation was detected 5 days
after CEA TCB-mediated killing of CEA-expressing tumor target cells
assessed on HPAFII (high CEA), BxPC-3 (medium CEA) and ASPC-1 (low
CEA) cells. HCT-116 (CEA negative tumor cell line) and the
untargeted TCB were used as negative controls. The experimental
conditions for the proliferation assay were similar to the ones
described in Example 9, but only 10 000 target cells were plated
per well of a 96-flat bottom well plate. To assess T cell
proliferation, freshly-isolated PBMCs were labeled using CFSE
(Sigma #21888). Briefly, CFSE stock solution was diluted to obtain
a working solution of 100 .mu.M. 90.times.10.sup.6 PBMC cells were
re-suspended in 90 ml pre-warmed PBS and supplemented with 90 .mu.l
of the CFSE working solution. Cells were mixed immediately and
incubated 15 min at 37.degree. C. 10 ml of pre-warmed FCS were
added to cells to stop the reaction. The cells were centrifuged for
10 min at 400 g, re-suspended in 50 ml medium and incubated for 30
min at 37.degree. C. After incubation, cells were washed once with
warm medium, counted, re-suspended in medium and added to target
cells for the killing assay and subsequent measurement of cell
proliferation and activation at an E:T of 10:1. Proliferation was
assessed 5 days after killing on CD4 and CD8 positive T cells by
quantification of the CFSE dye dilution. CD25 expression was
assessed on the same T cell subsets using the anti-human CD25
antibody. Briefly, after centrifugation (400.times.g for 4 min),
cells were resuspended, washed with FACS buffer and incubated with
25 .mu.l of the diluted CD4/CD8/CD25 antibody mix for 30 min at
4.degree. C. (APC/Cy7 anti-human CD4 #317418, APC anti-human CD8
#301014, PE/Cy7 anti-human CD25 #302612). Cells were then washed
three times to remove the unbound antibody, and finally resuspended
in 200 .mu.l FACS buffer containing propidium iodide (PI) to
exclude dead cells for the FACS measurement. Fluorescence was
measured using BD FACS CantoII. The results show that the CEA TCB
induced a strong and target-specific proliferation of CD8.sup.+ and
CD4.sup.+ T cells (FIG. 13, A-D) as well as their activation as
detected by up-regulation of the CD25 activation marker (FIG. 13,
E-H).
Example 11
[0339] Cytokine Secretion by Human Effector Cells after T
Cell-Mediated Killing of CEA-Expressing Tumor Cells Induced by CEA
TCB
[0340] Cytokine secretion by human PBMCs after T cell-mediated
killing of CEA-expressing MKN45 tumor cells induced by the CEA TCB
was assessed by FACS analysis (CBA kit) of cell supernatants 48 h
after killing.
[0341] The experimental conditions were identical to the ones
described in Example 9. At the end of the incubation time, the
plate was centrifuged for 5 min at 350.times.g, the supernatant
transferred into a new 96-well plate and stored at -20.degree. C.
until subsequent analysis. (A) IFN-.gamma., (B) TNF.alpha., (C)
Granzyme B, (D) IL-2, (E) IL-6 and (F) IL-10 secreted into cell
supernatants were detected using the BD CBA Human Soluble Protein
Flex Set, according to the manufacturer's instructions on a FACS
CantoII. The following kits were used: BD CBA human IL-2 BD Flex
Set #BD 558270; BD CBA human Granzyme B BD Flex Set #BD 560304; BD
CBA human TNF Flex Set #BD 558273; BD CBA human IFN-.gamma. Flex
Set #BD 558269; BD CBA human IL-4 Flex Set #BD 558272; BD CBA human
IL-10 Flex Set #BD 558274.
[0342] The results show that the CEA TCB mediated killing (but not
the killing mediated by untargeted TCB control) induced secretion
of IFN-.gamma., TNF.alpha., Granzyme B, IL-2, IL-6 and IL-10 (FIG.
14, A-F).
Example 12
[0343] T Cell-Mediated Killing of Target Cells in Presence of
Increasing Concentrations of Shed CEA (sCEA)
[0344] T cell-mediated killing of CEA-expressing tumor target cells
(LS180) induced by CEA TCB antibody in presence of increasing
concentrations of shed CEA (sCEA 2.5 ng/ml-5 .mu.g/ml) was
assessed. Human PBMCs were used as effector cells and killing
detected 24 h and 48 h after incubation with the bispecific
antibody and sCEA. Briefly, target cells were harvested with
Trypsin/EDTA, washed, and plated at density of 25 000 cells/well
using flat-bottom 96-well plates. Cells were left to adhere
overnight. Peripheral blood mononuclear cells (PBMCs) were prepared
by Histopaque density centrifugation of enriched lymphocyte
preparations (buffy coats) obtained from healthy human donors.
Fresh blood was diluted with sterile PBS and layered over
Histopaque gradient (Sigma, #H8889). After centrifugation
(450.times.g, 30 minutes, room temperature), the plasma above the
PBMC-containing interphase was discarded and PBMCs transferred in a
new Falcon tube subsequently filled with 50 ml of PBS. The mixture
was centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant discarded and the PBMC pellet washed twice with sterile
PBS (centrifugation steps 350.times.g, 10 minutes). The resulting
PBMC population was counted automatically (ViCell) and kept in
RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine
(Biochrom, K0302) in cell incubator (37.degree. C., 5% CO.sub.2)
until further use (no longer than 24 h). For the killing assay, the
CEA TCB antibody was used at a fixed concentration of 1 nM and sCEA
was spiked into the experiment at a concentration range of 2.5 ng-5
.mu.g/ml. PBMCs were added to target cells at the final E:T ratio
of 10:1. Target cell killing was assessed after 24 h and 48 h of
incubation by quantification of LDH (lactate dehydrogenase)
released into cell supernatants by apoptotic/necrotic cells (LDH
detection kit, Roche Applied Science, #11 644 793 001). Maximal
lysis of the target cells (=100%) was achieved by incubation of
target cells with 1% Triton X-100. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells without bispecific
antibody. The killing mediated by CEA TCB in absence of sCEA was
set at 100% and the killing obtained in presence of increasing
concentrations of sCEA was normalized to it. Results show that sCEA
had only a minor impact on CEA TCB-mediated killing of
CEA-expressing target cells (FIG. 15A, B). No effect on T cell
killing was detected up to 0.2 .mu.g/ml of sCEA. The sCEA
concentrations above 0.2 .mu.g/ml had only a minor impact on
overall killing (10-50% reduction).
Example 13
T Cell-Mediated Killing of Target Cells Using Human and Cynomolgus
PBMCs as Effector Cells
[0345] T cell-mediated killing of A549 (lung adenocarcinoma) cells
overexpressing human CEA (A549-hCEA), assessed 21 h and 40 h after
incubation with CEA TCB antibody and human PBMCs or cynomolgus
PBMCs as effector cells was assessed. Briefly, target cells were
harvested with Trypsin/EDTA, washed, and plated at density of 25
000 cells/well using flat-bottom 96-well plates. Cells were left to
adhere for several hours. Peripheral blood mononuclear cells
(PBMCs) were prepared by Histopaque density centrifugation of
enriched lymphocyte preparations (buffy coats) obtained from
healthy human donors or healthy cynomolgus monkey. For the later, a
90% Histopaque-PBS density gradient was used. Fresh blood was
diluted with sterile PBS and layered over Histopaque gradient
(Sigma, #H8889). After centrifugation (450.times.g, 30 minutes,
room temperature for human PBMCs, respective 520.times.g, 30 min,
room temperature for cynomolgus PBMCs), the plasma above the
PBMC-containing interphase was discarded and PBMCs transferred in a
new Falcon tube subsequently filled with 50 ml of PBS. The mixture
was centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant discarded and the PBMC pellet washed twice with sterile
PBS (centrifugation steps 350.times.g, 10 minutes). For the
preparation of the cynomolgus PBMCs, an additional low-speed
centrifugation step was performed at 150.times.g for 15 min. The
resulting PBMC population was counted automatically (ViCell) and
kept in RPMI1640 medium containing 10% FCS and 1%
L-alanyl-L-glutamine (Biochrom, K0302) in cell incubator
(37.degree. C., 5% CO.sub.2) until further use (up to 4h). For the
killing assay, the antibodies were added at indicated
concentrations (range of 6 pM-100 nM in triplicates). PBMCs were
added to target cells at the final E:T ratio of 10:1. Target cell
killing was assessed after 21 h and 40 h of incubation by
quantification of LDH (lactate dehydrogenase) released into cell
supernatants by apoptotic/necrotic cells (LDH detection kit, Roche
Applied Science, #11 644 793 001). Maximal lysis of the target
cells (=100%) was achieved by incubation of target cells with 1%
Triton X-100. Minimal lysis (=0%) refers to target cells
co-incubated with effector cells without bispecific antibody.
Results show that CEA TCB mediates target-specific killing of
CEA-positive target cells using both human (FIG. 16, A, C) and
cynomolgus (FIG. 16, B, D) effector cells (PBMCs). The EC.sub.50
values related to 40 h of killing, calculated using GraphPadPrism5
are 306 pM for human PBMCs and 102 pM for cynomolgus PBMCs.
Example 14
T Cell-Mediated Killing of CEA-Expressing Human Colorectal Cancer
Cell Lines Induced by CEA TCB Antibody
[0346] T cell-mediated killing of CEA-expressing human colorectal
cancer cell lines 48 h after incubation with human PBMCs and CEA
TCB antibody at 0.8 nM, 4 nM and 20 nM was assessed. Briefly, PBMCs
were isolated from leukocyte cones obtained from single healthy
donors. Cells were diluted with PBS (1:10) and layered on
Lymphoprep in 50 mL Falcon tubes. After centrifugation (1800 rpm
for 25 min), the PBMC layer was withdrawn from the interface and
washed 4.times. with PBS. PBMCs were counted, frozen in 10% DMSO in
FCS under controlled-rate freezing conditions at 40.times.10.sup.6
cells/mL and stored in liquid nitrogen until further use. For the
T-cell killing assay, tumor cells were plated directly into 96-well
plates from frozen stocks. Cells were warmed quickly and
transferred immediately into pre-warmed medium, centrifuged, and
re-suspended in complete medium (DMEM, Iscoves or RPMI-1640, all
supplemented with 10% FCS and 1% penicillin/streptomycin) and
plated at a density of 2.5.times.10.sup.4 cells/well. Plates were
then incubated at 37.degree. C. in a humidified 10% CO.sub.2
incubator and medium replaced the next day by 100 .mu.L of RPMI 2%
FCS with 1% glutamine and 50 .mu.L CEA TCB (final concentrations
ranging from 6.4 to 20000 pM, 1:5 titration steps, in duplicate
wells for each condition). Fresh-thawed PBMCs were used for the
assay (thawed from frozen vials within 2 hours of the assay start)
and 50 .mu.L (3.times.10.sup.5) was added to each well to give an
effector:target (E:T) ratio of 10:1. Triton X100 (50 .mu.L of 4%)
was added to 150 .mu.L of target cells to obtain maximum release
values. Plates were incubated at 37.degree. C. for 48 h and the
killing activity determined using the Lactose Dehydrogenase
Cytotoxicity Detection Kit (Roche) in accordance with the
manufacturer's instructions. Percentage of specific cell lysis was
calculated as [sample release-spontaneous release]/[maximum
release-spontaneous release].times.100. FIG. 17, A-C shows the
correlation between CEA expression (receptor copy number quantified
using QIFIKIT, see below) and % killing for 31 colorectal cancer
cell lines (listed on x axis). FIG. 17, D shows the correlation
between CEA expression and % specific lysis at 20 nM of CEA TCB
(Spearman correlation=0.7289, p<0.0001, n=31), indicating that
tumor cells displaying high CEA receptor copy numbers (>50 000)
are efficiently lysed by CEA TCB whereas a cluster of cells
displaying low CEA receptor copy numbers (.ltoreq.10 000) are not
being lysed by CEA TCB under the same experimental conditions. FIG.
17, E shows the correlation between CEA expression and EC.sub.50 of
CEA TCB. Although the correlation is not statistically significant
(Spearman correlation=-0.3994, p=0.1006, R.sup.2=0.1358) the graph
clearly shows a pattern of better CEA TCB potency (i.e. lower
EC.sub.50 values) on tumor cell lines expressing high CEA receptor
copy numbers.
[0347] For the analysis of CEA surface expression on cancer cell
lines, the Qifikit (DakoCytomation, Glostrup, Denmark) was used to
calibrate the fluorescent signals and determine the number of
binding sites per cell. Cells were incubated on ice for 30 min with
a mouse anti-human CEACAM5 monoclonal antibody (0.5 .mu.g for
5.times.105 cells, clone: CI-P83-1, sc-23928, Santa Cruz), washed
twice with PBS1X-BSA 0.1% followed by a 45 min incubation with
polyclonal fluorescein isothiocyanate-conjugated goat anti-mouse
antibody provided with the Qifikit. Dead cells were excluded from
the analysis using 4',6-diamidino-2-phenylindole (DAPI) staining.
Samples were analysed on a CyAn.TM. ADP Analyzer (Beckman Coulter).
All mean fluorescence intensities (MFIs) were obtained after data
analyses using Summit 4.3 software. These MFIs were used to
determine the relative number of antibody binding sites on the cell
lines (named as CEA copy number on the results) using the equation
obtained from the calibration curve (Qifikit calibration
beads).
[0348] The colorectal cancer cell lines used for the T-cell killing
assays and CEA surface expression quantification were seeded from
cryovials. The method used to maintain the frozen stock was as
described in Bracht et al. (Bracht et al. (2010), Br J Cancer 103,
340-346).
Example 15
[0349] In Vivo Anti-Tumor Efficacy of CEA TCB in a LS174T-Fluc2
Human Colon Carcinoma Co-Grafted with Human PBMC (E:T Ratio
5:1)
[0350] NOG (NOD/Shi-scid/IL-2R.gamma.null) mice (n=12) were
injected subcutaneously with 1.times.10.sup.6LS174T-fluc2 cells
pre-mixed with human PBMC in a total volume of 100 .mu.l in PBS,
E:T ratio 5:1. LS174T-fluc2 cells have been engineered to express
luciferase, which allows monitoring tumor progression by
bioluminescence (BLI) in a non-invasive and highly sensitive
manner. To assess early and delayed treatment effects, mice
received bi-weekly i.v. injections of either 0.5 or 2.5 mg/kg of
the CEA TCB starting at day 1 (early treatment) or day 7 (delayed
treatment) after tumor cell/PBMCs co-grafting s.c. As a control,
one group of mice received bi-weekly i.v. injections of 2.5 mg/kg
of a control TCB that had the same format as CEA TCB (in this case
the MCSP TCB served as untargeted control since LS174T-fluc2 cells
do not express MCSP), and an extra control group received only PBS
(vehicle) starting at day 1. Tumor volume was measured once a week
by digital caliper. Furthermore, mice were injected i.p. once
weekly with D-Luciferin and the bioluminescent light emission of
living tumor cells was measured with IVIS Spectrum (Perkin Elmer).
Treatment was administered until 19 days after tumor cell
inoculation, which corresponds to the day of study termination. The
results of the experiment are shown in FIG. 18A-D. Results show
average and SEM from 12 mice of tumor volume measured by caliper (A
and C) and by bioluminescence (Total Flux, B and D) in the
different study groups ((A, B) early treatment, (C, D) delayed
treatment).
Example 16
[0351] In Vivo Anti-Tumor Efficacy of CEA TCB in a LS174T-Fluc2
Human Colon Carcinoma Co-Grafted with Human PBMC (E:T Ratio
1:1)
[0352] NOG (NOD/Shi-scid/IL-2R.gamma.null) mice (n=10) were
injected subcutaneously with 1.times.10.sup.6 LS174T-fluc2 cells
(see Example 15) pre-mixed with human PBMC in a total volume of 100
.mu.l in PBS, E:T ratio 1:1. To assess early and delayed treatment
effects, mice received bi-weekly i.v. injections of 2.5 mg/kg of
the CEA TCB starting at day 1 (early treatment) or day 7 (delayed
treatment) after tumor cell inoculation. As control, one group of
mice received bi-weekly i.v. injections of 2.5 mg/kg of the MCSP
TCB (see also Example 15), and an extra control group received only
PBS (vehicle) starting at day 1. Tumor volume was measured once
weekly by digital caliper. Furthermore, mice were injected i.p.
once weekly with D-Luciferin and the bioluminescent light emission
of living tumor cells was measured with IVIS Spectrum (Perkin
Elmer). Treatment was administered until 23 days after tumor cell
inoculation, which corresponds to the day of study termination. The
results of the experiment are shown in FIG. 19. Results show
average and SEM of tumor volume measured by caliper (A) as well as
by bioluminescence (B) in the different study groups (n=10).
Example 17
[0353] In Vivo Efficacy of Murinized CEA TCB in a Panco2-huCEA
Orthotopic Tumor Model in Immunocompetent huCD3E/huCEA Transgenic
Mice
[0354] huCD3c/huCEA transgenic mice (n=10) received an
intra-pancreatic injection of 2.times.10.sup.5 Panco2-huCEA cells
in a total volume of 10 .mu.l in PBS. As murine cells do not
express CEA, the murine pancreatic carcinoma cell line Panco2 was
engineered to overexpress human CEA as the target antigen for the
CEA TCB. Mice were injected twice weekly i.v. with 0.5 mg/kg of the
murinized CEA TCB or PBS as a control group (vehicle) and survival
was monitored. Animals were controlled daily for clinical symptoms
and detection of adverse effects. Termination criteria for animals
were visible sickness: scruffy fur, arched back, breathing
problems, impaired locomotion. The result as overall survival is
shown in FIG. 20. Result shows percent of surviving animals per
time point. The significance of the treatment group to the PBS
control group was compared using a paired Student t test
(p=0.078).
Example 18
Affinity of the CEA TCB to CEA and CD3 by Surface Plasmon Resonance
(SPR)
[0355] Surface plasmon resonance (SPR) experiments were performed
on a Biacore T100 at 25.degree. C. with HBS-EP as running buffer
(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant
P20, Biacore, Freiburg/Germany).
[0356] For affinity measurements CEA TCB was captured on a CM5
sensorchip surface with immobilized anti human Fab (GE Healthcare
#28-9583-25). Capture IgG was coupled to the sensorchip surface by
direct immobilization of around 10,000 resonance units (RU) at pH
5.0 using the standard amine coupling kit (Biacore,
Freiburg/Germany).
[0357] To analyze the interaction to human CD3.epsilon. stalk-Fc
(knob)-Avi/CD3.delta.-stalk-Fc(hole) (SEQ ID NOs 120 and 121,
respectively), CEA TCB was captured for 30 s at 50 nM with 10
.mu.l/min. CD3.epsilon./CD3.delta. was passed at a concentration of
0.68-500 nM with a flowrate of 30 .mu.l/min through the flow cells
over 360 s. The dissociation was monitored for 360 s.
[0358] The K.sub.D value of the interaction between CEA TCB and the
recombinant tumor target antigen human NAB A-avi-his (containing
the B3 domain of human CEA (CEACAM5) surrounded by the N, A1 and A2
domain of human CEACAM1 with a C-terminal avi 6his tag; see SEQ ID
NO: 119) was determined by capturing the TCB molecule for 40 s at
10 .mu.l/min. The antigen was flown over the flow cell for 240 s in
a concentration range from 0.68 to 500 nM at a flow rate of 30
.mu.l/min. The dissociation was measured over 240 s.
[0359] Bulk refractive index differences were corrected for by
subtracting the response obtained on a reference flow cell. Here,
the antigens were flown over a surface with immobilized anti-human
Fab antibody but on which HBS-EP has been injected rather than
CEA.
[0360] Kinetic constants were derived using the Biacore T200
Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rate
equations for 1:1 Langmuir binding by numerical integration. The
half-life (t.sub.1/2) of the interaction was calculated using
following formula: t.sub.1/2=ln 2/k.sub.off.
[0361] The CEA TCB binds to the tumor target and
CD3.epsilon./CD3.delta. in the nM-range with K.sub.D values of 62
nM for the human NABA and 75.3 nM for the human
CD3.epsilon./CD3.delta.. The half-life of the monovalent binding to
NABA is 5.3 minutes, the half-life of the binding to
CD3.epsilon./CD3.delta. is 5.7 minutes. The kinetic values are
summarized in Table 9.
TABLE-US-00012 TABLE 9 Affinity of CEA TCB to human NABA and human
CD3.epsilon./CD3.delta. (T = 25.degree. C). Antigen TCB k.sub.on
[1/Ms] k.sub.off [1/s] t.sub.1/2 [min] K.sub.D [nM] Human CEA TCB
3.49 .times. 10.sup.4 2.18 .times. 10.sup.-3 5.3 62.4 NABA Human
CD3.epsilon./CD3.delta. CEA TCB 2.69 .times. 10.sup.4 2.03 .times.
10.sup.-3 5.7 75.3
Example 19
Affinity of the MSCP TCB to MCSP and CD3 by Surface Plasmon
Resonance (SPR)
[0362] Surface plasmon resonance (SPR) experiments were performed
on a Biacore T100 at 25.degree. C. with HBS-EP as running buffer
(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant
P20, Biacore, Freiburg/Germany).
[0363] For affinity measurements MCSP TCB was captured on a CM5
sensorchip surface with immobilized anti human Fab (GE Healthcare
#28-9583-25). Capture IgG was coupled to the sensorchip surface by
direct immobilization of around 7,500 resonance units (RU) at pH
5.0 using the standard amine coupling kit (Biacore,
Freiburg/Germany). MCSP TCB was captured for 60 s at 30 nM with 10
.mu.l/min. Human and cynomolgus MCSP D3 (see SEQ ID NOs 118 and
117, respectively) were passed at a concentration of 0.024-50 nM
with a flowrate of 30 .mu.l/min through the flow cells over 90 s.
The concentration range for human and cynomolgus CD3c stalk-Fc
(knob)-Avi/CD3.delta.-stalk-Fc(hole) was 1.17-600 nM. Since the
interaction with murine MCSP D3 (SEQ ID NO: 122) was expected to be
weak the concentration range for this antigen was chosen between
3.9 and 500 nM. The dissociation for all interactions was monitored
for 120 s. Bulk refractive index differences were corrected for by
subtracting the response obtained on a reference flow cell. Here,
the antigens were flown over a surface with immobilized anti-human
Fab antibody but on which HBS-EP has been injected rather than MCSP
TCB.
[0364] Kinetic constants were derived using the Biacore T200
Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rate
equations for 1:1 Langmuir binding by numerical integration. The
interaction for the MCSP TCB with the murine MCSP D3 was determined
in steady state. The half-life (t.sub.1/2) of the interaction was
calculated using following formular: t.sub.1/2=ln 2/k.sub.off.
[0365] The MCSP TCB binds to the tumor target in pM-range with
K.sub.D values of 0.15 nM for the human and 0.12 nM for the
cynomolgus antigen. Recombinant CD3.epsilon./CD3.delta. is bound by
the MCSP TCB with a K.sub.D value of 78 nM (human) and 104 nM
(cynomolgus). The half-life of the monovalent binding is up to 260
minutes for the tumor target and 2.9 minutes for the CD3e/CD3d.
Upon affinity maturation the MCSP antibody obtained some binding to
recombinant murine MCSP D3. K.sub.D value for this interaction is
in mM range (1.6 mM). The kinetic values are summarized in Table
10.
TABLE-US-00013 TABLE 10 Affinity of MCSP TCB to the human,
cynomolgus and murine MCSP D3 and human and cynomolgus
CD3.epsilon./CD3.delta. (T = 25.degree. C.). k.sub.on [1/Ms]
k.sub.off [1/s] t.sub.1/2 [min] K.sub.D [nM] Human MCSP D3 3.89
.times. 10.sup.5 5.63 .times. 10-.sup.5 205 0.15 Cynomolgus MCSP D3
3.70 .times. 10.sup.5 4.39 .times. 10-.sup.5 263 0.12 Murine MCSP
D3 nd nd nd 1570* Human CD3.epsilon./CD3.delta. 4.99 .times.
10.sup.4 3.92 .times. 10.sup.-3 2.9 78.7 Cynomolgus CD3.epsilon./
4.61 .times. 10.sup.4 4.78 .times. 10.sup.-3 2.4 104 CD3.delta.
*determined by steady state measurement
Example 20
Thermal Stability of CEA TCB
[0366] Thermal stability of the CEA TCB was monitored by Dynamic
Light Scattering (DLS). 30 .mu.g of filtered protein sample with a
protein concentration of 0.5 mg/ml was applied in duplicate to a
Dynapro plate reader (Wyatt Technology Corporation; USA). The
temperature was ramped from 25 to 75.degree. C. at 0.05.degree.
C./min, with the radius and total scattering intensity being
collected. The result is shown in FIG. 21. The aggregation
temperature of the CEA TCB was measured at 55.degree. C.
Example 21
Thermal Stability of MCSP TCB
[0367] Thermal stability of the MCSP TCB was monitored by Dynamic
Light Scattering (DLS). 30 .mu.g of filtered protein sample with a
protein concentration of 0.5 mg/ml was applied in duplicate to a
Dynapro plate reader (Wyatt Technology Corporation; USA). The
temperature was ramped from 25 to 75.degree. C. at 0.05.degree.
C./min, with the radius and total scattering intensity being
collected.
[0368] The result is shown in FIG. 22. The aggregation temperature
of the MCSP TCB was measured at 55.degree. C.
Example 22
T Cell-Mediated Killing of MCSP-Expressing Tumor Target Cells
Induced by MCSP TCB and MCSP 1+1 CrossMab Antibodies
[0369] T cell-mediated killing of target cells induced by MCSP TCB
and MCSP 1+1 CrossMab TCB (a T cell activating bispecific antibody
having the same CD3 and MCSP binding sequences as the MCSP TCB,
with the molecular format shown in FIG. 1D) antibodies was assessed
on A375 (high MCSP), MV-3 (medium MCSP) and HCT-116 (low MCSP)
tumor target cells. LS180 (MCSP negative tumor cell line) was used
as negative control. Tumor cell killing was assessed 24 h and 48 h
post incubation of target cells with the antibodies and effector
cells (human PBMCs). Briefly, target cells were harvested with
Trypsin/EDTA, washed, and plated at density of 25 000 cells/well
using flat-bottom 96-well plates. Cells were left to adhere
overnight. Peripheral blood mononuclear cells (PBMCs) were prepared
by Histopaque density centrifugation of enriched lymphocyte
preparations (buffy coats) obtained from healthy human donors.
Fresh blood was diluted with sterile PBS and layered over
Histopaque gradient (Sigma, #H8889). After centrifugation
(450.times.g, 30 minutes, room temperature), the plasma above the
PBMC-containing interphase was discarded and PBMCs transferred in a
new Falcon tube subsequently filled with 50 ml of PBS. The mixture
was centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant discarded and the PBMC pellet washed twice with sterile
PBS (centrifugation steps 350.times.g, 10 minutes). The resulting
PBMC population was counted automatically (ViCell) and kept in
RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine
(Biochrom, K0302) in cell incubator (37.degree. C., 5% CO.sub.2)
until further use (no longer than 24 h). For the killing assay, the
antibodies were added at indicated concentrations (range of 0.01
pM-10 nM in triplicates). PBMCs were added to target cells at the
final E:T ratio of 10:1. Target cell killing was assessed after 24
h and 48 h of incubation by quantification of LDH (lactate
dehydrogenase) released into cell supernatants by
apoptotic/necrotic cells (LDH detection kit, Roche Applied Science,
#11 644 793 001). Maximal lysis of the target cells (=100%) was
achieved by incubation of target cells with 1% Triton X-100.
Minimal lysis (=0%) refers to target cells co-incubated with
effector cells without bispecific antibody. The results show that
MCSP TCB antibody is more potent than the MCSP 1+1 CrossMab TCB as
it induced stronger killing of MCSP-positive target cells at both
time points and on all tumor target cells (FIG. 23A-H). The
EC.sub.50 values related to killing assays, calculated using
GraphPadPrism5, are given in Table 11.
TABLE-US-00014 TABLE 11 MCSP receptor copy number and EC.sub.50
values (pM) for T-cell mediated killing of MCSP-expressing tumor
cells induced by MCSP TCB antibody (n.d. = not determined). MCSP
receptor EC50 [pM] EC50 [pM] Cell line copy number 24 h 48 h A375
387 058 0.1 n.d. MV-3 260 000 1.0 0.7 HCT-116 36770 ~6.2e-008 ~0.09
LS180 negative ~764 n.d.
Example 23
[0370] CD25 and CD69 Upregulation on CD8.sup.+ and CD4.sup.+
Effector Cells after T Cell-Mediated Killing of MCSP-Expressing
Tumor Cells Induced by MCSP TCB and MCSP 1+1 CrossMab
Antibodies
[0371] Activation of CD8.sup.+ and CD4.sup.+ T cells after T-cell
killing of MCSP-expressing tumor cells (A375 and MV-3) mediated by
the MCSP TCB and MCSP 1+1 CrossMab antibodies was assessed by FACS
analysis using antibodies recognizing T cell activation markers
CD25 (late activation marker) and CD69 (early activation marker).
The antibody and the killing assay conditions were essentially as
described above (Example 22), using the same antibody concentration
range (0.01 pM-10 nM in triplicates), E:T ratio 10:1 and an
incubation time of 48 h.
[0372] After the incubation, PBMCs were transferred to a
round-bottom 96-well plate, centrifuged at 350.times.g for 5 min
and washed twice with PBS containing 0.1% BSA. Surface staining for
CD8 (FITC anti-human CD8, BD #555634), CD4 (PECy7 anti-human CD4,
BD #557852), CD69 (PE anti-human CD69, Biolegend #310906) and CD25
(APC anti-human CD25, BD #555434) was performed according to the
suppliers' indications. Cells were washed twice with 150 .mu.l/well
PBS containing 0.1% BSA and fixed for 15 min at 4.degree. C. using
100 .mu.l/well fixation buffer (BD #554655). After centrifugation,
the samples were resuspended in 200 .mu.l/well PBS 0.1% BSA
containing DAPI to exclude dead cells for the FACS measurement.
Samples were analyzed at BD FACS Fortessa. The results show that
MCSP TCB induced a strong and target-specific upregulation of
activation markers (CD25, CD69) on CD8.sup.+ T cells (FIGS. 24A, B
(for A375 cells) and E, F (for MV-3 cells)) and CD4.sup.+ T cells
(FIGS. 24C, D (for A375 cells) and G, H (for MV-3 cells)) after
killing. As for the killing results, the activation of T cells was
stronger with MCSP TCB than with MCSP 1+1 CrossMab.
Example 24
Preparation of DP47 GS TCB (2+1 Crossfab-IgG P329G LALA
Inverted="Untargeted TCB") Containing DP47 GS as Non Binding
Antibody and Humanized CH2527 as Anti CD3 Antibody
[0373] The "untargeted TCB" was used as a control in the above
experiments. The bispecific antibody engages CD3.epsilon. but does
not bind to any other antigen and therefore cannot crosslink T
cells to any target cells (and subsequently cannot induce any
killing). It was therefore used as negative control in the assays
to monitor any unspecific T cell activation.
[0374] The variable region of heavy and light chain DNA sequences
were subcloned in frame with either the constant heavy chain or the
constant light chain pre-inserted into the respective recipient
mammalian expression vector. The antibody expression is driven by
an MPSV promoter and carries a synthetic polyA signal sequence at
the 3' end of the CDS. In addition each vector contains an EBV OriP
sequence.
[0375] The molecule was produced by co-transfecting HEK293 EBNA
cells with the mammalian expression vectors using polyethylenimine
(PEI). The cells were transfected with the corresponding expression
vectors in a 1:2:1:1 ratio ("vector heavy chain Fc(hole)":"vector
light chain":"vector light chain Crossfab": "vector heavy chain
Fc(knob)-FabCrossfab").
[0376] For transfection HEK293 EBNA cells were cultivated in
suspension serum-free in CD CHO culture medium. For the production
in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24
hours before transfection. For transfection cells were centrifuged
for 5 min at 210.times.g, supernatant is replaced by pre-warmed 20
ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO
medium to a final amount of 200 .mu.g DNA. After addition of 540
.mu.l PEI solution the mixture was vortexed for 15 s and
subsequently incubated for 10 min at room temperature. Afterwards
cells were mixed with the DNA/PEI solution, transferred to a 500 ml
shake flask and incubated for 3 hours at 37.degree. C. in an
incubator with a 5% CO.sub.2 atmosphere. After the incubation time
160 ml F17 medium was added and cell were cultivated for 24 hours.
One day after transfection 1 mM valproic acid and 7% Feed 1 (Lonza)
was added. After 7 days cultivation supernatant was collected for
purification by centrifugation for 15 min at 210.times.g, the
solution was sterile filtered (0.22 .mu.m filter) and sodium azide
in a final concentration of 0.01% w/v was added, and kept at
4.degree. C.
[0377] The secreted protein was purified from cell culture
supernatants by affinity chromatography using Protein A.
Supernatant was loaded on a HiTrap Protein A HP column (CV=5 mL, GE
Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM
sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was
removed by washing with at least 10 column volumes 20 mM sodium
phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5.
Target protein was eluted during a gradient over 20 column volumes
from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM
sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was
neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target
protein was concentrated and filtrated prior loading on a HiLoad
Superdex 200 column (GE Healthcare) equilibrated with 20 mM
histidine, 140 mM sodium chloride solution of pH 6.0.
[0378] The protein concentration of purified protein samples was
determined by measuring the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence.
[0379] Purity and molecular weight of molecules were analyzed by
CE-SDS analyses in the presence and absence of a reducing agent.
The Caliper LabChip GXII system (Caliper lifescience) was used
according to the manufacturer's instruction. 2 .mu.g sample was
used for analyses.
[0380] The aggregate content of antibody samples was analyzed using
a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine
monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C.
TABLE-US-00015 TABLE 12 Summary production and purification of DP47
GS TCB. Aggregate after 1.sup.st Titer Yield purification HMW LMW
Monomer Construct [mg/l] [mg/l] step [%] [%] [%] [%] DP47 GS TCB
103.7 8.04 8 2.3 6.9 91.8
[0381] FIG. 25 and Table 13 show CE-SDS analyses of the DP47 GS TCB
(2+1 Crossfab-IgG P329G LALA inverted) containing DP47 GS as
non-binding antibody and humanized CH2527 as anti-CD3 antibody.
(SEQ ID NOs: 59, 60, 61 and 62).
TABLE-US-00016 TABLE 13 CE-SDS analyses of DP47 GS TCB. Peak kDa
Corresponding Chain DP47 GS TCB 1 165.22 Molecule with 2 missing
light non reduced (A) chains 2 181.35 Molecule with 1 missing light
chain 3 190.58 Correct molecule without N- linked glycosylation 4
198.98 Correct molecule DP47 GS TCB 1 27.86 Light chain DP47 GS
reduced (B) 2 35.74 Light chain huCH2527 3 63.57 Fc(hole) 4 93.02
Fc(knob)
[0382] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
1221455PRTArtificial SequenceHC CD3 CH2527 (VH_3-23(12)) 1Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr 115 120 125Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 195 200 205Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu 210 215 220Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro225 230 235 240Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295
300Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp305 310 315 320Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 325 330 335Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 340 345 350Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys385 390 395 400Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410
415Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 435 440 445Leu Ser Leu Ser Pro Gly Lys 450
4552215PRTArtificial SequenceLC CD3 CH2527 (VL_7-46(13)) 2Gln Ala
Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr
Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25
30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly
35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg
Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser
Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln Pro 100 105 110Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu Leu 115 120 125Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala145 150 155 160Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170
175Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg
180 185 190Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
Lys Thr 195 200 205Val Ala Pro Thr Glu Cys Ser 210
2153125PRTArtificial SequenceVH CD3 CH2527 (VH_3-23(12)) 3Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 12545PRTArtificial SequenceHCDR1 CD3 CH2527
(VH_3-23(12)) 4Thr Tyr Ala Met Asn1 5519PRTArtificial SequenceHCDR2
CD3 CH2527 (VH_3-23(12)) 5Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys Gly614PRTArtificial
SequenceHCDR3 CD3 CH2527 (VH_3-23(12)) 6His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr1 5 107109PRTArtificial SequenceVL CD3
CH2527 (VL_7-46(13)) 7Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys
Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg
Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly
Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu
Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105814PRTArtificial
SequenceLCDR1 CD3 CH2527 (VL_7-46(13)) 8Gly Ser Ser Thr Gly Ala Val
Thr Thr Ser Asn Tyr Ala Asn1 5 1097PRTArtificial SequenceLCDR2 CD3
CH2527 (VL_7-46(13)) 9Gly Thr Asn Lys Arg Ala Pro1
5109PRTArtificial SequenceLCDR3 CD3 CH2527 (VL_7-46(13)) 10Ala Leu
Trp Tyr Ser Asn Leu Trp Val1 511442PRTArtificial SequenceHC MCSP
M4-3 (C1)$ 11Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Thr Ser Gly 20 25 30Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly
Lys Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn
Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Arg Asp
Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asp Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135
140Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 195 200 205Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250
255Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 340 345 350Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375
380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 44012214PRTArtificial SequenceLC MCSP ML2 (G3) 12Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Ala Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21013112PRTArtificial
SequenceVH MCSP M4-3 (C1) 13Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Thr Ser Gly 20 25 30Tyr Tyr Trp Asn Trp Ile Arg Gln
His Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile Thr Phe Asp
Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile
Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asp Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
110146PRTArtificial SequenceHCDR1 MCSP M4-3 (C1) 14Ser Gly Tyr Tyr
Trp Asn1 51516PRTArtificial SequenceHCDR2 MCSP M4-3 (C1) 15Tyr Ile
Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10
15163PRTArtificial SequenceHCDR3 MCSP M4-3 (C1) 16Phe Asp
Tyr117107PRTArtificial SequenceVL MCSP ML2 (G3) 17Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ala Leu Pro
Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1051811PRTArtificial SequenceLCDR1 MCSP ML2 (G3) 18Arg Ala Ser Gln
Gly Ile Arg Asn Tyr Leu Asn1 5 10197PRTArtificial SequenceLCDR2
MCSP ML2 (G3) 19Tyr Thr Ser Ser Leu His Ser1 5209PRTArtificial
SequenceLCDR3 MCSP ML2 (G3) 20Gln Gln Tyr Ser Ala Leu Pro Trp Thr1
521451PRTArtificial SequenceHC CEA CH1A1A 98-99 21Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55
60Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr
Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45022215PRTArtificial SequenceLC CEA 2F1 22Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu
85 90 95Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
Ala 100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200
205Ser Phe Asn Arg Gly Glu Cys 210 21523121PRTArtificial SequenceVH
CEA CH1A1A 98-99 23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu
Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe
Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120245PRTArtificial SequenceHCDR1 CEA
CH1A1A 98-99 24Glu Phe Gly Met Asn1 52517PRTArtificial
SequenceHCDR2 CEA CH1A1A 98-99 25Trp Ile Asn Thr Lys Thr Gly Glu
Ala Thr Tyr Val Glu Glu Phe Lys1 5 10 15Gly2612PRTArtificial
SequenceHCDR3 CEA CH1A1A 98-99 26Trp Asp Phe Ala Tyr Tyr Val Glu
Ala Met Asp Tyr1 5 1027108PRTArtificial SequenceVL CEA 2F1 27Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr
Tyr Thr Tyr Pro Leu 85 90 95Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 1052811PRTArtificial SequenceLCDR1 CEA 2F1 28Lys Ala
Ser Ala Ala Val Gly Thr Tyr Val Ala1 5 10297PRTArtificial
SequenceLCDR2 CEA 2F1 29Ser Ala Ser Tyr Arg Lys Arg1
53010PRTArtificial SequenceLCDR3 CEA 2F1 30His Gln Tyr Tyr Thr Tyr
Pro Leu Phe Thr1 5 1031111PRTArtificial SequenceVL CD3 CH2527
(VL_7-43(11)) 31Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala
Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly
Gln Ala Pro Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro
Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala
Ala Leu Thr Leu Ser Gly Val65 70 75 80Gln Pro Glu Asp Glu Ala Glu
Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Ser Ser 100 105 11032125PRTArtificial
SequenceVH CD3 CH2527 (VHcomboA49SV93A) 32Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12533125PRTArtificial SequenceVH CD3 CH2527
(VHcomboA49SV93AR94K) 33Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Lys
His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12534112PRTArtificial SequenceVH MCSP M4-3 (D6) 34Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly 20 25 30Tyr Tyr
Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile
Gly Tyr Ile Thr Phe Asp Gly Lys Asn Asn Tyr Asn Pro Ser Leu 50 55
60Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser65
70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 100 105 1103515PRTArtificial SequenceHCDR2 MCSP M4-3 (D6)
35Ile Thr Phe Asp Gly Lys Asn Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10
1536112PRTArtificial SequenceVH MCSP M4-3 (A7) 36Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Asp Gly 20 25 30Tyr Tyr
Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile
Gly Tyr Ile Thr Phe Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu 50 55
60Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser65
70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 100 105 110376PRTArtificial SequenceHCDR1 MCSP M4-3 (A7)
37Asp Gly Tyr Tyr Trp Asn1 53815PRTArtificial SequenceHCDR2 MCSP
M4-3 (A7) 38Ile Thr Phe Asp Gly Arg Asn Asn Tyr Asn Pro Ser Leu Lys
Ser1 5 10 1539112PRTArtificial SequenceVH MCSP M4-3 (B7) 39Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly 20 25
30Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
35 40 45Ile Gly Tyr Ile Thr Phe Asp Gly Ile Asn Asn Tyr Asn Pro Ser
Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 100 105 1104015PRTArtificial SequenceHCDR2 MCSP
M4-3 (B7) 40Ile Thr Phe Asp Gly Ile Asn Asn Tyr Asn Pro Ser Leu Lys
Ser1 5 10 1541112PRTArtificial SequenceVH MCSP M4-3 (B8) 41Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly 20 25
30Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
35 40 45Ile Gly Tyr Ile Thr Phe Asp Gly Arg Asn Asn Tyr Asn Pro Ser
Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln
Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 100 105 11042112PRTArtificial SequenceVH MCSP
M4-3 42Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr
Ser Gly 20 25 30Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly
Leu Glu Trp 35 40 45Ile Gly Tyr Ile Thr Tyr Asp Gly Ser Asn Asn Tyr
Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser
Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asp Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 11043107PRTArtificial
SequenceVL MCSP ML2 (E10) 43Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Tyr Gly Ile Arg Gly Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Ser Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr His Cys Gln Gln Tyr Ser Lys Leu Pro Trp 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 1054411PRTArtificial
SequenceLCDR1 MCSP ML2 (E10) 44Arg Ala Ser Tyr Gly Ile Arg Gly Tyr
Leu Asn1 5 10459PRTArtificial SequenceLCDR3 MCSP ML2 (E10) 45Gln
Gln Tyr Ser Lys Leu Pro Trp Thr1 546107PRTArtificial SequenceVL
MCSP ML2 (E10-G3) 46Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Tyr
Gly Ile Arg Gly Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Ser Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
His Cys Gln Gln Tyr Ser Ala Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 10547107PRTArtificial SequenceVL MCSP
ML2 (C5) 47Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Gly Ile
Arg Glu Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Gly Ser Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Ser Glu Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 1054811PRTArtificial SequenceLCDR1 MCSP ML2
(C5) 48Arg Ala Ser Arg Gly Ile Arg Glu Tyr Leu Asn1 5
10497PRTArtificial SequenceLCDR2 MCSP ML2 (C5) 49Tyr Thr Gly Ser
Leu His Ser1 5509PRTArtificial SequenceLCDR3 MCSP ML2 (C5) 50Gln
Gln Tyr Ser Glu Leu Pro Trp Thr1 551107PRTArtificial SequenceVL
MCSP ML2 (C5-G3) 51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg
Gly Ile Arg Glu Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Gly Ser Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Ser Ala Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 10552107PRTArtificial SequenceVL MCSP
ML2 52Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Ser Lys Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 10553214PRTArtificial SequenceLC CD3 CH2527
(Crossfab, VL-CH1) 53Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys
Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg
Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly
Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu
Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala 100 105 110Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 115 120
125Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
130 135 140Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly145 150 155 160Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu 165 170 175Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr 180 185 190Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 195 200 205Val Glu Pro Lys
Ser Cys 21054685PRTArtificial SequenceMCSP M4-3 (C1) (VH-CH1) - CD3
CH2527 (Crossfab VH-Ck) - Fc(knob) P329GLALA 54Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Gly 20 25 30Tyr Tyr Trp
Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly
Tyr Ile Thr Phe Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60Lys
Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser65 70 75
80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 100 105 110Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 115 120 125Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 130 135 140Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser145 150 155 160Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200
205Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro225 230 235 240Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser 245 250 255Thr Tyr Ala Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu 260 265 270Trp Val Ser Arg Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr 275 280 285Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys 290 295 300Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala305 310 315
320Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
325 330 335Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala 340 345 350Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 355 360 365Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 370 375 380Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser385 390 395 400Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 405 410 415Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 420 425 430His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 435 440
445Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr His Thr Cys
450 455 460Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
Phe Leu465 470 475 480Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 485 490 495Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 500 505 510Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys 515 520 525Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 530 535 540Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys545 550 555
560Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys
565 570 575Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys 580 585 590Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
Cys Leu Val Lys 595 600 605Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 610 615 620Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly625 630 635 640Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 645 650 655Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 660 665 670His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680
68555442PRTArtificial SequenceMCSP M4-3 (C1) (VH-CH1) - Fc(hole)
P329GLALA 55Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
Thr Ser Gly 20 25 30Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys
Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile Thr Phe Asp Gly Ser Asn Asn
Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Arg Asp Thr
Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser145 150
155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 195 200 205Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265
270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn305 310 315 320Lys Ala Leu Gly Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro Gln Val
Cys Thr Leu Pro Pro Ser Arg Asp Glu 340 345 350Leu Thr Lys Asn Gln
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 355 360 365Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe385 390
395 400Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
44056214PRTArtificial SequenceLC CD3 CH2527 (Crossfab, VL-CH1)
56Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Ser Ser Ala 100 105 110Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser 115 120 125Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135 140Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly145 150 155
160Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
165 170 175Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr 180 185 190Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys 195 200 205Val Glu Pro Lys Ser Cys
21057694PRTArtificial SequenceCEA CH1A1A 98/99 - CD3 CH2527
(Crossfab VH-Ck) - Fc(knob) P329GLALA 57Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn
Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg
Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Leu225 230 235 240Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 245 250 255Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr Ala Met Asn Trp Val 260 265 270Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser Arg Ile Arg Ser 275 280 285Lys Tyr Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 290 295 300Phe Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met305 310 315 320Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His 325 330
335Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln
340 345 350Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro
Ser Val 355 360 365Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser 370 375 380Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln385 390 395 400Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val 405 410 415Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 420 425 430Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 435 440 445Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 450 455
460Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu465 470 475 480Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp 485 490 495Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp 500 505 510Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly 515 520 525Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 530 535 540Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp545 550 555 560Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly 565 570
575Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
580 585 590Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
Lys Asn 595 600 605Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile 610 615 620Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr625 630 635 640Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys 645 650 655Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 660 665 670Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 675 680 685Ser
Leu Ser Pro Gly Lys 69058451PRTArtificial SequenceCEA CH1A1A 98/99
(VH-CH1) - Fc(hole) P329GLALA 58Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys
Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr
Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355
360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45059215PRTArtificial SequenceLC DP47 GS 59Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
85 90 95Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala 100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200
205Ser Phe Asn Arg Gly Glu Cys 210 21560214PRTArtificial SequenceLC
CD3 CH2527 (Crossfab, VL-CH1) 60Gln Ala Val Val Thr Gln Glu Pro Ser
Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser
Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln
Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn
Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu
Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln Pro Glu
Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala 100 105
110Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
115 120 125Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe 130 135 140Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly145 150 155 160Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu 165 170 175Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr 180 185 190Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 195 200 205Val Glu Pro
Lys Ser Cys 21061688PRTArtificial SequenceDP47 GS (VH-CH1) - CD3
CH2527 (Crossfab VH-Ck) - Fc(knob) P329GLALA 61Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Gly Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200
205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser
210 215 220Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu225 230 235 240Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe 245 250 255Thr Phe Ser Thr Tyr Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys 260 265 270Gly Leu Glu Trp Val Ser Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala 275 280 285Thr Tyr Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 290 295 300Asp Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu305 310 315
320Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
325 330 335Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val 340 345 350Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser 355 360 365Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn 370 375 380Asn Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala385 390 395 400Leu Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 405 410 415Asp Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 420 425 430Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 435 440
445Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr
450 455 460His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
Pro Ser465 470 475 480Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 485 490 495Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 500 505 510Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 515 520 525Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 530 535 540Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr545 550 555
560Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
565 570 575Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 580 585 590Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Trp Cys 595 600 605Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 610 615 620Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp625 630 635 640Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 645 650 655Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 660 665 670Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675 680
68562445PRTArtificial SequenceDP47 GS (VH-CH1) - Fc(hole) P329GLALA
62Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Ser Gly Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155
160Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly 180 185 190Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys 195 200 205Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280
285Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys305 310 315 320Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr Leu Pro Pro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Ser Cys Ala Val Lys 355 360 365Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390 395
400Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 445631365DNAArtificial SequenceHC CD3 CH2527
(VH_3-23(12)) 63gaggtgcagc tgctggaatc tggcggcgga ctggtgcagc
ctggcggatc tctgagactg 60agctgtgccg ccagcggctt caccttcagc acctacgcca
tgaactgggt gcgccaggcc 120cctggcaaag gcctggaatg ggtgtcccgg
atcagaagca agtacaacaa ctacgccacc 180tactacgccg acagcgtgaa
gggccggttc accatcagcc gggacgacag caagaacacc 240ctgtacctgc
agatgaacag cctgcgggcc gaggacaccg ccgtgtacta ttgtgtgcgg
300cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca
gggcaccctc 360gtgaccgtgt catctgctag caccaagggc ccatcggtct
tccccctggc accctcctcc 420aagagcacct ctgggggcac agcggccctg
ggctgcctgg tcaaggacta cttccccgaa 480ccggtgacgg tgtcgtggaa
ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540gtcctacagt
cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc
600ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac
caaggtggac 660aagaaagttg agcccaaatc ttgtgacaaa actcacacat
gcccaccgtg cccagcacct 720gaactcctgg ggggaccgtc agtcttcctc
ttccccccaa aacccaagga caccctcatg 780atctcccgga cccctgaggt
cacatgcgtg gtggtggacg tgagccacga agaccctgag 840gtcaagttca
actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg
900gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct
gcaccaggac 960tggctgaatg gcaaggagta caagtgcaag gtctccaaca
aagccctccc agcccccatc 1020gagaaaacca tctccaaagc caaagggcag
ccccgagaac cacaggtgta caccctgccc 1080ccatcccggg atgagctgac
caagaaccag gtcagcctga cctgcctggt caaaggcttc 1140tatcccagcg
acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag
1200accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa
gctcaccgtg 1260gacaagagca ggtggcagca ggggaacgtc ttctcatgct
ccgtgatgca tgaggctctg 1320cacaaccact acacgcagaa gagcctctcc
ctgtctccgg gtaaa 136564645DNAArtificial SequenceLC CD3 CH2527
(VL_7-46(13)) 64caggccgtcg tgacccagga acccagcctg acagtgtctc
ctggcggcac cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc accagcaact
acgccaactg ggtgcaggaa 120aagcccggcc aggccttcag aggactgatc
ggcggcacca acaagagagc ccctggcacc 180cctgccagat tcagcggatc
tctgctggga ggaaaggccg ccctgacact gtctggcgcc 240cagccagaag
atgaggccga gtactactgc gccctgtggt acagcaacct gtgggtgttc
300ggcggaggca ccaagctgac agtcctaggt caacccaagg ctgcccccag
cgtgaccctg 360ttccccccca gcagcgagga actgcaggcc aacaaggcca
ccctggtctg cctgatcagc 420gacttctacc caggcgccgt gaccgtggcc
tggaaggccg acagcagccc cgtgaaggcc 480ggcgtggaga ccaccacccc
cagcaagcag agcaacaaca agtacgccgc cagcagctac 540ctgagcctga
cccccgagca gtggaagagc cacaggtcct acagctgcca ggtgacccac
600gagggcagca ccgtggagaa aaccgtggcc cccaccgagt gcagc
64565375DNAArtificial SequenceVH CD3 CH2527 (VH_3-23(12))
65gaggtgcagc tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg
60agctgtgccg ccagcggctt caccttcagc acctacgcca tgaactgggt gcgccaggcc
120cctggcaaag gcctggaatg ggtgtcccgg atcagaagca agtacaacaa
ctacgccacc 180tactacgccg acagcgtgaa gggccggttc accatcagcc
gggacgacag caagaacacc 240ctgtacctgc agatgaacag cctgcgggcc
gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact tcggcaacag
ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt catct
3756615DNAArtificial SequenceHCDR1 CD3 CH2527 (VH_3-23(12))
66acctacgcca tgaac 156757DNAArtificial SequenceHCDR2 CD3 CH2527
(VH_3-23(12)) 67cggatcagaa gcaagtacaa caactacgcc acctactacg
ccgacagcgt gaagggc 576842DNAArtificial SequenceHCDR3 CD3 CH2527
(VH_3-23(12)) 68cacggcaact tcggcaacag ctatgtgtct tggtttgcct ac
4269327DNAArtificial SequenceVL CD3 CH2527 (VL_7-46(13))
69caggccgtcg tgacccagga acccagcctg acagtgtctc ctggcggcac cgtgaccctg
60acatgtggca gttctacagg cgccgtgacc accagcaact acgccaactg ggtgcaggaa
120aagcccggcc aggccttcag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg
ccctgacact gtctggcgcc 240cagccagaag atgaggccga gtactactgc
gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac agtccta
3277042DNAArtificial SequenceLCDR1 CD3 CH2527 (VL_7-46(13))
70ggcagttcta caggcgccgt gaccaccagc aactacgcca ac
427121DNAArtificial SequenceLCDR2 CD3 CH2527 (VL_7-46(13))
71ggcaccaaca agagagcccc t 217227DNAArtificial SequenceLCDR3 CD3
CH2527 (VL_7-46(13)) 72gccctgtggt acagcaacct gtgggtg
27731326DNAArtificial SequenceHC MCSP M4-3 (C1) 73caggtgcaat
tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgcaccg
tgtccggcgg cagcatcacc agcggctatt attggaactg gattcggcag
120caccccggca agggcctgga atggatcggc tacatcactt tcgacggctc
taacaactac 180aaccccagcc tgaagtccag agtgaccatc agccgggaca
ccagcaagaa ccagttcagc 240ctgaagctgt ccagcgtgac agccgccgac
accgccgtgt actactgcgc cgacttcgac 300tactggggcc agggcaccct
ggtcaccgtg tccagcgcta gcaccaaggg cccatcggtc 360ttccccctgg
caccctcctc caagagcacc tctgggggca cagcggccct gggctgcctg
420gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc
cctgaccagc 480ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac
tctactccct cagcagcgtg 540gtgaccgtgc cctccagcag cttgggcacc
cagacctaca tctgcaacgt gaatcacaag 600cccagcaaca ccaaggtgga
caagaaagtt gagcccaaat cttgtgacaa aactcacaca 660tgcccaccgt
gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca
720aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt
ggtggtggac 780gtgagccacg aagaccctga ggtcaagttc aactggtacg
tggacggcgt ggaggtgcat 840aatgccaaga caaagccgcg ggaggagcag
tacaacagca cgtaccgtgt ggtcagcgtc 900ctcaccgtcc tgcaccagga
ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 960aaagccctcc
cagcccccat
cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1020ccacaggtgt
acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg
1080acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga
gagcaatggg 1140cagccggaga acaactacaa gaccacgcct cccgtgctgg
actccgacgg ctccttcttc 1200ctctacagca agctcaccgt ggacaagagc
aggtggcagc aggggaacgt cttctcatgc 1260tccgtgatgc atgaggctct
gcacaaccac tacacgcaga agagcctctc cctgtctccg 1320ggtaaa
132674642DNAArtificial SequenceLC MCSP ML2 (G3) 74gacatccaga
tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacctgcc
gggccagcca gggcatccgg aactacctga actggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctactac accagcagcc tgcacagcgg
cgtgcctagc 180cggtttagcg gcagcggctc cggcaccgac tacaccctga
ccattagctc cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
tactctgctc tgccgtggac cttcggccag 300ggaacaaagg tggagatcaa
gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gt 64275336DNAArtificial SequenceVH MCSP M4-3
(C1) 75caggtgcaat tgcaggaaag cggccctggc ctggtcaagc ccagccagac
cctgagcctg 60acctgcaccg tgtccggcgg cagcatcacc agcggctatt attggaactg
gattcggcag 120caccccggca agggcctgga atggatcggc tacatcactt
tcgacggctc taacaactac 180aaccccagcc tgaagtccag agtgaccatc
agccgggaca ccagcaagaa ccagttcagc 240ctgaagctgt ccagcgtgac
agccgccgac accgccgtgt actactgcgc cgacttcgac 300tactggggcc
agggcaccct ggtcaccgtg tccagc 3367618DNAArtificial SequenceHCDR1
MCSP M4-3 (C1) 76agcggctatt attggaac 187748DNAArtificial
SequenceHCDR2 MCSP M4-3 (C1) 77tacatcactt tcgacggctc taacaactac
aaccccagcc tgaagtcc 48789DNAArtificial SequenceHCDR3 MCSP M4-3 (C1)
78ttcgactac 979321DNAArtificial SequenceVL MCSP ML2 (G3)
79gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc
60atcacctgcc gggccagcca gggcatccgg aactacctga actggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctactac accagcagcc tgcacagcgg
cgtgcctagc 180cggtttagcg gcagcggctc cggcaccgac tacaccctga
ccattagctc cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
tactctgctc tgccgtggac cttcggccag 300ggaacaaagg tggagatcaa g
3218033DNAArtificial SequenceLCDR1 MCSP ML2 (G3) 80cgggccagcc
agggcatccg gaactacctg aac 338121DNAArtificial SequenceLCDR2 MCSP
ML2 (G3) 81tacaccagca gcctgcacag c 218227DNAArtificial
SequenceLCDR3 MCSP ML2 (G3) 82cagcagtact ctgctctgcc gtggacc
27831353DNAArtificial SequenceHC CEA CH1A1A 98-99 83caggtgcagc
tggtgcagtc tggcgccgaa gtgaagaaac ctggagctag tgtgaaggtg 60tcctgcaagg
ccagcggcta caccttcacc gagttcggca tgaactgggt ccgacaggct
120ccaggccagg gcctcgaatg gatgggctgg atcaacacca agaccggcga
ggccacctac 180gtggaagagt tcaagggcag agtgaccttc accacggaca
ccagcaccag caccgcctac 240atggaactgc ggagcctgag aagcgacgac
accgccgtgt actactgcgc cagatgggac 300ttcgcctatt acgtggaagc
catggactac tggggccagg gcaccaccgt gaccgtgtct 360agcgctagca
ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct
420gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc gtgcacacct
tcccggctgt cctacagtcc 540tcaggactct actccctcag cagcgtggtg
accgtgccct ccagcagctt gggcacccag 600acctacatct gcaacgtgaa
tcacaagccc agcaacacca aggtggacaa gaaagttgag 660cccaaatctt
gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg
720ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 840tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 900aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 960aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
1020tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc
atcccgggat 1080gagctgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc 1200gtgctggact ccgacggctc
cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1320acgcagaaga gcctctccct gtctccgggt aaa 135384645DNAArtificial
SequenceLC CEA 2F1 84gatatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtgggaga cagagtcacc 60atcacttgca aggccagtgc ggctgtgggt acgtatgttg
cgtggtatca gcagaaacca 120gggaaagcac ctaagctcct gatctattcg
gcatcctacc gcaaaagggg agtcccatca 180aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagatttcg
caacttacta ctgtcaccaa tattacacct atcctctatt cacgtttggc
300cagggcacca agctcgagat caagcgtacg gtggctgcac catctgtctt
catcttcccg 360ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 420tatcccagag aggccaaagt acagtggaag
gtggataacg ccctccaatc gggtaactcc 480caggagagtg tcacagagca
ggacagcaag gacagcacct acagcctcag cagcaccctg 540acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag
600ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt
64585363DNAArtificial SequenceVH CEA CH1A1A 98-99 85caggtgcagc
tggtgcagtc tggcgccgaa gtgaagaaac ctggagctag tgtgaaggtg 60tcctgcaagg
ccagcggcta caccttcacc gagttcggca tgaactgggt ccgacaggct
120ccaggccagg gcctcgaatg gatgggctgg atcaacacca agaccggcga
ggccacctac 180gtggaagagt tcaagggcag agtgaccttc accacggaca
ccagcaccag caccgcctac 240atggaactgc ggagcctgag aagcgacgac
accgccgtgt actactgcgc cagatgggac 300ttcgcctatt acgtggaagc
catggactac tggggccagg gcaccaccgt gaccgtgtct 360agc
3638615DNAArtificial SequenceHCDR1 CEA CH1A1A 98-99 86gagttcggca
tgaac 158751DNAArtificial SequenceHCDR2 CEA CH1A1A 98-99
87tggatcaaca ccaagaccgg cgaggccacc tacgtggaag agttcaaggg c
518836DNAArtificial SequenceHCDR3 CEA CH1A1A 98-99 88tgggacttcg
cctattacgt ggaagccatg gactac 3689324DNAArtificial SequenceVL CEA
2F1 89gatatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga
cagagtcacc 60atcacttgca aggccagtgc ggctgtgggt acgtatgttg cgtggtatca
gcagaaacca 120gggaaagcac ctaagctcct gatctattcg gcatcctacc
gcaaaagggg agtcccatca 180aggttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagatttcg caacttacta
ctgtcaccaa tattacacct atcctctatt cacgtttggc 300cagggcacca
agctcgagat caag 3249033DNAArtificial SequenceLCDR1 CEA 2F1
90aaggccagtg cggctgtggg tacgtatgtt gcg 339121DNAArtificial
SequenceLCDR2 CEA 2F1 91tcggcatcct accgcaaaag g 219230DNAArtificial
SequenceLCDR3 CEA 2F1 92caccaatatt acacctatcc tctattcacg
3093642DNAArtificial SequenceLC CD3 CH2527 (Crossfab, VL-CH1)
93caggccgtcg tgacccagga acccagcctg acagtgtctc ctggcggcac cgtgaccctg
60acatgtggca gttctacagg cgccgtgacc accagcaact acgccaactg ggtgcaggaa
120aagcccggcc aggccttcag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg
ccctgacact gtctggcgcc 240cagccagaag atgaggccga gtactactgc
gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac
agtgctgagc agcgcttcca ccaaaggccc ttccgtgttt 360cctctggctc
ctagctccaa gtccacctct ggaggcaccg ctgctctcgg atgcctcgtg
420aaggattatt ttcctgagcc tgtgacagtg tcctggaata gcggagcact
gacctctgga 480gtgcatactt tccccgctgt gctgcagtcc tctggactgt
acagcctgag cagcgtggtg 540acagtgccca gcagcagcct gggcacccag
acctacatct gcaacgtgaa ccacaagccc 600agcaacacca aggtggacaa
gaaggtggaa cccaagtctt gt 642942055DNAArtificial SequenceMCSP M4-3
(C1) (VH-CH1) - CD3 CH2527 (Crossfab VH-Ck) - Fc(knob) P329GLALA
94caggtgcaat tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgcaccg tgtccggcgg cagcatcacc agcggctatt attggaactg gattcggcag
120caccccggca agggcctgga atggatcggc tacatcactt tcgacggctc
taacaactac 180aaccccagcc tgaagtccag agtgaccatc agccgggaca
ccagcaagaa ccagttcagc 240ctgaagctgt ccagcgtgac agccgccgac
accgccgtgt actactgcgc cgacttcgac 300tactggggcc agggcaccct
ggtcaccgtg tccagcgcta gcacaaaggg ccccagcgtg 360ttccctctgg
cccctagcag caagagcaca tctggcggaa cagccgccct gggctgcctc
420gtgaaggact actttcccga gcctgtgacc gtgtcctgga actctggcgc
cctgacaagc 480ggcgtgcaca cctttccagc cgtgctgcag agcagcggcc
tgtactctct gagcagcgtg 540gtcaccgtgc ctagcagcag cctgggcacc
cagacctaca tctgcaacgt gaaccacaag 600cccagcaaca ccaaagtgga
caagaaggtg gagcccaaga gctgtgatgg cggaggaggg 660tccggaggcg
gaggatccga ggtgcagctg ctggaatctg gcggcggact ggtgcagcct
720ggcggatctc tgagactgag ctgtgccgcc agcggcttca ccttcagcac
ctacgccatg 780aactgggtgc gccaggcccc tggcaaaggc ctggaatggg
tgtcccggat cagaagcaag 840tacaacaact acgccaccta ctacgccgac
agcgtgaagg gccggttcac catcagccgg 900gacgacagca agaacaccct
gtacctgcag atgaacagcc tgcgggccga ggacaccgcc 960gtgtactatt
gtgtgcggca cggcaacttc ggcaacagct atgtgtcttg gtttgcctac
1020tggggccagg gcaccctcgt gaccgtgtca agcgctagcg tggccgctcc
ctccgtgttt 1080atctttcccc catccgatga acagctgaaa agcggcaccg
cctccgtcgt gtgtctgctg 1140aacaattttt accctaggga agctaaagtg
cagtggaaag tggataacgc actgcagtcc 1200ggcaactccc aggaatctgt
gacagaacag gactccaagg acagcaccta ctccctgtcc 1260tccaccctga
cactgtctaa ggctgattat gagaaacaca aagtctacgc ctgcgaagtc
1320acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga
gtgtgacaag 1380acccacacct gtcccccttg tcctgcccct gaagctgctg
gcggcccttc tgtgttcctg 1440ttccccccaa agcccaagga caccctgatg
atcagccgga cccccgaagt gacctgcgtg 1500gtggtggatg tgtcccacga
ggaccctgaa gtgaagttca attggtacgt ggacggcgtg 1560gaagtgcaca
acgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg
1620gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta
caagtgcaag 1680gtctccaaca aagccctcgg cgcccccatc gagaaaacca
tctccaaagc caaagggcag 1740ccccgagaac cacaggtgta caccctgccc
ccatgccggg atgagctgac caagaaccag 1800gtcagcctgt ggtgcctggt
caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1860agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
1920tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca
ggggaacgtc 1980ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacgcagaa gagcctctcc 2040ctgtctccgg gtaaa 2055951326DNAArtificial
SequenceMCSP M4-3 (C1) (VH-CH1) - Fc(hole) P329GLALA 95caggtgcaat
tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgcaccg
tgtccggcgg cagcatcacc agcggctatt attggaactg gattcggcag
120caccccggca agggcctgga atggatcggc tacatcactt tcgacggctc
taacaactac 180aaccccagcc tgaagtccag agtgaccatc agccgggaca
ccagcaagaa ccagttcagc 240ctgaagctgt ccagcgtgac agccgccgac
accgccgtgt actactgcgc cgacttcgac 300tactggggcc agggcaccct
ggtcaccgtg tccagcgcta gcaccaaggg cccctccgtg 360ttccccctgg
cccccagcag caagagcacc agcggcggca cagccgctct gggctgcctg
420gtcaaggact acttccccga gcccgtgacc gtgtcctgga acagcggagc
cctgacctcc 480ggcgtgcaca ccttccccgc cgtgctgcag agttctggcc
tgtatagcct gagcagcgtg 540gtcaccgtgc cttctagcag cctgggcacc
cagacctaca tctgcaacgt gaaccacaag 600cccagcaaca ccaaggtgga
caagaaggtg gagcccaaga gctgcgacaa aactcacaca 660tgcccaccgt
gcccagcacc tgaagctgca gggggaccgt cagtcttcct cttcccccca
720aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt
ggtggtggac 780gtgagccacg aagaccctga ggtcaagttc aactggtacg
tggacggcgt ggaggtgcat 840aatgccaaga caaagccgcg ggaggagcag
tacaacagca cgtaccgtgt ggtcagcgtc 900ctcaccgtcc tgcaccagga
ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 960aaagccctcg
gcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa
1020ccacaggtgt gcaccctgcc cccatcccgg gatgagctga ccaagaacca
ggtcagcctc 1080tcgtgcgcag tcaaaggctt ctatcccagc gacatcgccg
tggagtggga gagcaatggg 1140cagccggaga acaactacaa gaccacgcct
cccgtgctgg actccgacgg ctccttcttc 1200ctcgtgagca agctcaccgt
ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1260tccgtgatgc
atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1320ggtaaa
132696642DNAArtificial SequenceLC CD3 CH2527 (Crossfab, VL-CH1)
96caggccgtcg tgacccagga acccagcctg acagtgtctc ctggcggcac cgtgaccctg
60acatgtggca gttctacagg cgccgtgacc accagcaact acgccaactg ggtgcaggaa
120aagcccggcc aggccttcag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg
ccctgacact gtctggcgcc 240cagccagaag atgaggccga gtactactgc
gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac
agtgctgagc agcgcttcca ccaaaggccc ttccgtgttt 360cctctggctc
ctagctccaa gtccacctct ggaggcaccg ctgctctcgg atgcctcgtg
420aaggattatt ttcctgagcc tgtgacagtg tcctggaata gcggagcact
gacctctgga 480gtgcatactt tccccgctgt gctgcagtcc tctggactgt
acagcctgag cagcgtggtg 540acagtgccca gcagcagcct gggcacccag
acctacatct gcaacgtgaa ccacaagccc 600agcaacacca aggtggacaa
gaaggtggaa cccaagtctt gt 642972082DNAArtificial SequenceCEA CH1A1A
98/99 - CD3 CH2527 (Crossfab VH-Ck) - Fc(knob) P329GLALA
97caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgccag cgtgaaggtg
60tcctgcaagg ccagcggcta caccttcacc gagttcggca tgaactgggt ccgacaggcc
120cctggacagg gcctggaatg gatgggctgg atcaacacca agaccggcga
ggccacctac 180gtggaagagt tcaagggcag agtgaccttc accaccgaca
ccagcaccag caccgcctac 240atggaactgc ggagcctgag aagcgacgac
accgccgtgt actactgcgc cagatgggac 300ttcgcctact atgtggaagc
catggactac tggggccagg gcaccaccgt gaccgtgtct 360agtgctagca
caaagggccc cagcgtgttc cctctggccc ctagcagcaa gagcacatct
420ggcggaacag ccgccctggg ctgcctggtc aaggactact ttcccgagcc
cgtgacagtg 480tcctggaact ctggcgccct gacaagcggc gtgcacacct
ttccagccgt gctgcagagc 540agcggcctgt actctctgag cagcgtggtc
accgtgccta gctctagcct gggcacccag 600acctacatct gcaacgtgaa
ccacaagccc agcaacacca aggtggacaa gaaggtggaa 660cccaagagct
gcgatggcgg aggcggctcc ggaggcggag gatccgaggt gcagctgctg
720gaatctggcg gcggactggt gcagcctggc ggatctctga gactgagctg
tgccgccagc 780ggcttcacct tcagcaccta cgccatgaac tgggtgcgcc
aggcccctgg caaaggcctg 840gaatgggtgt cccggatcag aagcaagtac
aacaactacg ccacctacta cgccgacagc 900gtgaagggcc ggttcaccat
cagccgggac gacagcaaga acaccctgta cctgcagatg 960aacagcctgc
gggccgagga caccgccgtg tactattgtg tgcggcacgg caacttcggc
1020aacagctatg tgtcttggtt tgcctactgg ggccagggca ccctcgtgac
cgtgtcaagc 1080gctagcgtgg ccgctccctc cgtgtttatc tttcccccat
ccgatgaaca gctgaaaagc 1140ggcaccgcct ccgtcgtgtg tctgctgaac
aatttttacc ctagggaagc taaagtgcag 1200tggaaagtgg ataacgcact
gcagtccggc aactcccagg aatctgtgac agaacaggac 1260tccaaggaca
gcacctactc cctgtcctcc accctgacac tgtctaaggc tgattatgag
1320aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc
cgtcacaaag 1380agcttcaaca ggggagagtg tgacaagacc cacacctgtc
ccccttgtcc tgcccctgaa 1440gctgctggcg gcccttctgt gttcctgttc
cccccaaagc ccaaggacac cctgatgatc 1500agccggaccc ccgaagtgac
ctgcgtggtg gtggatgtgt cccacgagga ccctgaagtg 1560aagttcaatt
ggtacgtgga cggcgtggaa gtgcacaacg ccaagacaaa gccgcgggag
1620gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg 1680ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag
ccctcggcgc ccccatcgag 1740aaaaccatct ccaaagccaa agggcagccc
cgagaaccac aggtgtacac cctgccccca 1800tgccgggatg agctgaccaa
gaaccaggtc agcctgtggt gcctggtcaa aggcttctat 1860cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
1920acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct
caccgtggac 1980aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg
tgatgcatga ggctctgcac 2040aaccactaca cgcagaagag cctctccctg
tctccgggta aa 2082981353DNAArtificial SequenceCEA CH1A1A 98/99
(VH-CH1) - Fc(hole) P329GLALA 98caggtgcagc tggtgcagtc tggcgccgaa
gtgaagaaac ctggagctag tgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc
gagttcggca tgaactgggt ccgacaggct 120ccaggccagg gcctcgaatg
gatgggctgg atcaacacca agaccggcga ggccacctac 180gtggaagagt
tcaagggcag agtgaccttc accacggaca ccagcaccag caccgcctac
240atggaactgc ggagcctgag aagcgacgac accgccgtgt actactgcgc
cagatgggac 300ttcgcctatt acgtggaagc catggactac tggggccagg
gcaccaccgt gaccgtgtct 360agcgctagca ccaagggccc ctccgtgttc
cccctggccc ccagcagcaa gagcaccagc 420ggcggcacag ccgctctggg
ctgcctggtc aaggactact tccccgagcc cgtgaccgtg 480tcctggaaca
gcggagccct gacctccggc gtgcacacct tccccgccgt gctgcagagt
540tctggcctgt atagcctgag cagcgtggtc accgtgcctt ctagcagcct
gggcacccag 600acctacatct gcaacgtgaa ccacaagccc agcaacacca
aggtggacaa gaaggtggag 660cccaagagct gcgacaaaac tcacacatgc
ccaccgtgcc cagcacctga agctgcaggg 720ggaccgtcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780cctgaggtca
catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
840tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 900aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 960aaggagtaca agtgcaaggt ctccaacaaa
gccctcggcg cccccatcga gaaaaccatc 1020tccaaagcca aagggcagcc
ccgagaacca caggtgtgca ccctgccccc atcccgggat 1080gagctgacca
agaaccaggt cagcctctcg tgcgcagtca aaggcttcta tcccagcgac
1140atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 1200gtgctggact ccgacggctc cttcttcctc gtgagcaagc
tcaccgtgga caagagcagg 1260tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 1320acgcagaaga gcctctccct
gtctccgggt aaa 135399645DNAArtificial SequenceLC DP47 GS
99gaaatcgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcttgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggagcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg atccgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccgct gacgttcggc 300caggggacca
aagtggaaat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 645100642DNAArtificial
SequenceLC CD3 CH2527 (Crossfab, VL-CH1) 100caggccgtcg tgacccagga
acccagcctg acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca gttctacagg
cgccgtgacc accagcaact acgccaactg ggtgcaggaa 120aagcccggcc
aggccttcag aggactgatc ggcggcacca acaagagagc ccctggcacc
180cctgccagat tcagcggatc tctgctggga ggaaaggccg ccctgacact
gtctggcgcc 240cagccagaag atgaggccga gtactactgc gccctgtggt
acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac agtgctgagc
agcgcttcca ccaaaggccc ttccgtgttt 360cctctggctc ctagctccaa
gtccacctct ggaggcaccg ctgctctcgg atgcctcgtg 420aaggattatt
ttcctgagcc tgtgacagtg tcctggaata gcggagcact gacctctgga
480gtgcatactt tccccgctgt gctgcagtcc tctggactgt acagcctgag
cagcgtggtg 540acagtgccca gcagcagcct gggcacccag acctacatct
gcaacgtgaa ccacaagccc 600agcaacacca aggtggacaa gaaggtggaa
cccaagtctt gt 6421012064DNAArtificial SequenceDP47 GS (VH-CH1) -
CD3 CH2527 (Crossfab VH-Ck) - Fc(knob) P329GLALA 101gaggtgcaat
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcagatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaaggcagc 300ggatttgact actggggcca
aggaaccctg gtcaccgtct cgagtgctag cacaaagggc 360cccagcgtgt
tccctctggc ccctagcagc aagagcacat ctggcggaac agccgccctg
420ggctgcctcg tgaaggacta ctttcccgag cctgtgaccg tgtcctggaa
ctctggcgcc 480ctgacaagcg gcgtgcacac ctttccagcc gtgctgcaga
gcagcggcct gtactctctg 540agcagcgtgg tcaccgtgcc tagcagcagc
ctgggcaccc agacctacat ctgcaacgtg 600aaccacaagc ccagcaacac
caaagtggac aagaaggtgg agcccaagag ctgtgatggc 660ggaggagggt
ccggaggcgg aggatccgag gtgcagctgc tggaatctgg cggcggactg
720gtgcagcctg gcggatctct gagactgagc tgtgccgcca gcggcttcac
cttcagcacc 780tacgccatga actgggtgcg ccaggcccct ggcaaaggcc
tggaatgggt gtcccggatc 840agaagcaagt acaacaacta cgccacctac
tacgccgaca gcgtgaaggg ccggttcacc 900atcagccggg acgacagcaa
gaacaccctg tacctgcaga tgaacagcct gcgggccgag 960gacaccgccg
tgtactattg tgtgcggcac ggcaacttcg gcaacagcta tgtgtcttgg
1020tttgcctact ggggccaggg caccctcgtg accgtgtcaa gcgctagcgt
ggccgctccc 1080tccgtgttta tctttccccc atccgatgaa cagctgaaaa
gcggcaccgc ctccgtcgtg 1140tgtctgctga acaattttta ccctagggaa
gctaaagtgc agtggaaagt ggataacgca 1200ctgcagtccg gcaactccca
ggaatctgtg acagaacagg actccaagga cagcacctac 1260tccctgtcct
ccaccctgac actgtctaag gctgattatg agaaacacaa agtctacgcc
1320tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa
caggggagag 1380tgtgacaaga cccacacctg tcccccttgt cctgcccctg
aagctgctgg cggcccttct 1440gtgttcctgt tccccccaaa gcccaaggac
accctgatga tcagccggac ccccgaagtg 1500acctgcgtgg tggtggatgt
gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 1560gacggcgtgg
aagtgcacaa cgccaagaca aagccgcggg aggagcagta caacagcacg
1620taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 1680aagtgcaagg tctccaacaa agccctcggc gcccccatcg
agaaaaccat ctccaaagcc 1740aaagggcagc cccgagaacc acaggtgtac
accctgcccc catgccggga tgagctgacc 1800aagaaccagg tcagcctgtg
gtgcctggtc aaaggcttct atcccagcga catcgccgtg 1860gagtgggaga
gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac
1920tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag
gtggcagcag 1980gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 2040agcctctccc tgtctccggg taaa
20641021335DNAArtificial SequenceDP47 GS (VH-CH1) - Fc(hole)
P329GLALA 102gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcagatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaaggcagc 300ggatttgact
actggggcca aggaaccctg gtcaccgtct cgagtgctag caccaagggc
360ccctccgtgt tccccctggc ccccagcagc aagagcacca gcggcggcac
agccgctctg 420ggctgcctgg tcaaggacta cttccccgag cccgtgaccg
tgtcctggaa cagcggagcc 480ctgacctccg gcgtgcacac cttccccgcc
gtgctgcaga gttctggcct gtatagcctg 540agcagcgtgg tcaccgtgcc
ttctagcagc ctgggcaccc agacctacat ctgcaacgtg 600aaccacaagc
ccagcaacac caaggtggac aagaaggtgg agcccaagag ctgcgacaaa
660actcacacat gcccaccgtg cccagcacct gaagctgcag ggggaccgtc
agtcttcctc 720ttccccccaa aacccaagga caccctcatg atctcccgga
cccctgaggt cacatgcgtg 780gtggtggacg tgagccacga agaccctgag
gtcaagttca actggtacgt ggacggcgtg 840gaggtgcata atgccaagac
aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900gtcagcgtcc
tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag
960gtctccaaca aagccctcgg cgcccccatc gagaaaacca tctccaaagc
caaagggcag 1020ccccgagaac cacaggtgtg caccctgccc ccatcccggg
atgagctgac caagaaccag 1080gtcagcctct cgtgcgcagt caaaggcttc
tatcccagcg acatcgccgt ggagtgggag 1140agcaatgggc agccggagaa
caactacaag accacgcctc ccgtgctgga ctccgacggc 1200tccttcttcc
tcgtgagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc
1260ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa
gagcctctcc 1320ctgtctccgg gtaaa 1335103207PRTHomo sapiens 103Met
Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1 5 10
15Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr
20 25 30Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu
Thr 35 40 45Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn
Asp Lys 50 55 60Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser
Asp Glu Asp65 70 75 80His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu
Gln Ser Gly Tyr Tyr 85 90 95Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu
Asp Ala Asn Phe Tyr Leu 100 105 110Tyr Leu Arg Ala Arg Val Cys Glu
Asn Cys Met Glu Met Asp Val Met 115 120 125Ser Val Ala Thr Ile Val
Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140Leu Leu Leu Val
Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys145 150 155 160Pro
Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170
175Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg
180 185 190Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg
Ile 195 200 205104198PRTMacaca fascicularis 104Met Gln Ser Gly Thr
Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1 5 10 15Ile Gly Val Trp
Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr 20 25 30Gln Thr Pro
Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45Cys Ser
Gln His Leu Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys 50 55 60Asn
Lys Glu Asp Ser Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu65 70 75
80Met Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro
85 90 95Glu Asp Ala Ser His His Leu Tyr Leu Lys Ala Arg Val Cys Glu
Asn 100 105 110Cys Met Glu Met Asp Val Met Ala Val Ala Thr Ile Val
Ile Val Asp 115 120 125Ile Cys Ile Thr Leu Gly Leu Leu Leu Leu Val
Tyr Tyr Trp Ser Lys 130 135 140Asn Arg Lys Ala Lys Ala Lys Pro Val
Thr Arg Gly Ala Gly Ala Gly145 150 155 160Gly Arg Gln Arg Gly Gln
Asn Lys Glu Arg Pro Pro Pro Val Pro Asn 165 170 175Pro Asp Tyr Glu
Pro Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly 180 185 190Leu Asn
Gln Arg Arg Ile 19510515PRTArtificial Sequencelinker 105Glu Pro Lys
Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
1510615PRTArtificial Sequencelinker 106Glu Pro Lys Ser Cys Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15107227PRTHomo sapiens
107Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22510819PRTArtificial
Sequenceleader peptide 108Met Asp Trp Thr Trp Arg Ile Leu Phe Leu
Val Ala Ala Ala Thr Gly1 5 10 15Ala His Ser10957DNAArtificial
Sequenceleader peptide 109atggactgga cctggagaat cctcttcttg
gtggcagcag ccacaggagc ccactcc 5711057DNAArtificial Sequenceleader
peptide 110atggactgga cctggaggat cctcttcttg gtggcagcag ccacaggagc
ccactcc 5711122PRTArtificial Sequenceleader peptide 111Met Asp Met
Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro
Gly Ala Arg Cys 2011266DNAArtificial Sequenceleader peptide
112atggacatga gggtccccgc tcagctcctg ggcctcctgc tgctctggtt
cccaggtgcc 60aggtgt 6611319PRTArtificial Sequenceleader peptide
113Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1
5 10 15Val His Ser11457DNAArtificial Sequenceleader peptide
114atgggatgga gctgtatcat cctcttcttg gtagcaacag ctaccggtgt gcattcc
5711557DNAArtificial Sequenceleader peptide 115atgggctggt
cctgcatcat cctgtttctg gtggctaccg ccactggagt gcattcc
5711657DNAArtificial Sequenceleader peptide 116atgggctggt
cctgcatcat cctgtttctg gtcgccacag ccaccggcgt gcactct
57117643PRTMacaca fascicularis 117Leu Ser Leu Glu Gly Ser Arg Thr
Leu Thr Val Cys Pro Gly Ser Val1 5 10 15Gln Pro Leu Ser Ser Gln Thr
Leu Arg Ala Ser Ser Ser Ala Gly Thr 20 25 30Asp Pro Gln Leu Leu Leu
Tyr Arg Val Val Arg Gly Pro Gln Leu Gly 35 40 45Arg Leu Phe His Ala
Gln Gln Asp Ser Thr Gly Glu Ala Leu Val Asn 50 55 60Phe Thr Gln Ala
Glu Val Tyr Ala Gly Asn Ile Leu Tyr Glu His Glu65 70 75 80Met Pro
Thr Glu Pro Phe Trp Glu Ala His Asp Thr Leu Glu Leu Gln 85 90 95Leu
Ser Ser Pro Pro Ala Arg Asp Val Ala Ala Thr Leu Ala Val Ala 100 105
110Val Ser Phe Glu Ala Ala Cys Pro Gln Arg Pro Ser His Leu Trp Lys
115 120 125Asn Lys Gly Leu Trp Val Pro Glu Gly Gln Arg Ala Lys Ile
Thr Met 130 135 140Ala Ala Leu Asp Ala Ser Asn Leu Leu Ala Ser Val
Pro Ser Pro Gln145 150 155 160Arg Leu Glu His Asp Val Leu Phe Gln
Val Thr Gln Phe Pro Ser Arg 165 170 175Gly Gln Leu Leu Val Ser Glu
Glu Pro Leu His Ala Gly Gln Pro His 180 185 190Phe Leu Gln Ser Gln
Leu Ala Ala Gly Gln Leu Val Tyr Ala His Gly 195 200 205Gly Gly Gly
Thr Gln Gln Asp Gly Phe His Phe Arg Ala His Leu Gln 210 215 220Gly
Pro Ala Gly Ala Thr Val Ala Gly Pro Gln Thr Ser Glu Ala Phe225 230
235 240Ala Ile Thr Val Arg Asp Val Asn Glu Arg Pro Pro Gln Pro Gln
Ala 245 250 255Ser Val Pro Leu Arg Ile Thr Arg Gly Ser Arg Ala Pro
Ile Ser Arg 260 265 270Ala Gln Leu Ser Val Val Asp Pro Asp Ser Ala
Pro Gly Glu Ile Glu 275 280 285Tyr Glu Val Gln Arg Ala Pro His Asn
Gly Phe Leu Ser Leu Val Gly 290 295 300Gly Gly Pro Gly Pro Val Thr
His Phe Thr Gln Ala Asp Val Asp Ser305 310 315 320Gly Arg Leu Ala
Phe Val Ala Asn Gly Ser Ser Val Ala Gly Val Phe 325 330 335Gln Leu
Ser Met Ser Asp Gly Ala Ser Pro Pro Leu Pro Met Ser Leu 340 345
350Ala Val Asp Ile Leu Pro Ser Ala Ile Glu Val Gln Leu Gln Ala Pro
355 360 365Leu Glu Val Pro Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln
Gln Gln 370 375 380Leu Arg Val Val Ser Asp Arg Glu Glu Pro Glu Ala
Ala Tyr Arg Leu385 390 395 400Ile Gln Gly Pro Lys Tyr Gly His Leu
Leu Val Gly Gly Arg Pro Ala 405 410 415Ser Ala Phe Ser Gln Leu Gln
Ile Asp Gln Gly Glu Val Val Phe Ala 420 425 430Phe Thr Asn Phe Ser
Ser Ser His Asp His Phe Arg Val Leu Ala Leu 435 440 445Ala Arg Gly
Val Asn Ala Ser Ala Val Val Asn Ile Thr Val Arg Ala 450 455 460Leu
Leu His Val Trp Ala Gly Gly Pro Trp Pro Gln Gly Ala Thr Leu465 470
475 480Arg Leu Asp Pro Thr Ile Leu Asp Ala Gly Glu Leu Ala Asn Arg
Thr 485 490 495Gly Ser Val Pro His Phe Arg Leu Leu Glu Gly Pro Arg
His Gly Arg 500 505 510Val Val Arg Val Pro Arg Ala Arg Thr Glu Pro
Gly Gly Ser Gln Leu 515 520 525Val Glu Gln Phe Thr Gln Gln Asp Leu
Glu Asp Gly Arg Leu Gly Leu 530 535 540Glu Val Gly Arg Pro Glu Gly
Arg Ala Pro Ser Pro Thr Gly Asp Ser545 550 555 560Leu Thr Leu Glu
Leu Trp Ala Gln Gly Val Pro Pro Ala Val Ala Ser 565 570 575Leu Asp
Phe Ala Thr Glu Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val 580 585
590Ala Leu Leu Ser Val Pro Glu Ala Thr Arg Met Glu Ala Gly Lys Pro
595 600 605Glu Ser Ser Thr Pro Thr Gly Glu Pro Gly Pro Met Ala Ser
Ser Pro 610 615 620Val Pro Ala Val Ala Lys Gly Gly Phe Leu Gly Phe
Leu Glu Ala Asn625 630 635 640Met Phe Ser118643PRTHomo sapiens
118Leu Ser Leu Lys Gly Ser Gln Thr Leu Thr Val Cys Pro Gly Ser Val1
5 10 15Gln Pro Leu Ser Ser Gln Thr Leu Arg Ala Ser Ser Ser Ala Gly
Thr 20 25 30Asp Pro Gln Leu Leu Leu Tyr Arg Val Val Arg Gly Pro Gln
Leu Gly 35 40 45Arg Leu Phe His Ala Gln Gln Asp Ser Thr Gly Glu Ala
Leu Val Asn 50 55 60Phe Thr Gln Ala Glu Val Tyr Ala Gly Asn Ile Leu
Tyr Glu His Glu65 70 75 80Met Pro Pro Glu Pro Phe Trp Glu Ala His
Asp Thr Leu Glu Leu Gln 85 90 95Leu Ser Ser Pro Pro Ala Arg Asp Val
Ala Ala Thr Leu Ala Val Ala 100 105 110Val Ser Phe Glu Ala Ala Cys
Pro Gln His Pro Ser His Leu Trp Lys 115 120 125Asn Lys Gly Leu Trp
Val Pro Glu Gly Gln Arg Ala Arg Ile Thr Val 130 135 140Ala Ala Leu
Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gln145 150
155 160Arg Ser Glu His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Ser
Arg 165 170 175Gly Gln Leu Leu Val Ser Glu Glu Pro Leu His Ala Gly
Gln Pro His 180 185 190Phe Leu Gln Ser Gln Leu Ala Ala Gly Gln Leu
Val Tyr Ala His Gly 195 200 205Gly Gly Gly Thr Gln Gln Asp Gly Phe
His Phe Arg Ala His Leu Gln 210 215 220Gly Pro Ala Gly Ala Ser Val
Ala Gly Pro Gln Thr Ser Glu Ala Phe225 230 235 240Ala Ile Thr Val
Arg Asp Val Asn Glu Arg Pro Pro Gln Pro Gln Ala 245 250 255Ser Val
Pro Leu Arg Leu Thr Arg Gly Ser Arg Ala Pro Ile Ser Arg 260 265
270Ala Gln Leu Ser Val Val Asp Pro Asp Ser Ala Pro Gly Glu Ile Glu
275 280 285Tyr Glu Val Gln Arg Ala Pro His Asn Gly Phe Leu Ser Leu
Val Gly 290 295 300Gly Gly Leu Gly Pro Val Thr Arg Phe Thr Gln Ala
Asp Val Asp Ser305 310 315 320Gly Arg Leu Ala Phe Val Ala Asn Gly
Ser Ser Val Ala Gly Ile Phe 325 330 335Gln Leu Ser Met Ser Asp Gly
Ala Ser Pro Pro Leu Pro Met Ser Leu 340 345 350Ala Val Asp Ile Leu
Pro Ser Ala Ile Glu Val Gln Leu Arg Ala Pro 355 360 365Leu Glu Val
Pro Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln Gln Gln 370 375 380Leu
Arg Val Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu385 390
395 400Ile Gln Gly Pro Gln Tyr Gly His Leu Leu Val Gly Gly Arg Pro
Thr 405 410 415Ser Ala Phe Ser Gln Phe Gln Ile Asp Gln Gly Glu Val
Val Phe Ala 420 425 430Phe Thr Asn Phe Ser Ser Ser His Asp His Phe
Arg Val Leu Ala Leu 435 440 445Ala Arg Gly Val Asn Ala Ser Ala Val
Val Asn Val Thr Val Arg Ala 450 455 460Leu Leu His Val Trp Ala Gly
Gly Pro Trp Pro Gln Gly Ala Thr Leu465 470 475 480Arg Leu Asp Pro
Thr Val Leu Asp Ala Gly Glu Leu Ala Asn Arg Thr 485 490 495Gly Ser
Val Pro Arg Phe Arg Leu Leu Glu Gly Pro Arg His Gly Arg 500 505
510Val Val Arg Val Pro Arg Ala Arg Thr Glu Pro Gly Gly Ser Gln Leu
515 520 525Val Glu Gln Phe Thr Gln Gln Asp Leu Glu Asp Gly Arg Leu
Gly Leu 530 535 540Glu Val Gly Arg Pro Glu Gly Arg Ala Pro Gly Pro
Ala Gly Asp Ser545 550 555 560Leu Thr Leu Glu Leu Trp Ala Gln Gly
Val Pro Pro Ala Val Ala Ser 565 570 575Leu Asp Phe Ala Thr Glu Pro
Tyr Asn Ala Ala Arg Pro Tyr Ser Val 580 585 590Ala Leu Leu Ser Val
Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro 595 600 605Glu Ser Ser
Thr Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro 610 615 620Glu
Pro Ala Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn625 630
635 640Met Phe Ser119428PRTArtificial SequenceNABA-avi-his 119Gln
Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly Lys Glu1 5 10
15Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly Tyr Ser
20 25 30Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val Gly
Tyr 35 40 45Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser
Gly Arg 50 55 60Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn
Val Thr Gln65 70 75 80Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile
Lys Ser Asp Leu Val 85 90 95Asn Glu Glu Ala Thr Gly Gln Phe His Val
Tyr Pro Glu Leu Pro Lys 100 105 110Pro Ser Ile Ser Ser Asn Asn Ser
Asn Pro Val Glu Asp Lys Asp Ala 115 120 125Met Ala Phe Thr Cys Glu
Pro Glu Thr Gln Asp Thr Thr Tyr Leu Trp 130 135 140Trp Ile Asn Asn
Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser145 150 155 160Asn
Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Thr 165 170
175Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Arg Ser
180 185 190Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp Thr Pro
Thr Ile 195 200 205Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn
Leu Asn Leu Ser 210 215 220Cys His Ser Ala Ser Asn Pro Ser Pro Gln
Tyr Ser Trp Arg Ile Asn225 230 235 240Gly Ile Pro Gln Gln His Thr
Gln Val Leu Phe Ile Ala Lys Ile Thr 245 250 255Pro Asn Asn Asn Gly
Thr Tyr Ala Cys Phe Val Ser Asn Leu Ala Thr 260 265 270Gly Arg Asn
Asn Ser Ile Val Lys Ser Ile Thr Val Ser Ala Leu Ser 275 280 285Pro
Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Thr 290 295
300Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp Thr
Gly305 310 315 320Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu
Pro Ser Ser Glu 325 330 335Arg Met Lys Leu Ser Gln Gly Asn Ile Thr
Leu Ser Ile Asn Pro Val 340 345 350Lys Arg Glu Asp Ala Gly Thr Tyr
Trp Cys Glu Val Phe Asn Pro Ile 355 360 365Ser Lys Asn Gln Ser Asp
Pro Ile Met Leu Asn Val Asn Tyr Asn Ala 370 375 380Leu Pro Gln Glu
Asn Leu Ile Asn Val Asp Leu Glu Val Leu Phe Gln385 390 395 400Gly
Pro Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu 405 410
415Trp His Glu Ala Arg Ala His His His His His His 420
425120360PRTArtificial SequenceCD3e stalk-Fc(knob)-Avi 120Gln Asp
Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys1 5 10 15Val
Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25
30Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp
35 40 45Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu
Lys 50 55 60Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr
Pro Arg65 70 75 80Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr
Leu Arg Ala Arg 85 90 95Val Ser Glu Asn Cys Val Asp Glu Gln Leu Tyr
Phe Gln Gly Gly Ser 100 105 110Pro Lys Ser Ala Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 115 120 125Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 130 135 140Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val145 150 155 160Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 165 170
175Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
180 185 190Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 195 200 205Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 210 215 220Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg225 230 235 240Glu Pro Gln Val Tyr Thr Leu
Pro Pro Cys Arg Asp Glu Leu Thr Lys 245 250 255Asn Gln Val Ser Leu
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 260 265 270Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 275 280 285Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 290 295
300Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser305 310 315 320Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser 325 330 335Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly
Leu Asn Asp Ile Phe Glu 340 345 350Ala Gln Lys Ile Glu Trp His Glu
355 360121325PRTArtificial SequenceCD3d stalk-Fc(hole) 121Phe Lys
Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys1 5 10 15Asn
Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser 20 25
30Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly
35 40 45Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser
Thr 50 55 60Val Gln Val His Tyr Arg Met Cys Arg Ser Glu Gln Leu Tyr
Phe Gln65 70 75 80Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 85 90 95Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 100 105 110Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 115 120 125His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 130 135 140Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr145 150 155 160Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 165 170
175Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
180 185 190Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 195 200 205Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 210 215 220Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val225 230 235 240Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 245 250 255Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 260 265 270Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 275 280 285Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 290 295
300Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln
Lys305 310 315 320Ile Glu Trp His Glu 325122658PRTMus musculus
122Leu Ser Leu Glu Gly Thr Arg Lys Leu Thr Val Cys Pro Glu Ser Val1
5 10 15Gln Pro Leu Ser Ser Gln Ser Leu Ser Ala Ser Ser Ser Thr Gly
Ala 20 25 30Asp Pro Arg His Leu Leu Tyr Arg Val Val Arg Gly Pro Gln
Leu Gly 35 40 45Arg Leu Leu His Ala Gln Gln Gly Ser Ala Glu Glu Val
Leu Val Asn 50 55 60Phe Thr Gln Ala Glu Val Asn Ala Gly Asn Ile Leu
Tyr Glu His Glu65 70 75 80Met Ser Ser Glu Pro Phe Trp Glu Ala His
Asp Thr Ile Gly Leu Leu 85 90 95Leu Ser Ser Pro Pro Ala Arg Asp Leu
Ala Ala Thr Leu Ala Val Met 100 105 110Val Ser Phe Asp Ala Ala Cys
Pro Gln Arg Pro Ser Arg Leu Trp Lys 115 120 125Asn Lys Gly Leu Trp
Val Pro Glu Gly Gln Arg Ala Lys Ile Thr Val 130 135 140Ala Ala Leu
Asp Ala Ala Asn Leu Leu Ala Ser Val Pro Ala Ser Gln145 150 155
160Arg Ser Arg His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Thr Arg
165 170 175Gly Gln Leu Leu Val Ser Glu Glu Pro Leu His Ala Arg Arg
Pro Tyr 180 185 190Phe Leu Gln Ser Glu Leu Ala Ala Gly Gln Leu Val
Tyr Ala His Gly 195 200 205Gly Gly Gly Thr Gln Gln Asp Gly Phe Arg
Phe Arg Ala His Leu Gln 210 215 220Gly Pro Thr Gly Thr Ser Val Ala
Gly Pro Gln Thr Ser Glu Ala Phe225 230 235 240Val Ile Thr Val Arg
Asp Val Asn Glu Arg Pro Pro Gln Pro Gln Ala 245 250 255Ser Ile Pro
Leu Arg Val Thr Arg Gly Ser Arg Ala Pro Val Ser Arg 260 265 270Ala
Gln Leu Ser Val Val Asp Pro Asp Ser Ala Pro Gly Glu Ile Glu 275 280
285Tyr Glu Val Gln Arg Ala Pro His Asn Gly Phe Leu Ser Leu Ala Gly
290 295 300Asp Asn Thr Gly Pro Val Thr His Phe Thr Gln Ala Asp Val
Asp Ala305 310 315 320Gly Arg Leu Ala Phe Val Ala Asn Gly Ser Ser
Val Ala Gly Val Phe 325 330 335Gln Leu Ser Met Ser Asp Gly Ala Ser
Pro Pro Ile Pro Met Ser Leu 340 345 350Ala Val Asp Val Leu Pro Ser
Thr Ile Glu Val Gln Leu Arg Ala Pro 355 360 365Leu Glu Val Pro Gln
Ala Leu Gly Arg Thr Ser Leu Ser Arg Gln Gln 370 375 380Leu Gln Val
Ile Ser Asp Arg Glu Glu Pro Asp Val Ala Tyr Arg Leu385 390 395
400Thr Gln Gly Pro Leu Tyr Gly Gln Leu Leu Val Gly Gly Gln Pro Ala
405 410 415Ser Ala Phe Ser Gln Leu Gln Val Asp Gln Gly Asp Val Val
Phe Val 420 425 430Phe Thr Asn Phe Ser Ser Ser Gln Asp His Phe Lys
Val Val Ala Leu 435 440 445Ala Arg Gly Val Asn Ala Ser Ala Thr Val
Asn Val Thr Val Gln Ala 450 455 460Leu Leu His Val Trp Ala Gly Gly
Pro Trp Pro Gln Gly Thr Thr Leu465 470 475 480Arg Leu Asp Pro Thr
Val Leu Asp Ala Ser Glu Leu Ala Asn Arg Thr 485 490 495Gly Ser Met
Pro His Phe Arg Leu Leu Ala Gly Pro Arg Tyr Gly Arg 500 505 510Val
Val Arg Val Ser Gln Gly Arg Thr Glu Ser Arg Ser Asn Gln Leu 515 520
525Val Glu His Phe Thr Gln Arg Asp Leu Glu Glu Gly Gln Leu Gly Leu
530 535 540Glu Val Gly Lys Pro Glu Gly Arg Ser Thr Gly Pro Ala Gly
Asp Arg545 550 555 560Leu Thr Leu Glu Leu Trp Ala Lys Gly Val Pro
Pro Ala Val Ala Leu 565 570 575Leu Asp Phe Ala Thr Glu Pro Tyr His
Ala Ala Lys Ser Tyr Ser Val 580 585 590Ala Leu Leu Ser Val Pro Glu
Ala Val Arg Thr Glu Thr Glu Lys Pro 595 600 605Gly Arg Ser Val Pro
Thr Gly Gln Pro Gly Gln Ala Ala Ser Ser Pro 610 615 620Val Pro Thr
Ala Ala Lys Gly Gly Val Asp Gly Leu Asn Asp Ile Phe625 630 635
640Glu Ala Gln Lys Ile Glu Trp His Glu Ala Arg Ala His His His His
645 650 655His His
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