U.S. patent application number 16/937536 was filed with the patent office on 2020-11-05 for bispecific t cell activating antigen binding molecules.
The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Marina BACAC, Tanja FAUTI, Sabine IMHOF-JUNG, Christian KLEIN, Stefan KLOSTERMANN, Ekkehard MOESSNER, Michael MOLHOJ, Christiane NEUMANN, Joerg Thomas REGULA, Wolfgang SCHAEFER, Pablo UMANA.
Application Number | 20200347147 16/937536 |
Document ID | / |
Family ID | 1000004961497 |
Filed Date | 2020-11-05 |
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United States Patent
Application |
20200347147 |
Kind Code |
A1 |
BACAC; Marina ; et
al. |
November 5, 2020 |
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; (Zurich,
CH) ; FAUTI; Tanja; (Zurich, CH) ; IMHOF-JUNG;
Sabine; (Planegg, DE) ; KLEIN; Christian;
(Bonstetten, CH) ; KLOSTERMANN; Stefan; (Neuried,
DE) ; MOESSNER; Ekkehard; (Kreuzlingen, CH) ;
MOLHOJ; Michael; (Munich, DE) ; NEUMANN;
Christiane; (Niederweningen, CH) ; REGULA; Joerg
Thomas; (Munich, DE) ; SCHAEFER; Wolfgang;
(Mannheim, DE) ; UMANA; Pablo; (Wollerau,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Family ID: |
1000004961497 |
Appl. No.: |
16/937536 |
Filed: |
July 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15281484 |
Sep 30, 2016 |
10766967 |
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16937536 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 16/2809 20130101; C07K 2317/52 20130101; C07K 16/468 20130101;
C07K 2317/94 20130101; C07K 2317/66 20130101; C07K 2317/565
20130101; C07K 2317/522 20130101; C07K 2317/56 20130101; C07K
2317/35 20130101; C07K 2317/92 20130101; C07K 2317/73 20130101;
C07K 2319/00 20130101; C07K 16/3007 20130101; C07K 2317/622
20130101; C07K 2317/31 20130101; A61K 2039/505 20130101; C07K
2317/55 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28; C07K 16/46 20060101
C07K016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2015 |
EP |
15188035.8 |
Claims
1. A T cell activating bispecific antigen binding molecule
comprising (a) a first antigen binding moiety which specifically
binds to a first antigen; (b) a second antigen binding moiety which
specifically binds to a second antigen; wherein the first antigen
is an activating T cell antigen and the second antigen is CEA, or
the first antigen is CEA and the second antigen is an activating T
cell antigen; and wherein the antigen binding moiety which
specifically binds to CEA comprises a heavy chain variable region,
particularly a humanized heavy chain variable region, comprising
the heavy chain complementarity determining region (HCDR) 1 of SEQ
ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO:
16, and a light chain variable region, particularly a humanized
light chain variable region, comprising the light chain
complementarity determining region (LCDR) 1 of SEQ ID NO: 17, the
LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.
2. The T cell activating bispecific antigen binding molecule
according to claim 1, wherein the antigen binding moiety which
specifically binds to CEA 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: 22 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: 23.
3. The T cell activating bispecific antigen binding molecule
according to claim 1 or 2, wherein the first and/or the second
antigen binding moiety is a Fab molecule.
4. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-3, wherein the second antigen
binding moiety is a Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH or the
constant domains CL and CH1 of the Fab light chain and the Fab
heavy chain are replaced by each other.
5. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-4, wherein the first antigen is
CEA and the second antigen is an activating T cell antigen.
6. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-5, wherein the activating T cell
antigen is CD3, particularly CD3 epsilon.
7. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-6, wherein the antigen binding
moiety which specifically binds to the activating T cell antigen
comprises 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.
8. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-7, wherein the antigen binding
moiety which specifically binds to the activating T cell antigen
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.
9. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-8, wherein the first antigen
binding moiety under (a) is a first Fab molecule which specifically
binds to a first antigen, the second antigen binding moiety under
(b) is a second Fab molecule which specifically binds to a second
antigen wherein the variable domains VL and VH of the Fab light
chain and the Fab heavy chain are replaced by each other; and i) in
the constant domain CL of the first Fab molecule under a) the amino
acid at position 124 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat), and
wherein in the constant domain CH1 of the first Fab molecule under
a) the amino acid at position 147 or the amino acid at position 213
is substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index); or ii) in the constant
domain CL of the second Fab molecule under b) the amino acid at
position 124 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat), and wherein in
the constant domain CH1 of the second Fab molecule under b) the
amino acid at position 147 or the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
10. The T cell activating bispecific antigen binding molecule
according to claim 9, wherein in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and wherein in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 or the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
11. The T cell activating bispecific antigen binding molecule
according to claim 9 or 10, wherein in the constant domain CL of
the first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and wherein in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 is substituted independently by glutamic acid (E), or
aspartic acid (D) (numbering according to Kabat EU index).
12. The T cell activating bispecific antigen binding molecule
according to any one of claims 9-11, wherein in the constant domain
CL of the first Fab molecule under a) the amino acid at position
124 is substituted independently by lysine (K), arginine (R) or
histidine (H) (numbering according to Kabat) and the amino acid at
position 123 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat), and wherein in
the constant domain CH1 of the first Fab molecule under a) the
amino acid at position 147 is substituted independently by glutamic
acid (E), or aspartic acid (D) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
13. The T cell activating bispecific antigen binding molecule
according to any one of claims 9-12, wherein in the constant domain
CL of the first Fab molecule under a) the amino acid at position
124 is substituted by lysine (K) (numbering according to Kabat) and
the amino acid at position 123 is substituted by arginine (R)
(numbering according to Kabat), and wherein in the constant domain
CH1 of the first Fab molecule under a) the amino acid at position
147 is substituted by glutamic acid (E) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
by glutamic acid (E) (numbering according to Kabat EU index).
14. The T cell activating bispecific antigen binding molecule
according to any one of claims 9-12, wherein in the constant domain
CL of the first Fab molecule under a) the amino acid at position
124 is substituted by lysine (K) (numbering according to Kabat) and
the amino acid at position 123 is substituted by lysine (K)
(numbering according to Kabat), and wherein in the constant domain
CH1 of the first Fab molecule under a) the amino acid at position
147 is substituted by glutamic acid (E) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
by glutamic acid (E) (numbering according to Kabat EU index).
15. The T cell activating bispecific antigen binding molecule
according to claim 9, wherein in the constant domain CL of the
second Fab molecule under b) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and wherein in the constant
domain CH1 of the second Fab molecule under b) the amino acid at
position 147 or the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
16. The T cell activating bispecific antigen binding molecule
according to claim 9 or 15, wherein in the constant domain CL of
the second Fab molecule under b) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and wherein in the constant
domain CH1 of the second Fab molecule under b) the amino acid at
position 147 is substituted independently by glutamic acid (E), or
aspartic acid (D) (numbering according to Kabat EU index).
17. The T cell activating bispecific antigen binding molecule
according to any one of claim 9, and 16, wherein in the constant
domain CL of the second Fab molecule under b) the amino acid at
position 124 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) and the amino
acid at position 123 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat), and
wherein in the constant domain CH1 of the second Fab molecule under
b) the amino acid at position 147 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
18. The T cell activating bispecific antigen binding molecule
according to any one of claims 9 and 15-17, wherein in the constant
domain CL of the second Fab molecule under b) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by
arginine (R) (numbering according to Kabat), and wherein in the
constant domain CH1 of the second Fab molecule under b) the amino
acid at position 147 is substituted by glutamic acid (E) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index).
19. The T cell activating bispecific antigen binding molecule
according to any one of claims 9 and 15-17, wherein in the constant
domain CL of the second Fab molecule under b) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) (numbering according to Kabat), and wherein in the constant
domain CH1 of the second Fab molecule under b) the amino acid at
position 147 is substituted by glutamic acid (E) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index).
20. The T cell activating bispecific antigen binding molecule
according to any one of claims 1-19, further comprising c) a third
antigen binding moiety which specifically binds to the first
antigen.
21. The T cell activating bispecific antigen binding molecule
according to claim 20, wherein the third antigen binding moiety is
a Fab molecule.
22. The T cell activating bispecific antigen binding molecule
according to claim 20 or 21, wherein the third antigen binding
moiety is identical to the first antigen binding moiety.
23. The T cell activating bispecific antigen binding molecule
according to any one of claims 20-22, wherein the first and the
third antigen binding moiety specifically bind to a target cell
antigen, and the second antigen binding moiety specifically binds
to an activating T cell antigen, particularly CD3, more
particularly CD3 epsilon.
24. The T cell activating bispecific antigen binding molecule
according to any one of claims 1 to 23, additionally comprising d)
an Fc domain composed of a first and a second subunit capable of
stable association.
25. The T cell activating bispecific antigen binding molecule
according to any one of claims 1 to 24, wherein the first and the
second antigen binding moiety are fused to each other, optionally
via a peptide linker.
26. The T cell activating bispecific antigen binding molecule
according to any one of claims 1 to 25, wherein the first and the
second antigen binding moieties are Fab molecules 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.
27. The T cell activating bispecific antigen binding molecule of
any one of claims 1 to 25, wherein the first and the second antigen
binding moieties are Fab molecules 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.
28. The T cell activating bispecific antigen binding molecule of
claim 26 or 27, wherein the first and the second antigen binding
moieties are Fab molecules and 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.
29. The T cell activating bispecific antigen binding molecule
according to claim 24, wherein the first and the second antigen
binding moieties are Fab molecules 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.
30. The T cell activating bispecific antigen binding molecule
according to claim 24, wherein the first and the second antigen
binding moieties are Fab molecules 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.
31. The T cell activating bispecific antigen binding molecule
according to claim 24, wherein the first and the second antigen
binding moieties are Fab molecules and 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.
32. The T cell activating bispecific antigen binding molecule
according to any one of claims 24, 29 or 30, wherein the third
antigen binding moiety is a Fab molecule and 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.
33. The T cell activating bispecific antigen binding molecule of
claim 24, wherein the first, second and third antigen binding
moieties are Fab molecules and 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.
34. The T cell activating bispecific antigen binding molecule
according to claim 24, wherein the first, second and third antigen
binding moieties are Fab molecules and 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.
35. The T cell activating bispecific antigen binding molecule
according to claim 34, wherein the first and the third antigen
binding moiety and the Fc domain are part of an immunoglobulin
molecule, particularly an IgG class immunoglobulin.
36. The T cell activating bispecific antigen binding molecule
according to any one of claims 24-35, wherein the Fc domain is an
IgG, specifically an IgG or IgG.sub.4, Fc domain.
37. The T cell activating bispecific antigen binding molecule
according to any one of claims 24-36, wherein the Fc domain is a
human Fc domain.
38. The T cell activating bispecific antigen binding molecule
according to any one of claims 24-37, 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 39, wherein said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), and tryptophan (W), and said amino
acid residue having a smaller side chain volume is selected from
the group consisting of alanine (A), serine (S), threonine (T), and
valine (V).
41. The T cell activating bispecific antigen binding molecule of
claim 39 or 40, wherein 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), and optionally 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) (numberings according to Kabat EU index).
42. The T cell activating bispecific antigen binding molecule of
any one of claims 39-41, wherein in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C) or the glutamic acid residue at
position 356 is replaced with a cysteine residue (E356C), and in
the second subunit of the Fc domain additionally the tyrosine
residue at position 349 is replaced by a cysteine residue (Y349C)
(numberings according to Kabat EU index).
43. The T cell activating bispecific antigen binding molecule of
any one of claims 39-42, wherein the first subunit of the Fc domain
comprises amino acid substitutions S354C and T366W, and the second
subunit of the Fc domain comprises amino acid substitutions Y349C,
T366S, L368A and Y407V (numbering according to Kabat EU index).
44. The T cell activating bispecific antigen binding molecule
according to any one of claims 24-43, 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.
45. The T cell activating bispecific antigen binding molecule
according to any one of claims 24-44, wherein the Fc domain
comprises one or more amino acid substitution that reduces binding
to an Fc receptor and/or effector function.
46. The T cell activating bispecific antigen binding molecule
according to claim 45, wherein said one or more amino acid
substitution is at one or more position selected from the group of
L234, L235, and P329 (Kabat EU index numbering).
47. The T cell activating bispecific antigen binding molecule
according to any one of claims 24-46, 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
(Kabat EU index numbering).
48. The T cell activating bispecific antigen binding molecule of
any one of claims 44 to 47, wherein the Fc receptor is an Fc.gamma.
receptor.
49. The T cell activating bispecific antigen binding molecule of
any one of claims 44 to 48, wherein the effector function is
antibody-dependent cell-mediated cytotoxicity (ADCC).
50. One or more isolated polynucleotide encoding the T cell
activating bispecific antigen binding molecule of any one of claims
1 to 49.
51. One or more vector, particularly expression vector, comprising
the polynucleotide(s) of claim 50.
52. A host cell comprising the polynucleotide(s) of claim 50 or the
vector(s) of claim 51.
53. A method of producing a T cell activating bispecific antigen
binding molecule capable of specific binding to CEA and an
activating T cell antigen, comprising the steps of a) culturing the
host cell of claim 52 under conditions suitable for the expression
of the T cell activating bispecific antigen binding molecule and b)
optionally recovering the T cell activating bispecific antigen
binding molecule.
54. A T cell activating bispecific antigen binding molecule
produced by the method of claim 53.
55. A pharmaceutical composition comprising the T cell activating
bispecific antigen binding molecule of any one of claims 1 to 49 or
54 and a pharmaceutically acceptable carrier.
56. 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 any one of claims 1 to 49 or 54 in a
pharmaceutically acceptable form.
57. The method of claim 56, wherein said disease is cancer.
58. A method for inducing lysis of a target cell, comprising
contacting a target cell with the T cell activating bispecific
antigen binding molecule of any one of claims 1-49 or 54 in the
presence of a T cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/281,484, filed on Sep. 30, 2016, which
claims priority to European Patent Application No. EP 15188035.8,
filed Oct. 2, 2015, the disclosure of which are incorporated herein
by reference in their entirety.
SEQUENCE LISTING
[0002] The present application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 20, 2020, is named
51177-014002_Sequence_Listing_7_20_20_ST25.txt and is 84,348 bytes
in size.
FIELD OF THE INVENTION
[0003] 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
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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)).
[0008] 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.
[0009] 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.
[0010] Different approaches have been taken to overcome the chain
association issue in bispecific antibodies (see e.g. Klein et al.,
mAbs 6, 653-663 (2012)). For example, the `knobs-into-holes`
strategy aims at forcing the pairing of two different antibody
heavy chains by introducing mutations into the CH3 domains to
modify the contact interface. On one chain bulky amino acids are
replaced by amino acids with short side chains to create a `hole`.
Conversely, amino acids with large side chains are introduced into
the other CH3 domain, to create a `knob`. By coexpressing these two
heavy chains (and two identical light chains, which have to be
appropriate for both heavy chains), high yields of heterodimer
(`knob-hole`) versus homodimer (`hole-hole` or `knob-knob`) are
observed (Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621;
and WO 96/027011). The percentage of heterodimer could be further
increased by remodeling the interaction surfaces of the two CH3
domains using a phage display approach and the introduction of a
disulfide bridge to stabilize the heterodimers (Merchant, A. M., et
al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol.
Biol. 270 (1997) 26-35). New approaches for the knobs-into-holes
technology are described in e.g. in EP 1870459 A1.
[0011] The `knobs-into-holes` strategy does, however, not solve the
problem of heavy chain-light chain mispairing, which occurs in
bispecific antibodies comprising different light chains for binding
to the different target antigens.
[0012] A strategy to prevent heavy chain-light chain mispairing is
to exchange domains between the heavy and light chains of one of
the binding arms of a bispecific antibody (see WO 2009/080251, WO
2009/080252, WO 2009/080253, WO 2009/080254 and Schaefer, W. et al,
PNAS, 108 (2011) 11187-11191, which relate to bispecific IgG
antibodies with a domain crossover).
[0013] Exchanging the heavy and light chain variable domains VH and
VL in one of the binding arms of the bispecific antibody
(WO2009/080252, see also Schaefer, W. et al, PNAS, 108 (2011)
11187-11191) clearly reduces the side products caused by the
mispairing of a light chain against a first antigen with the wrong
heavy chain against the second antigen (compared to approaches
without such domain exchange). Nevertheless, these antibody
preparations are not completely free of side products. The main
side product is based on a Bence Jones-type interaction (Schaefer,
W. et al, PNAS, 108 (2011) 11187-11191; in Fig. S1I of the
Supplement). A further reduction of such side products is thus
desirable to improve e.g. the yield of such bispecific
antibodies.
[0014] The choice of target antigens and appropriate binders for
both the T cell antigen and the target cell antigen is a further
crucial aspect in the generation of T cell bispecific (TCB)
antibodies for therapeutic application. Carcinoembryonic antigen
(CEA) is an attractive target antigen as the prevalence of CEA
expression is generally high in tumors, but low in normal tissues.
Accordingly, numerous antibodies have been raised against this
target, one of which is the murine antibody T84.66 (Wagener et al.,
J Immunol 130, 2308 (1983), Neumaier et al., J Immunol 135, 3604
(1985)), which has also been chimerized (WO 1991/01990) and
humanized (WO 2005/086875). WO 2007/071426 or WO 2014/131712
describe bispecific antibodies targeting CD3 on T cells and
carcinoembryonic antigen (CEA) on target cells.
[0015] The present invention provides novel, improved bispecific
antigen binding molecules designed for T cell activation and
re-direction, targeting CD3 and CEA, that combine good efficacy and
produceability with low toxicity and favorable pharmacokinetic
properties.
SUMMARY OF THE INVENTION
[0016] The present inventors have developed a novel T cell
activating bispecific antigen binding molecule with unexpected,
improved properties using a novel humanized anti-CEA antibody.
[0017] Thus, in a first aspect the present invention provides a T
cell activating bispecific antigen binding molecule comprising
(a) a first antigen binding moiety which specifically binds to a
first antigen; (b) a second antigen binding moiety which
specifically binds to a second antigen; wherein the first antigen
is an activating T cell antigen and the second antigen is CEA, or
the first antigen is CEA and the second antigen is an activating T
cell antigen; and wherein the antigen binding moiety which
specifically binds to CEA comprises a heavy chain variable region,
particularly a humanized heavy chain variable region, comprising
the heavy chain complementarity determining region (HCDR) 1 of SEQ
ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO:
16, and a light chain variable region, particularly a humanized
light chain variable region, comprising the light chain
complementarity determining region (LCDR) 1 of SEQ ID NO: 17, the
LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.
[0018] In one embodiment, the antigen binding moiety which
specifically binds to CEA 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: 22 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: 23.
[0019] In particular embodiments, the first and/or the second
antigen binding moiety is a Fab molecule. In a particular
embodiment, the second antigen binding moiety is a Fab molecule
which specifically binds to a second antigen, and wherein the
variable domains VL and VH or the constant domains CL and CH1 of
the Fab light chain and the Fab heavy chain are replaced by each
other (i.e. according to such embodiment, the second Fab molecule
is a crossover Fab molecule wherein the variable or constant
domains of the Fab light chain and the Fab heavy chain are
exchanged).
[0020] In particular embodiments, the first (and the third, if any)
Fab molecule is a conventional Fab molecule. In a further
particular embodiment, not more than one Fab molecule capable of
specific binding to an activating T cell antigen 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 the activating T cell antigen).
[0021] In one embodiment, the first antigen is CEA and the second
antigen is an activating T cell antigen.
[0022] In a more specific embodiment, the activating T cell antigen
is CD3, particularly CD3 epsilon.
[0023] In a particular embodiment, the T cell activating bispecific
antigen binding molecule of the invention comprises
(a) a first Fab molecule which specifically binds to a first
antigen; (b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH or the
constant domains CL and CH1 of the Fab light chain and the Fab
heavy chain are replaced by each other; wherein the first antigen
is CEA and the second antigen is an activating T cell antigen;
wherein the first Fab molecule under (a) comprises a heavy chain
variable region, particularly a humanized heavy chain variable
region, comprising the heavy chain complementarity determining
region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and
the HCDR 3 of SEQ ID NO: 16, and a light chain variable region,
particularly a humanized light chain variable region, comprising
the light chain complementarity determining region (LCDR) 1 of SEQ
ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO:
19.
[0024] According to a further aspect of the invention, the ratio of
a desired bispecific antibody compared to undesired side products,
in particular Bence Jones-type side products occurring in
bispecific antibodies with a VH/VL domain exchange in one of their
binding arms, can be improved by the introduction of charged amino
acids with opposite charges at specific amino acid positions in the
CH1 and CL domains (sometimes referred to herein as "charge
modifications").
[0025] Thus, in some embodiments the first antigen binding moiety
under (a) is a first Fab molecule which specifically binds to a
first antigen, the second antigen binding moiety under (b) is a
second Fab molecule which specifically binds to a second antigen
wherein the variable domains VL and VH of the Fab light chain and
the Fab heavy chain are replaced by each other;
and [0026] i) in the constant domain CL of the first Fab molecule
under a) the amino acid at position 124 is substituted
independently by lysine (K), arginine (R) or histidine (H)
(numbering according to Kabat), and wherein in the constant domain
CH1 of the first Fab molecule under a) the amino acid at position
147 or the amino acid at position 213 is substituted independently
by glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index); or [0027] ii) in the constant domain CL of the
second Fab molecule under b) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and wherein in the constant
domain CH1 of the second Fab molecule under b) the amino acid at
position 147 or the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0028] In one such embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in one preferred embodiment
independently by lysine (K) or arginine (R)), and in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 or the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0029] In a further embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and in the constant domain CH1
of the first Fab molecule under a) the amino acid at position 147
is substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0030] In yet another embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in one preferred embodiment
independently by lysine (K) or arginine (R)) and the amino acid at
position 123 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) (in one
preferred embodiment independently by lysine (K) or arginine (R)),
and in the constant domain CH1 of the first Fab molecule under a)
the amino acid at position 147 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0031] In a particular embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by lysine (K) (numbering
according to Kabat), and in the constant domain CH1 of the first
Fab molecule under a) the amino acid at position 147 is substituted
by glutamic acid (E) (numbering according to Kabat EU index) and
the amino acid at position 213 is substituted by glutamic acid (E)
(numbering according to Kabat EU index).
[0032] In another particular embodiment, in the constant domain CL
of the first Fab molecule under a) the amino acid at position 124
is substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by arginine (R)
(numbering according to Kabat), and in the constant domain CH1 of
the first Fab molecule under a) the amino acid at position 147 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index).
[0033] In an alternative embodiment, in the constant domain CL of
the second Fab molecule under b) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in one preferred embodiment
independently by lysine (K) or arginine (R)), and in the constant
domain CH1 of the second Fab molecule under b) the amino acid at
position 147 or the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0034] In a further embodiment, in the constant domain CL of the
second Fab molecule under b) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and in the constant domain CH1
of the second Fab molecule under b) the amino acid at position 147
is substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0035] In still another embodiment, in the constant domain CL of
the second Fab molecule under b) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in one preferred embodiment
independently by lysine (K) or arginine (R)) and the amino acid at
position 123 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) (in one
preferred embodiment independently by lysine (K) or arginine (R)),
and in the constant domain CH1 of the second Fab molecule under b)
the amino acid at position 147 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0036] In one embodiment, in the constant domain CL of the second
Fab molecule under b) the amino acid at position 124 is substituted
by lysine (K) (numbering according to Kabat) and the amino acid at
position 123 is substituted by lysine (K) (numbering according to
Kabat), and in the constant domain CH1 of the second Fab molecule
under b) the amino acid at position 147 is substituted by glutamic
acid (E) (numbering according to Kabat EU index) and the amino acid
at position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index).
[0037] In another embodiment, in the constant domain CL of the
second Fab molecule under b) the amino acid at position 124 is
substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by arginine (R)
(numbering according to Kabat), and in the constant domain CH1 of
the second Fab molecule under b) the amino acid at position 147 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index).
[0038] In a particular embodiment, the T cell activating bispecific
antigen binding molecule of the invention comprises
(a) a first Fab molecule which specifically binds to a first
antigen; (b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each other;
wherein the first antigen is CEA and the second antigen is an
activating T cell antigen; wherein the first Fab molecule under (a)
comprises a heavy chain variable region, particularly a humanized
heavy chain variable region, comprising the heavy chain
complementarity determining region (HCDR) 1 of SEQ ID NO: 14, the
HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, and a
light chain variable region, particularly a humanized light chain
variable region, comprising the light chain complementarity
determining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID
NO: 18 and the LCDR 3 of SEQ ID NO: 19; and wherein in the constant
domain CL of the first Fab molecule under a) the amino acid at
position 124 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) (in one
preferred embodiment independently by lysine (K) or arginine (R))
and the amino acid at position 123 is substituted independently by
lysine (K), arginine (R) or histidine (H) (numbering according to
Kabat) (in one preferred embodiment independently by lysine (K) or
arginine (R)), and in the constant domain CH1 of the first Fab
molecule under a) the amino acid at position 147 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0039] In some embodiments, the T cell activating bispecific
antigen binding molecule according to the invention further
comprises a third antigen binding moiety which specifically binds
to the first antigen. In particular embodiments, the third antigen
binding moiety is identical to the first antigen binding moiety. In
one embodiment, the third antigen binding moiety is a Fab
molecule.
[0040] In particular embodiments, the third and the first antigen
binding moiety are each a Fab molecule and the third Fab molecule
is identical to the first Fab molecule. In these embodiments, the
third Fab molecule thus comprises the same amino acid
substitutions, if any, as the first Fab molecule. Like the first
Fab molecule, the third Fab molecule particularly is a conventional
Fab molecule.
[0041] If a third antigen binding moiety is present, in a
particular embodiment the first and the third antigen moiety
specifically bind to CEA, and the second antigen binding moiety
specifically binds to an activating T cell antigen, particularly
CD3, more particularly CD3 epsilon.
[0042] In some embodiments of the T cell activating bispecific
antigen binding molecule according to the invention the first
antigen binding moiety under a) and the second antigen binding
moiety under b) are fused to each other, optionally via a peptide
linker. In particular embodiments, the first and the second antigen
binding moiety are each a Fab molecule. In a specific such
embodiment, the second Fab molecule is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the Fab heavy chain of the
first Fab molecule. In an alternative such embodiment, the first
Fab molecule is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the Fab heavy chain of the second Fab molecule.
In embodiments wherein either (i) the second Fab molecule is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the first Fab molecule or (ii) the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule,
additionally the Fab light chain of the Fab molecule and the Fab
light chain of the second Fab molecule may be fused to each other,
optionally via a peptide linker.
[0043] In particular embodiments, the T cell activating bispecific
antigen binding molecule according to the invention additionally
comprises an Fc domain composed of a first and a second subunit
capable of stable association.
[0044] The T cell activating bispecific antigen binding molecule
according to the invention can have different configurations, i.e.
the first, second (and optionally third) antigen binding moiety may
be fused to each other and to the Fc domain in different ways. The
components may be fused to each other directly or, preferably, via
one or more suitable peptide linkers. Where fusion of a Fab
molecule is to the N-terminus of a subunit of the Fc domain, it is
typically via an immunoglobulin hinge region.
[0045] In one embodiment, the first and the second antigen binding
moiety are each a Fab molecule 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. In
such embodiment, the first antigen binding moiety may be 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 or to the
N-terminus of the other one of the subunits of the Fc domain.
[0046] In one embodiment, the first and the second antigen binding
moiety are each a Fab molecule and 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. In
this embodiment, the T cell activating bispecific antigen binding
molecule essentially comprises an immunoglobulin molecule, wherein
in one of the Fab arms the heavy and light chain variable regions
VH and VL (or the constant regions CH1 and CL in embodiments
wherein no charge modifications as described herein are introduced
in CH1 and CL domains) are exchanged/replaced by each other (see
FIG. 1A, D).
[0047] In alternative embodiments, a third antigen binding moiety,
particularly a third Fab molecule, 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 such 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 Fab molecule. In this embodiment,
the T cell activating bispecific antigen binding molecule
essentially comprises an immunoglobulin molecule, wherein in one of
the Fab arms the heavy and light chain variable regions VH and VL
(or the constant regions CH1 and CL in embodiments wherein no
charge modifications as described herein are introduced in CH1 and
CL domains) are exchanged/replaced by each other, and wherein an
additional (conventional) Fab molecule is N-terminally fused to
said Fab arm (see FIG. 1B, E). In another such 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 this
embodiment, the T cell activating bispecific antigen binding
molecule essentially comprises an immunoglobulin molecule with an
additional Fab molecule N-terminally fused to one of the
immunoglobulin Fab arms, wherein in said additional Fab molecule
the heavy and light chain variable regions VH and VL (or the
constant regions CH1 and CL in embodiments wherein no charge
modifications as described herein are introduced in CH1 and CL
domains) are exchanged/replaced by each other (see FIG. 1C, F).
[0048] In a particular embodiment, the immunoglobulin molecule
comprised in the T cell activating bispecific antigen binding
molecule according to the invention 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.
[0049] In a particular embodiment, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH or the
constant domains CL and CH1 of the Fab light chain and the Fab
heavy chain are replaced by each other; c) a third Fab molecule
which specifically binds to the first antigen; and d) an Fc domain
composed of a first and a second subunit capable of stable
association; wherein the first antigen is CEA and the second
antigen is an activating T cell antigen, particularly CD3, more
particularly CD3 epsilon; wherein the third Fab molecule under c)
is identical to the first Fab molecule under a); wherein (i) the
first Fab molecule under a) is fused at the C-terminus of the Fab
heavy chain to the N-terminus of the Fab heavy chain of the second
Fab molecule under b), and the second Fab molecule under b) and the
third Fab molecule under c) 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 under d), or (ii) the second Fab molecule under b) is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the first Fab molecule under a), and the first
Fab molecule under a) and the third Fab molecule under c) 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 under d); and wherein the
first Fab molecule under a) and the third Fab molecule under c)
comprise a heavy chain variable region, particularly a humanized
heavy chain variable region, comprising the heavy chain
complementarity determining region (HCDR) 1 of SEQ ID NO: 14, the
HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, and a
light chain variable region, particularly a humanized light chain
variable region, comprising the light chain complementarity
determining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID
NO: 18 and the LCDR 3 of SEQ ID NO: 19.
[0050] In another embodiment, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH or the
constant domains CL and CH1 of the Fab light chain and the Fab
heavy chain are replaced by each other; c) an Fc domain composed of
a first and a second subunit capable of stable association; wherein
the first antigen is CEA and the second antigen is an activating T
cell antigen, particularly CD3, more particularly CD3 epsilon;
wherein (i) the first Fab molecule under a) is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule under b), and the second Fab
molecule under b) is fused at the C-terminus of the Fab heavy chain
to the N-terminus of one of the subunits of the Fc domain under c),
or (ii) the second Fab molecule under b) is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the Fab heavy chain of
the first Fab molecule under a), and the first Fab molecule under
a) is fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain under c); and
wherein the first Fab molecule under a) comprises a heavy chain
variable region, particularly a humanized heavy chain variable
region, comprising the heavy chain complementarity determining
region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and
the HCDR 3 of SEQ ID NO: 16, and a light chain variable region,
particularly a humanized light chain variable region, comprising
the light chain complementarity determining region (LCDR) 1 of SEQ
ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO:
19.
[0051] In a further embodiment, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH or the
constant domains CL and CH1 of the Fab light chain and the Fab
heavy chain are replaced by each other; and c) an Fc domain
composed of a first and a second subunit capable of stable
association; wherein (i) the first antigen is CEA and the second
antigen is an activating T cell antigen, particularly CD3, more
particularly CD3 epsilon; or (ii) the second antigen is CEA and the
first antigen is an activating T cell antigen, particularly CD3,
more particularly CD3 epsilon; wherein the first Fab molecule under
a) and the second Fab molecule under b) 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 under c); and wherein the Fab molecule
which specifically binds to CEA comprises a heavy chain variable
region, particularly a humanized heavy chain variable region,
comprising the heavy chain complementarity determining region
(HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR
3 of SEQ ID NO: 16, and a light chain variable region, particularly
a humanized light chain variable region, comprising the light chain
complementarity determining region (LCDR) 1 of SEQ ID NO: 17, the
LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.
[0052] In all of the different configurations of the T cell
activating bispecific antigen binding molecule according to the
invention, the amino acid substitutions described herein, if
present, may either be in the CH1 and CL domains of the first and
(if present) the third Fab molecule, or in the CH1 and CL domains
of the second Fab molecule. Preferably, they are in the CH1 and CL
domains of the first and (if present) the third Fab molecule. In
accordance with the concept of the invention, if amino acid
substitutions as described herein are made in the first (and, if
present, the third) Fab molecule, no such amino acid substitutions
are made in the second Fab molecule. Conversely, if amino acid
substitutions as described herein are made in the second Fab
molecule, no such amino acid substitutions are made in the first
(and, if present, the third) Fab molecule. No amino acid
substitutions are made in T cell activating bispecific antigen
binding molecules comprising a Fab molecule wherein the constant
domains CL and CH1 of the Fab light chain and the Fab heavy chain
are replaced by each other.
[0053] In particular embodiments of the T cell activating
bispecific antigen binding molecule according to the invention,
particularly wherein amino acid substitutions as described herein
are made in the first (and, if present, the third) Fab molecule,
the constant domain CL of the first (and, if present, the third)
Fab molecule is of kappa isotype. In other embodiments of the T
cell activating bispecific antigen binding molecule according to
the invention, particularly wherein amino acid substitutions as
described herein are made in the second Fab molecule, the constant
domain CL of the second Fab molecule is of kappa isotype. In some
embodiments, the constant domain CL of the first (and, if present,
the third) Fab molecule and the constant domain CL of the second
Fab molecule are of kappa isotype.
[0054] In a particular embodiment, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each other;
c) a third Fab molecule which specifically binds to the first
antigen; and d) an Fc domain composed of a first and a second
subunit capable of stable association; wherein the first antigen is
CEA and the second antigen is an activating T cell antigen,
particularly CD3, more particularly CD3 epsilon; wherein the third
Fab molecule under c) is identical to the first Fab molecule under
a); wherein in the constant domain CL of the first Fab molecule
under a) and the third Fab molecule under c) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) or arginine (R) (numbering according to Kabat), and wherein in
the constant domain CH1 of the first Fab molecule under a) and the
third Fab molecule under c) the amino acid at position 147 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index); wherein
(i) the first Fab molecule under a) is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the Fab heavy chain of the
second Fab molecule under b), and the second Fab molecule under b)
and the third Fab molecule under c) 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 under d), or (ii) the second Fab molecule
under b) is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first Fab molecule under
a), and the first Fab molecule under a) and the third Fab molecule
under c) 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 under d);
and wherein the first Fab molecule under a) and the third Fab
molecule under c) comprise a heavy chain variable region,
particularly a humanized heavy chain variable region, comprising
the heavy chain complementarity determining region (HCDR) 1 of SEQ
ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO:
16, and a light chain variable region, particularly a humanized
light chain variable region, comprising the light chain
complementarity determining region (LCDR) 1 of SEQ ID NO: 17, the
LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.
[0055] In an even more particular embodiment, the invention
provides a T cell activating bispecific antigen binding molecule
comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each other;
c) a third Fab molecule which specifically binds to the first
antigen; and d) an Fc domain composed of a first and a second
subunit capable of stable association; wherein the first antigen is
CEA and the second antigen is an activating T cell antigen,
particularly CD3, more particularly CD3 epsilon; wherein the third
Fab molecule under c) is identical to the first Fab molecule under
a); wherein in the constant domain CL of the first Fab molecule
under a) and the third Fab molecule under c) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by
arginine (R) (numbering according to Kabat), and wherein in the
constant domain CH1 of the first Fab molecule under a) and the
third Fab molecule under c) the amino acid at position 147 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index); wherein
the first Fab molecule under a) is fused at the C-terminus of the
Fab heavy chain to the N-terminus of the Fab heavy chain of the
second Fab molecule under b), and the second Fab molecule under b)
and the third Fab molecule under c) 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 under d); and wherein the first Fab
molecule under a) and the third Fab molecule under c) comprise a
heavy chain variable region, particularly a humanized heavy chain
variable region, comprising the heavy chain complementarity
determining region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID
NO: 15 and the HCDR 3 of SEQ ID NO: 16, and a light chain variable
region, particularly a humanized light chain variable region,
comprising the light chain complementarity determining region
(LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR
3 of SEQ ID NO: 19.
[0056] In another embodiment, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each other;
c) an Fc domain composed of a first and a second subunit capable of
stable association; wherein the first antigen is CEA and the second
antigen is an activating T cell antigen, particularly CD3, more
particularly CD3 epsilon; wherein in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by lysine (K) or arginine
(R) (numbering according to Kabat), and wherein in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 is substituted by glutamic acid (E) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index); wherein (i) the first Fab molecule under a) is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule under b), and the second Fab
molecule under b) is fused at the C-terminus of the Fab heavy chain
to the N-terminus of one of the subunits of the Fc domain under c),
or (ii) the second Fab molecule under b) is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the Fab heavy chain of
the first Fab molecule under a), and the first Fab molecule under
a) is fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain under c); and
wherein the first Fab molecule under a) comprises a heavy chain
variable region, particularly a humanized heavy chain variable
region, comprising the heavy chain complementarity determining
region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and
the HCDR 3 of SEQ ID NO: 16, and a light chain variable region,
particularly a humanized light chain variable region, comprising
the light chain complementarity determining region (LCDR) 1 of SEQ
ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO:
19.
[0057] In a further embodiment, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each other;
and c) an Fc domain composed of a first and a second subunit
capable of stable association; wherein (i) the first antigen is CEA
and the second antigen is an activating T cell antigen,
particularly CD3, more particularly CD3 epsilon; or (ii) the second
antigen is CEA and the first antigen is an activating T cell
antigen, particularly CD3, more particularly CD3 epsilon; wherein
in the constant domain CL of the first Fab molecule under a) the
amino acid at position 124 is substituted by lysine (K) (numbering
according to Kabat) and the amino acid at position 123 is
substituted by lysine (K) or arginine (R) (numbering according to
Kabat), and wherein in the constant domain CH1 of the first Fab
molecule under a) the amino acid at position 147 is substituted by
glutamic acid (E) (numbering according to Kabat EU index) and the
amino acid at position 213 is substituted by glutamic acid (E)
(numbering according to Kabat EU index); wherein the first Fab
molecule under a) and the second Fab molecule under b) 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 under c); and wherein the Fab
molecule which specifically binds to CEA comprises a heavy chain
variable region, particularly a humanized heavy chain variable
region, comprising the heavy chain complementarity determining
region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and
the HCDR 3 of SEQ ID NO: 16, and a light chain variable region,
particularly a humanized light chain variable region, comprising
the light chain complementarity determining region (LCDR) 1 of SEQ
ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO:
19.
[0058] In particular embodiments of the T cell activating
bispecific antigen binding molecule, 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.
[0059] 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.
[0060] 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.
[0061] In one embodiment, the one or more amino acid substitution
in the Fe 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 EU index 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 (Kabat EU index
numbering). 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
(Kabat EU index numbering). 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)
(Kabat EU index numbering).
[0062] 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).
[0063] In a specific embodiment of the T cell activating bispecific
antigen binding molecule according to the invention, the antigen
binding moiety which specifically binds to an activating T cell
antigen, particularly CD3, more particularly CD3 epsilon, comprises
a heavy chain variable region comprising the heavy chain
complementarity determining region (HCDR) 1 of SEQ ID NO: 4, the
HCDR 2 of SEQ ID NO: 5, the HCDR 3 of SEQ ID NO: 6, and a light
chain variable region comprising the light chain complementarity
determining region (LCDR) 1 of SEQ ID NO: 8, the LCDR 2 of SEQ ID
NO: 9 and the LCDR 3 of SEQ ID NO: 10. In an even more specific
embodiment, the antigen binding moiety which specifically binds to
an activating T cell antigen, particularly CD3, more particularly
CD3 epsilon, 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 some embodiments, the
antigen binding moiety which specifically binds to an activating T
cell antigen is a Fab molecule. In one specific embodiment, the
second antigen binding moiety, particularly Fab molecule, comprised
in the T cell activating bispecific antigen binding molecule
according to the invention specifically binds to CD3, more
particularly CD3 epsilon, and comprises 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. In an
even more specific embodiment, said second antigen binding moiety,
particularly Fab molecule, 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.
[0064] In a further specific embodiment of the T cell activating
bispecific antigen binding molecule according to the invention, the
antigen binding moiety, particularly Fab molecule, which
specifically binds to CEA comprises the heavy chain complementarity
determining region (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: 17, the light chain CDR 2 of SEQ ID NO:
18 and the light chain CDR 3 of SEQ ID NO: 19. In an even more
specific embodiment, the antigen binding moiety, particularly Fab
molecule, which specifically binds to CEA 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: 22 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: 23. In one specific embodiment, the first (and, if present,
the third) antigen binding moiety, particularly Fab molecule,
comprised in the T cell activating bispecific antigen binding
molecule according to the invention specifically binds to CEA, and
comprises the heavy chain complementarity determining region (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: 17, the light chain CDR 2 of SEQ ID NO: 18 and the light chain
CDR 3 of SEQ ID NO: 19. In an even more specific embodiment, said
first (and, if present, said third) antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 22 and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 23.
[0065] In a particular aspect, the invention provides a T cell
activating bispecific antigen binding molecule comprising
a) a first Fab molecule which specifically binds to a first
antigen; b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH or the
constant domains CL and CH1 of the Fab light chain and the Fab
heavy chain are replaced by each other; c) a third Fab molecule
which specifically binds to the first antigen; and d) an Fc domain
composed of a first and a second subunit capable of stable
association; wherein (i) the first antigen is CEA and the second
antigen is CD3, particularly CD3 epsilon; (ii) the first Fab
molecule under a) and the third Fab molecule under c) each comprise
the heavy chain complementarity determining region (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: 17, the
light chain CDR 2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ
ID NO: 19, and the second Fab molecule under b) comprises the heavy
chain 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; and (iii) the first Fab molecule under a)
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the Fab heavy chain of the second Fab molecule under b), and the
second Fab molecule under b) and the third Fab molecule under c)
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 under d).
[0066] In one embodiment, in the second Fab molecule under b) the
variable domains VL and VH are replaced by each other and further
(iv) in the constant domain CL of the first Fab molecule under a)
and the third Fab molecule under c) the amino acid at position 124
is substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by lysine (K) or arginine
(R), particularly by arginine (R) (numbering according to Kabat),
and in the constant domain CH1 of the first Fab molecule under a)
and the third Fab molecule under c) the amino acid at position 147
is substituted by glutamic acid (E) (numbering according to Kabat
EU index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index).
[0067] According to another aspect of the invention there is
provided one or more isolated polynucleotide(s) encoding a T cell
activating bispecific antigen binding molecule of the invention.
The invention further provides one or more expression vector(s)
comprising the isolated polynucleotide(s) of the invention, and a
host cell comprising the isolated polynucleotide(s) or the
expression vector(s) of the invention. In some embodiments the host
cell is a eukaryotic cell, particularly a mammalian cell.
[0068] 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.
[0069] The invention further provides a pharmaceutical composition
comprising the T cell activating bispecific antigen binding
molecule of the invention and a pharmaceutically acceptable
carrier.
[0070] 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.
[0071] 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.
[0072] 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
[0073] FIGS. 1A-Z. Exemplary configurations of the T cell
activating bispecific antigen binding molecules (TCBs) of the
invention. (A, D) Illustration of the "1+1 CrossMab" molecule. (B,
E) Illustration of the "2+1 IgG Crossfab" molecule with alternative
order of Crossfab and Fab components ("inverted"). (C, F)
Illustration of the "2+1 IgG Crossfab" molecule. (G, K)
Illustration of the "1+1 IgG Crossfab" molecule with alternative
order of Crossfab and Fab components ("inverted"). (H, L)
Illustration of the "1+1 IgG Crossfab" molecule. (I, M)
Illustration of the "2+1 IgG Crossfab" molecule with two CrossFabs.
(J, N) Illustration of the "2+1 IgG Crossfab" molecule with two
CrossFabs and alternative order of Crossfab and Fab components
("inverted"). (O, S) Illustration of the "Fab-Crossfab" molecule.
(P, T) Illustration of the "Crossfab-Fab" molecule. (Q, U)
Illustration of the "(Fab).sub.2-Crossfab" molecule. (R, V)
Illustration of the "Crossfab-(Fab).sub.2" molecule. (W, Y)
Illustration of the "Fab-(Crossfab).sub.2" molecule. (X, Z)
Illustration of the "(Crossfab).sub.2-Fab" molecule. Black dot:
optional modification in the Fc domain promoting
heterodimerization. ++, --: amino acids of opposite charges
optionally introduced in the CH1 and CL domains. Crossfab molecules
are depicted as comprising an exchange of VH and VL regions, but
may--in embodiments wherein no charge modifications are introduced
in CH1 and CL domains--alternatively comprise an exchange of the
CH1 and CL domains.
[0074] FIG. 2. Binding of different humanized variants of T84.66
IgGs to cells. EC50 values, based on binding curves, were
calculated by Graph Pad Prism and are presented in Table 1.
[0075] FIGS. 3A-F. Illustration of the TCBs prepared in the
Examples. (A, B) Illustration of "2+1 IgG CrossFab, inverted"
anti-CEA/anti-CD3 TCB molecules with charge modifications (VH/VL
exchange in CD3 binder, charge modification in CEA binder, molecule
A and B). (C) Illustration of "1+1 IgG CrossFab, inverted"
anti-CEA/anti-CD3 TCB molecule with charge modifications (VH/VL
exchange in CD3 binder, charge modification in CEA binder, molecule
C). (D) Illustration of "1+1 IgG CrossMab" anti-CEA/anti-CD3 TCB
molecule with charge modifications (VH/VL exchange in CD3 binder,
charge modification in CEA binder, molecule D). (E) Illustration of
"2+1 IgG CrossFab, inverted" anti-CEA/anti-CD3 TCB molecule with
charge modifications and longer linker (VH/VL exchange in CD3
binder, charge modification in CEA binder, molecule E). (F)
Illustration of "2+1 IgG CrossFab, inverted" anti-CEA/anti-CD3 TCB
molecule without charge modifications (VH/VL exchange in CD3
binder, molecule F). EE=147E, 213E; RK=123R, 124K.
[0076] FIGS. 4A-F. CE-SDS analyses of the TCB molecules prepared in
the Examples (final purified preparations). (A) Electropherogram of
"2+1 IgG CrossFab, inverted" with charge modifications (VH/VL
exchange in CD3 binder, charge modification in CEA binder, parental
murine CEA binder (T84.66); molecule A). (B) Electropherogram of
"2+1 IgG CrossFab, inverted" with charge modifications (VH/VL
exchange in CD3 binder, charge modification in CEA binder,
humanized CEA binder; molecule B). (C) Electropherogram of "1+1 IgG
CrossFab, inverted" with charge modifications (VH/VL exchange in
CD3 binder, charge modification in CEA binder, humanized CEA
binder; molecule C). (D) Electropherogram of "1+1 IgG CrossMab"
with charge modifications (VH/VL exchange in CD3 binder, charge
modification in CEA binder, humanized CEA binder; molecule D). (E)
Electropherogram of "2+1 IgG CrossFab inverted" with charge
modifications and longer linker (VH/VL exchange in CD3 binder,
charge modification in CEA binder, humanized CEA binder; molecule
E). (F) Electropherogram of "2+1 IgG CrossFab, inverted" without
charge modifications (VH/VL exchange in CD3 binder, humanized CEA
binder; molecule F). Lane A=non reduced, lane B=reduced.
[0077] FIGS. 5A-D. Binding of different CEA TCB formats to cells,
expressing either high levels of CEA (MKN45; A), medium levels of
CEA (LS174T; B) or low levels of CEA (HT29; C), or human CD3
(Jurkat cells; D). Median Fluorescence intensities (MFI) are
depicted. Error bars indicate SD of triplicates.
[0078] FIGS. 6A-F. T cell mediated lysis of tumor cells induced by
different CEA CD3 TCB molecules, as measured by LDH release after
24 h (A-C) or 48 h (D-F). Human PBMCs were used as effector cells
and MKN45 (A, D), BxPC-3 (B, E) or HT29 (C, F) cells were used as
target cells, at a final effector to target cell ratio of 10:1.
Depicted are triplicates with SD. EC50 values were calculated by
GraphPadPrism 5 and are given in Table 4 and 5.
[0079] FIGS. 7A-H. Up-regulation of CD25 on human CD4+ (A-D) and
CD8+ (E-H) T cells after T cell-mediated lysis of CEA-expressing
tumor cells induced by different CEA CD3 TCB molecules. Human PBMCs
were used as effector cells and MKN45 (A, E), BxPC-3 (B, F) or HT29
(C, G) cells were used as target cells, at a final effector to
target cell ratio of 10:1. Results without target cells are shown
in (D) and (H). Percentage of CD25-positive T cells was determined
by FACS after 48 h. Depicted are triplicates with SD.
[0080] FIGS. 8A-J. Up-regulation of CD69 on human CD4+ (A-E) and
CD8+ (F-J) T cells upon co-incubation with CEA-expressing tumor or
primary epithelial cells and different CEA CD3 TCB molecules. Human
PBMCs were used as effector cells and MKN45 (A, F), LS174T (B, G),
HT29 (C, H), CCD841 (D, I) cells were used as target cells, at a
final effector to target cell ratio of 10:1. Results without target
cells are shown in (E) and (J). Percentage of CD69-positive T cells
was determined by FACS after 48 h. Depicted are triplicates with
SD.
[0081] FIGS. 9A-D. T cell activation and tumor cell lysis induced
by different CEA CD3 TCB molecules, as measured by FACS (percent of
CD69-positive CD8+ T cells (A, B)), or LDH (C, D) after 48 h. Human
PBMCs were used as effector cells at a final effector to target
cell ratio of 10:1. Target cells were either high CEA expressing
MKN45 or low CEA-expressing primary epithelial cells CCD841 CoN. In
addition, T cell activation was assessed in the absence of targets
("no targets"). Depicted are triplicates with SD. A, C: CEA CD3 TCB
with parental chimeric T84.66 CEA binder (molecule A), B, D: CEA
CD3 TCB with humanized CEA binder (humanized variant 1; molecule
B).
[0082] FIGS. 10A-B. Jurkat-NFAT reporter cell assay to determine
early CD3-mediated activation of Jurkats upon simultaneous binding
of different CEA CD3 TCB molecules to target and Jurkat effector
cells. The intensity of CD3-mediated activation and signaling was
detected by measuring the relative luminescence signal (RLUs).
Depicted are triplicates with SD. (A) CEA CD3 TCB with parental
chimeric T84.66 CEA binder (molecule A), (B) CEA CD3 TCB with
humanized CEA binder (humanized variant 1; molecule B).
[0083] FIGS. 11A-H. T cell activation (A-D) and tumor cell lysis
(E-H) induced by different CEA CD3 TCB molecules, as measured by
FACS (A-D, percent of CD69-positive CD8+ T cells), or LDH (E-H)
after 48 h. Human PBMCs were used as effector cells at a final
effector to target cell ratio of 10:1. Target cells were either
medium CEA expressing BxPC-3 (A, E), low CEA-expressing NCI-H2122
(B, F) cells or very low CEA expressing COR-L105 (C, G) or primary
epithelial cells HBEpiC (D, H). Depicted are triplicates with
SD.
[0084] FIGS. 12A-H. Proliferation of CD8+ (A-D) and CD4+ (E-H) T
cells, induced by different CEA CD3 TCB molecules, as measured by
FACS after 5 days. Human PBMCs were used as effector cells at a
final effector to target cell ratio of 10:1. Target cells were
either high CEA expressing MKN45 (A, E), medium CEA expressing
LS174T (B, F), low CEA-expressing HT29 (C, G) cells or very low CEA
expressing primary epithelial cells CCD841 CoN (D, H). Depicted are
triplicates with SD.
[0085] FIGS. 13A-D. Binding of different CEA CD3 TCB molecules
(molecule B and molecule E) to MKN45 (A), LS174T (B), HT29 (C) and
Jurkat (D) cells, as measured by FACS.
[0086] FIGS. 14A-H. T cell mediated lysis of tumor cells induced by
different CEA CD3 TCB molecules (molecule B and molecule E), as
measured by LDH release after 24 h (A-D) or 48 h (E-H). Human PBMCs
were used as effector cells and MKN45 (A, E), LS174T (B, F), HT29
(C, G), HBEpiC (D, H) cells were used as target cells, at a final
effector to target cell ratio of 10:1. Depicted are triplicates
with SD. EC50 values were calculated by GraphPadPrism 5 and are
given in Table 6.
[0087] FIG. 15. Comparison of pharmacokinetics of CEA CD3 TCB
molecule B and CEA CD3 TCB molecule X after a single i.v. bolus
administration in NOG mice.
[0088] FIGS. 16A-D. Binding of different CEA CD3 TCB molecules to
human CEA, expressed on MKN45 (A), LS174T (B) and HT29 (C) cells,
or to human CD3, expressed on Jurkat cells (D). Depicted are
triplicates and SD. EC50 values, based on binding curves, were
calculated by Graph Pad Prism and are presented in Table 6.
[0089] FIGS. 17A-E. Determination of antigen-dependent tumor cell
lysis, induced by different CEA CD3 TCB molecules in the presence
of various CEA-positive tumor cells: (A) HCC1954, (B) NCI-H596, (C)
NCI-H2122, (D) Kato III, (E) CX-1. Tumor cell lysis was determined
by quantification of LDH released from apoptotic/necrotic tumor
cells. Depicted are triplicates with SD. EC50 values of tumor cell
lysis were calculated by Graph Pad Prism and are presented in Table
7.
[0090] FIGS. 18A-B. Antigen-dependent T cell activation and tumor
lysis induced by CEA CD3 TCB molecule B in the presence of
CEA-positive tumor cells, but not in the presence of low
CEA-expressing primary epithelia cells. Tumor cell lysis was
determined by quantification of LDH released from
apoptotic/necrotic tumor cells (A). T cell activation was
determined by FACS measurement of up-regulation of the early
activation marker CD69 on CD4+ effector cells (B). Depicted are
triplicates with SD. EC50 values of tumor cell lysis and T-cell
activation were calculated by Graph Pad Prism and are presented in
Table 9 and Table 11.
[0091] FIGS. 19A-B. Antigen-dependent T cell activation and tumor
lysis induced by CEA CD3 TCB molecule B in the presence of
CEA-positive tumor cells, but not in the presence of low
CEA-expressing primary epithelial cells. Tumor cell lysis was
determined by quantification of LDH released from
apoptotic/necrotic tumor cells (A). T cell activation was
determined by FACS measurement of up-regulation of the late
activation marker CD25 on CD4+ effector cells (B). Depicted are
triplicates with SD. EC50 values of tumor cell lysis were
calculated by Graph Pad Prism and are presented in Table 10.
[0092] FIGS. 20A-B. (A) Binding of CEA CD3 TCB molecule B to
transient HEK293T transfectants, expressing either human CEACAM5,
CEACAM1 or CEACAM6. Depicted are triplicates with SD. (B) Binding
of an anti-CD66 antibody to transient HEK293T transfectants shows
the transfection efficacy, respectively expression levels of the
three CEACAM family members. Depicted are MFI (median fluorescence
signals), based on triplicates and SD.
[0093] FIGS. 21A-C. Anti-tumor activity of CEA CD3 TCB molecule B
versus CEA CD3 TCB molecule X in the MKN45 model in fully humanized
NOG mice. Different doses and schedules of administration were
tested: (A) 2.5 mg/kg twice/week, (B) 2.5 mg/kg once/week, (C) 0.5
mg/kg once/week. Black arrow indicates start of therapy. Treatment
was administered for 9 weeks. *p<0.05 **p<0.01; ***p<0.001
Two-tailed unpaired t-test at study termination (day 70)
(8<n<9).
[0094] FIG. 22. Simulated PK of different dose levels and schedules
used in the dose-range efficacy study (Example 12). A 2-compartment
model was used and the simulation was based on the SDPK data.
[0095] FIGS. 23A-B. (A) Anti-tumor activity of CEA CD3 TCB molecule
B versus CEA CD3 TCB molecule X in the MKN45 model in fully
humanized NSG mice. Different doses and schedules of administration
were tested. Black arrow indicates start of therapy. Treatment was
administered for 4 weeks. (B) 2-way ANOVA multiple comparison
analysis at study termination (day 38). *p<0.05 **p<0.01;
***p<0.001; ****p<0.0001 (9<n<14).
[0096] FIG. 24. Anti-tumor activity of CEA CD3 TCB molecule B
versus CEA CD3 TCB molecule X (2.5 mg/kg, once a week) in the
HPAF-II model in NOG mice with huPBMC transfer. Grey arrow
indicates human PBMC (huPBMC) injection and black arrow indicates
start of therapy. Treatment was administered for 3 weeks.
*p<0.05; **p<0.01; ****p<0.0001 Two-tailed unpaired t-test
at study termination (day 32) (n=10).
[0097] FIG. 25. Comparison of pharmacokinetics of CEA CD3 TCB
molecule B and CEA CD3 TCB molecule X after iv bolus administration
in cynomolgus monkeys. The pharmacokinetics of individual
representative animals is depicted.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0098] Terms are used herein as generally used in the art, unless
otherwise defined in the following.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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..
[0104] 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. 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. An exemplary human protein
useful as antigen is CD3, particularly the epsilon subunit of CD3
(see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1,
SEQ ID NO: 1 for the human sequence; or UniProt no. Q95LI5 (version
49), NCBI GENBANK.RTM. no. BAB71849.1, SEQ ID NO: 2 for the
cynomolgus [Macaca fascicularis] sequence), or 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). In certain embodiments the T cell
activating bispecific antigen binding molecule of the invention
binds to an epitope of CD3 or CEA that is conserved among the CD3
or CEA antigens from different species.
[0105] 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.RTM.
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.-8 M or less, e.g. from 10.sup.-8 M to 10.sup.-13 M,
e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0106] "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).
[0107] "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.
[0108] An "activating T cell antigen" as used herein refers to an
antigenic determinant expressed on the surface of a T lymphocyte,
particularly a cytotoxic T lymphocyte, which is capable of inducing
T cell activation upon interaction with an antigen binding
molecule. Specifically, interaction of an antigen binding molecule
with an activating T cell antigen may induce T cell activation by
triggering the signaling cascade of the T cell receptor complex. In
a particular embodiment the activating T cell antigen is CD3,
particularly the epsilon subunit of CD3 (see UniProt no. P07766
(version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 1 for the
human sequence; or UniProt no. Q95LI5 (version 49), NCBI
GENBANK.RTM. no. BAB71849.1, SEQ ID NO: 2 for the cynomolgus
[Macaca fascicularis] sequence).
[0109] "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.
[0110] 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. In a particular embodiment, the target cell
antigen is CEA, particularly human CEA.
[0111] As used herein, the terms "first", "second" or "third" with
respect to Fab molecules 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] By a "crossover" Fab molecule (also termed "Crossfab") is
meant a Fab molecule wherein the variable domains or the constant
domains of the Fab heavy and light chain are exchanged (i.e.
replaced by each other), i.e. the crossover Fab molecule comprises
a peptide chain composed of the light chain variable domain VL and
the heavy chain constant domain 1 CH1 (VL-CH1, in N- to C-terminal
direction), and a peptide chain composed of the heavy chain
variable domain VH and the light chain constant domain CL (VH-CL,
in N- to C-terminal direction). For clarity, in a crossover Fab
molecule wherein the variable domains of the Fab light chain and
the Fab heavy chain are exchanged, the peptide chain comprising the
heavy chain constant domain 1 CH1 is referred to herein as the
"heavy chain" of the (crossover) Fab molecule. Conversely, in a
crossover Fab molecule wherein the constant domains of the Fab
light chain and the Fab heavy chain are exchanged, the peptide
chain comprising the heavy chain variable domain VH is referred to
herein as the "heavy chain" of the (crossover) Fab molecule.
[0116] 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 domains
(VH-CH1, in N- to C-terminal direction), and a light chain composed
of the light chain variable and constant domains (VL-CL, in N- to
C-terminal direction).
[0117] 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 domain (VH), also
called a variable heavy domain or a heavy chain variable region,
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 domain (VL), also called a variable
light domain or a light chain variable region, followed by a
constant light (CL) domain, also called a light chain constant
region.
[0118] The heavy chain of an immunoglobulin may be assigned to one
of five types, called a (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.
[0119] 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.
[0120] 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 B1). 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.
[0121] 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 domain (VL) and an antibody heavy chain variable
domain (VH).
[0122] 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.
[0123] 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., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991) 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. The CDR sequences given herein are
generally according to the Kabat definition.
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.
[0124] 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
in connection with variable region seqeunces, "Kabat numbering"
refers to the numbering system set forth by Kabat et al., Sequences
of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991).
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.
[0125] As used herein, the amino acid positions of all constant
regions and domains of the heavy and light chain are numbered
according to the Kabat numbering system described in Kabat, et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)
and is referred to as "numbering according to Kabat" or "Kabat
numbering" herein. Specifically the Kabat numbering system (see
pages 647-660 of Kabat, et al., Sequences of Proteins of
Immunological Interest, 5th ed., Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) is used for the light
chain constant domain CL of kappa and lambda isotype and the Kabat
EU index numbering system (see pages 661-723) is used for the heavy
chain constant domains (CH1, Hinge, CH2 and CH3), which is herein
further clarified by referring to "numbering according to Kabat EU
index" in this case.
[0126] 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.
[0127] "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.
[0128] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. Such variable domains are referred to
herein as "humanized variable region". A humanized antibody
optionally may comprise at least a portion of an antibody constant
region derived from a human antibody. A "humanized form" of an
antibody, e.g., a non-human antibody, refers to an antibody that
has undergone humanization. Other forms of "humanized antibodies"
encompassed by the present invention are those in which the
constant region has been additionally modified or changed from that
of the original antibody to generate the properties according to
the invention, especially in regard to C1q binding and/or Fc
receptor (FcR) binding.
[0129] 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.
[0130] 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, antibodies produced by host cells may undergo
post-translational cleavage of one or more, particularly one or
two, amino acids from the C-terminus of the heavy chain. Therefore
an antibody produced by a host cell by expression of a specific
nucleic acid molecule encoding a full-length heavy chain may
include the full-length heavy chain, or it may include a cleaved
variant of the full-length heavy chain (also referred to herein as
a "cleaved variant heavy chain"). This may be the case where the
final two C-terminal amino acids of the heavy chain are glycine
(G446) and lysine (K447, numbering according to Kabat EU index).
Therefore, the C-terminal lysine (Lys447), or the C-terminal
glycine (Gly446) and lysine (K447), of the Fc region may or may not
be present. Amino acid sequences of heavy chains including Fc
domains (or a subunit of an Fc domain as defined herein) are
denoted herein without C-terminal glycine-lysine dipeptide if not
indicated otherwise. In one embodiment of the invention, a heavy
chain including a subunit of an Fc domain as specified herein,
comprised in a T cell activating bispecific antigen binding
molecule according to the invention, comprises an additional
C-terminal glycine-lysine dipeptide (G446 and K447, numbering
according to EU index of Kabat). In one embodiment of the
invention, a heavy chain including a subunit of an Fc domain as
specified herein, comprised in a T cell activating bispecific
antigen binding molecule according to the invention, comprises an
additional C-terminal glycine residue (G446, numbering according to
EU index of Kabat). Compositions of the invention, such as the
pharmaceutical compositions described herein, comprise a population
of T cell activating bispecific antigen binding molecules of the
invention. The population of T cell activating bispecific antigen
binding molecule may comprise molecules having a full-length heavy
chain and molecules having a cleaved variant heavy chain. The
population of T cell activating bispecific antigen binding
molecules may consist of a mixture of molecules having a
full-length heavy chain and molecules having a cleaved variant
heavy chain, wherein at least 50%, at least 60%, at least 70%, at
least 80% or at least 90% of the T cell activating bispecific
antigen binding molecules have a cleaved variant heavy chain. In
one embodiment of the invention a composition comprising a
population of T cell activating bispecific antigen binding
molecules of the invention comprises an T cell activating
bispecific antigen binding molecule comprising a heavy chain
including a subunit of an Fc domain as specified herein with an
additional C-terminal glycine-lysine dipeptide (G446 and K447,
numbering according to EU index of Kabat). In one embodiment of the
invention a composition comprising a population of T cell
activating bispecific antigen binding molecules of the invention
comprises an T cell activating bispecific antigen binding molecule
comprising a heavy chain including a subunit of an Fc domain as
specified herein with an additional C-terminal glycine residue
(G446, numbering according to EU index of Kabat). In one embodiment
of the invention such a composition comprises a population of T
cell activating bispecific antigen binding molecules comprised of
molecules comprising a heavy chain including a subunit of an Fc
domain as specified herein; molecules comprising a heavy chain
including a subunit of a Fc domain as specified herein with an
additional C-terminal glycine residue (G446, numbering according to
EU index of Kabat); and molecules comprising a heavy chain
including a subunit of an Fc domain as specified herein with an
additional C-terminal glycine-lysine dipeptide (G446 and K447,
numbering according to EU index of Kabat). 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 (see also
above). 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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,
G.sub.329, P329G, or Pro329Gly.
[0135] 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.
[0136] 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.
[0137] "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
receding paragraph using the ALIGN-2 computer program.
[0138] 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.
[0139] 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. 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).
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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).
[0144] 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).
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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
[0152] The invention provides a T cell activating bispecific
antigen binding molecule with favorable properties for therapeutic
application, in particular with improved efficacy and safety (e.g.
with respect to unspecific activation of T cells or selectivity
towards tumor cells over normal cells), and improved produceability
(e.g. with respect to purity, yield).
[0153] The inventors have discovered that T cell activating
bispecific antigen binding molecules comprising an antigen binding
moiety with the binding specificity of the anti-CEA antibody T84.66
(Wagener et al., J Immunol 130, 2308-(1983), Neumaier et al., J
Immunol 135, 3604 (1985)) provide unexpectedly high potency in
mediating killing of CEA-expressing tumor cells by T cells.
Moreover, T cell activating bispecific antigen binding molecules
comprising a novel humanized version of antibody T84.66 were
surprisingly found to exhibit improved selectivity towards tumor
cells over normal cells as compared to a T cell activating
bispecific antigen binding molecule comprising the parental T84.66
binder.
Charge Modifications
[0154] The T cell activating bispecific antigen binding molecules
of the invention may comprise amino acid substitutions in Fab
molecules comprised therein which are particularly efficient in
reducing mispairing of light chains with non-matching heavy chains
(Bence-Jones-type side products), which can occur in the production
of Fab-based bi-/multispecific antigen binding molecules with a
VH/VL exchange in one (or more, in case of molecules comprising
more than two antigen-binding Fab molecules) of their binding arms
(see also PCT publication no. WO 2015/150447, particularly the
examples therein, incorporated herein by reference in its
entirety).
[0155] Accordingly, in particular embodiments, the T cell
activating bispecific antigen binding molecule of the invention
comprises
(a) a first Fab molecule which specifically binds to a first
antigen (b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each other,
wherein the first antigen is an activating T cell antigen and the
second antigen is CEA, or the first antigen is CEA and the second
antigen is an activating T cell antigen; and wherein [0156] i) in
the constant domain CL of the first Fab molecule under a) the amino
acid at position 124 is substituted by a positively charged amino
acid (numbering according to Kabat), and wherein in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 or the amino acid at position 213 is substituted by a
negatively charged amino acid (numbering according to Kabat EU
index); or [0157] ii) in the constant domain CL of the second Fab
molecule under b) the amino acid at position 124 is substituted by
a positively charged amino acid (numbering according to Kabat), and
wherein in the constant domain CH1 of the second Fab molecule under
b) the amino acid at position 147 or the amino acid at position 213
is substituted by a negatively charged amino acid (numbering
according to Kabat EU index).
[0158] The T cell activating bispecific antigen binding molecule
does not comprise both modifications mentioned under i) and ii).
The constant domains CL and CH1 of the second Fab molecule are not
replaced by each other (i.e. remain unexchanged).
[0159] In one embodiment of the T cell activating bispecific
antigen binding molecule according to the invention, in the
constant domain CL of the first Fab molecule under a) the amino
acid at position 124 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat) (in
one preferred embodiment independently by lysine (K) or arginine
(R)), and in the constant domain CH1 of the first Fab molecule
under a) the amino acid at position 147 or the amino acid at
position 213 is substituted independently by glutamic acid (E), or
aspartic acid (D) (numbering according to Kabat EU index).
[0160] In a further embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and in the constant domain CH1
of the first Fab molecule under a) the amino acid at position 147
is substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0161] In a particular embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in one preferred embodiment
independently by lysine (K) or arginine (R)) and the amino acid at
position 123 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) (in one
preferred embodiment independently by lysine (K) or arginine (R)),
and in the constant domain CH1 of the first Fab molecule under a)
the amino acid at position 147 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0162] In a more particular embodiment, in the constant domain CL
of the first Fab molecule under a) the amino acid at position 124
is substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by lysine (K) or arginine
(R) (numbering according to Kabat), and in the constant domain CH1
of the first Fab molecule under a) the amino acid at position 147
is substituted by glutamic acid (E) (numbering according to Kabat
EU index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index).
[0163] In an even more particular embodiment, in the constant
domain CL of the first Fab molecule under a) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by
arginine (R) (numbering according to Kabat), and in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 is substituted by glutamic acid (E) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index).
[0164] In particular embodiments, the constant domain CL of the
first Fab molecule under a) is of kappa isotype.
[0165] Alternatively, the amino acid substitutions according to the
above embodiments may be made in the constant domain CL and the
constant domain CH1 of the second Fab molecule under b) instead of
in the constant domain CL and the constant domain CH1 of the first
Fab molecule under a). In particular such embodiments, the constant
domain CL of the second Fab molecule under b) is of kappa
isotype.
[0166] The T cell activating bispecific antigen binding molecule
according to the invention may further comprise a third Fab
molecule which specifically binds to the first antigen. In
particular embodiments, said third Fab molecule is identical to the
first Fab molecule under a). In these embodiments, the amino acid
substitutions according to the above embodiments will be made in
the constant domain CL and the constant domain CH1 of each of the
first Fab molecule and the third Fab molecule. Alternatively, the
amino acid substitutions according to the above embodiments may be
made in the constant domain CL and the constant domain CH1 of the
second Fab molecule under b), but not in the constant domain CL and
the constant domain CH1 of the first Fab molecule and the third Fab
molecule.
[0167] In particular embodiments, the T cell activating bispecific
antigen binding molecule according to the invention further
comprises an Fc domain composed of a first and a second subunit
capable of stable association.
T Cell Activating Bispecific Antigen Binding Molecule Formats
[0168] 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 FIG.
1.
[0169] In particular embodiments, the antigen binding moieties
comprised in the T cell activating bispecific antigen binding
molecule are Fab molecules. In such embodiments, the first, second,
third etc. antigen binding moiety may be referred to herein as
first, second, third etc. Fab molecule, respectively. Furthermore,
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.
[0170] In some embodiments, the second Fab 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.
[0171] In one such embodiment, the first Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule. In a specific such
embodiment, the T cell activating bispecific antigen binding
molecule essentially consists of the first and the second Fab
molecule, the Fc domain composed of a first and a second subunit,
and optionally one or more peptide linkers, wherein the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule, and
the second Fab 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. Such a configuration is schematically depicted in FIGS.
1G and 1K. Optionally, the Fab light chain of the first Fab
molecule and the Fab light chain of the second Fab molecule may
additionally be fused to each other.
[0172] In another such embodiment, the first Fab molecule 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 the first and the second Fab
molecule, the Fc domain composed of a first and a second subunit,
and optionally one or more peptide linkers, wherein the first and
the second Fab 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. Such a configuration is schematically depicted in FIGS. 1A
and 1D. The first and the second Fab molecule may be fused to the
Fc domain directly or through a peptide linker. In a particular
embodiment the first and the second Fab molecule 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, particularly where the Fc domain is an IgG.sub.1 Fc
domain.
[0173] In other embodiments, the first Fab molecule 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.
[0174] In one such embodiment, the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule. In a specific such
embodiment, the T cell activating bispecific antigen binding
molecule essentially consists of the first and the second Fab
molecule, the Fc domain composed of a first and a second subunit,
and optionally one or more peptide linkers, wherein the second Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first Fab molecule, and
the first Fab 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. Such a configuration is schematically depicted in FIGS.
1H and 1L. Optionally, the Fab light chain of the first Fab
molecule and the Fab light chain of the second Fab molecule may
additionally be fused to each other.
[0175] The Fab molecules 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 an
integer from 1 to 10, typically from 2 to 4. In one embodiment said
peptide linker has a length of at least 5 amino acids, in one
embodiment a length of 5 to 100, in a further embodiment of 10 to
50 amino acids. In one embodiment said peptide linker is
(GxS).sub.n or (GxS).sub.nG.sub.m with G=glycine, S=serine, and
(x=3, n=3, 4, or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and
m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a further
embodiment x=4 and n=2. In one embodiment said peptide linker is
(G.sub.4S).sub.2. A particularly suitable peptide linker for fusing
the Fab light chains of the first and the second Fab molecule 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 Fab fragments comprises the sequence (D)-(G.sub.4S).sub.2
(SEQ ID NOs 11 and 12). Another suitable such linker comprises the
sequence (G.sub.4S).sub.4. Additionally, linkers may comprise (a
portion of) an immunoglobulin hinge region. Particularly where a
Fab molecule is fused to the N-terminus of an Fe domain subunit, it
may be fused via an immunoglobulin hinge region or a portion
thereof, with or without an additional peptide linker.
[0176] A T cell activating bispecific antigen binding molecule with
a single antigen binding moiety (such as a Fab molecule) capable of
specific binding to a target cell antigen (for example as shown in
FIG. 1A, D, G, H, K, L) 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 availablity.
[0177] 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 (such as Fab
moelcules) specific for a target cell antigen (see examples shown
in FIG. 1B, 1C, 1E, 1F, 1I, 1J, 1M or 1N), for example to optimize
targeting to the target site or to allow crosslinking of target
cell antigens.
[0178] Accordingly, in particular embodiments, the T cell
activating bispecific antigen binding molecule of the invention
further comprises a third Fab molecule which specifically binds to
the first antigen. The first antigen preferably is the target cell
antigen, i.e. CEA. In one embodiment, the third Fab molecule is a
conventional Fab molecule. In one embodiment, the third Fab
molecule is identical to the first Fab molecule (i.e. the first and
the third Fab molecule comprise the same heavy and light chain
amino acid sequences and have the same arrangement of domains (i.e.
conventional or crossover)). In a particular embodiment, the second
Fab molecule specifically binds to an activating T cell antigen,
particularly CD3, and the first and third Fab molecule specifically
bind to CEA.
[0179] In alternative embodiments, the T cell activating bispecific
antigen binding molecule of the invention further comprises a third
Fab molecule which specifically binds to the second antigen. In
these embodiments, the second antigen preferably is the target cell
antigen, i.e. CEA. In one such embodiment, the third Fab molecule
is a crossover Fab molecule (a Fab molecule wherein the variable
domains VH and VL or the constant domains CL and CH1 of the Fab
heavy and light chains are exchanged/replaced by each other). In
one such embodiment, the third Fab molecule is identical to the
second Fab molecule (i.e. the second and the third Fab molecule
comprise the same heavy and light chain amino acid sequences and
have the same arrangement of domains (i.e. conventional or
crossover)). In one such embodiment, the first Fab molecule
specifically binds to an activating T cell antigen, particularly
CD3, and the second and third Fab molecule specifically bind to
CEA.
[0180] In one embodiment, the third Fab molecule 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.
[0181] In a particular embodiment, the second and the third Fab
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 Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second Fab
molecule. In a specific such embodiment, the T cell activating
bispecific antigen binding molecule essentially consists of the
first, the second and the third Fab molecule, the Fc domain
composed of a first and a second subunit, and optionally one or
more peptide linkers, wherein the first Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule, and the second Fab molecule
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 Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the second subunit of the Fc domain. Such a
configuration is schematically depicted in FIGS. 1B and 1E
(particular embodiments, wherein the third Fab molecule is a
conventional Fab molecule and preferably identical to the first Fab
molecule), and FIGS. 1I and 1M (alternative embodiments, wherein
the third Fab molecule is a crossover Fab molecule and preferably
identical to the second Fab molecule). The second and the third Fab
molecule may be fused to the Fc domain directly or through a
peptide linker. In a particular embodiment the second and the third
Fab molecule 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,
particularly where the Fc domain is an IgG.sub.1 Fc domain.
Optionally, the Fab light chain of the first Fab molecule and the
Fab light chain of the second Fab molecule may additionally be
fused to each other.
[0182] In another embodiment, the first and the third Fab 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
Fab molecule is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the Fab heavy chain of the first Fab molecule. In
a specific such embodiment, the T cell activating bispecific
antigen binding molecule essentially consists of the first, the
second and the third Fab molecule, the Fc domain composed of a
first and a second subunit, and optionally one or more peptide
linkers, wherein the second Fab molecule is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the Fab heavy chain of
the first Fab molecule, and the first Fab molecule 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 Fab molecule is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the second subunit of the Fc domain. Such a configuration is
schematically depicted in FIGS. 1C and 1F (particular embodiments,
wherein the third Fab molecule is a conventional Fab molecule and
preferably identical to the first Fab molecule) and in FIGS. 1J and
1N (alternative embodiments, wherein the third Fab molecule is a
crossover Fab molecule and preferably identical to the second Fab
molecule). The first and the third Fab molecule may be fused to the
Fc domain directly or through a peptide linker. In a particular
embodiment the first and the third Fab molecule 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, particularly where the Fc domain is an IgG.sub.1 Fc
domain. Optionally, the Fab light chain of the first Fab molecule
and the Fab light chain of the second Fab molecule may additionally
be fused to each other.
[0183] In configurations of the T cell activating bispecific
antigen binding molecule wherein a Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of each of the
subunits of the Fc domain through an immunoglobulin hinge regions,
the two Fab molecules, the hinge regions and the Fc domain
essentially form 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.
[0184] In some of the T cell activating bispecific antigen binding
molecule of the invention, the Fab light chain of the first Fab
molecule and the Fab light chain of the second Fab molecule are
fused to each other, optionally via a peptide linker. Depending on
the configuration of the first and the second Fab molecule, the Fab
light chain of the first Fab molecule may be fused at its
C-terminus to the N-terminus of the Fab light chain of the second
Fab molecule, or the Fab light chain of the second Fab molecule may
be fused at its C-terminus to the N-terminus of the Fab light chain
of the first Fab molecule. Fusion of the Fab light chains of the
first and the second Fab molecule 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.
[0185] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab light chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab heavy chain constant region of the second Fab molecule (i.e.
the second Fab molecule 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.(2)-CH1.sub.(2)-CH2-CH3(-CH4)), and a polypeptide wherein
the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-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 second Fab molecule shares a carboxy-terminal peptide bond
with the Fab light chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In certain embodiments
the polypeptides are covalently linked, e.g., by a disulfide
bond.
[0186] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab light chain constant region of the second Fab molecule (i.e.
the second Fab molecule 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)-CL.sub.(2)-CH2-CH3(-CH4)), and a polypeptide wherein
the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-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 second Fab molecule shares a carboxy-terminal peptide bond
with the Fab heavy chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In certain embodiments
the polypeptides are covalently linked, e.g., by a disulfide
bond.
[0187] In some embodiments, the T cell activating bispecific
antigen binding molecule comprises a polypeptide wherein the Fab
light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
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 first Fab molecule, which in turn shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VL.sub.(2)-CH1.sub.(2)-VH.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
first Fab molecule shares a carboxy-terminal peptide bond with the
Fab light chain variable region of the second Fab molecule which in
turn shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule (i.e. the second
Fab molecule 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.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)).
[0188] In some of these embodiments the T cell activating
bispecific antigen binding molecule further comprises a crossover
Fab light chain polypeptide of the second Fab molecule, wherein the
Fab heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)), and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In others of these embodiments the T cell
activating bispecific antigen binding molecule further comprises a
polypeptide wherein the Fab heavy chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab light chain constant region of the second Fab molecule which in
turn shares a carboxy-terminal peptide bond with the Fab light
chain polypeptide of the first Fab molecule
(VH.sub.(2)-CL.sub.(2)-VL.sub.(1)-CL.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule
(VL.sub.(1)-CL.sub.(1)-VH.sub.(2)-CL.sub.(2)), as appropriate.
[0189] 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 Fab molecule 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 Fab molecule (VL.sub.(3)-CL.sub.(3). In
certain embodiments the polypeptides are covalently linked, e.g.,
by a disulfide bond.
[0190] In some embodiments, the T cell activating bispecific
antigen binding molecule comprises a polypeptide wherein the Fab
heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (i.e. the second Fab molecule
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 first Fab molecule, which in turn shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CL.sub.(2)-VH.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
first Fab molecule shares a carboxy-terminal peptide bond with the
Fab heavy chain variable region of the second Fab molecule which in
turn shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule (i.e. the second
Fab molecule 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)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)-CH2-CH3(-CH4)).
[0191] In some of these embodiments the T cell activating
bispecific antigen binding molecule further comprises a crossover
Fab light chain polypeptide of the second Fab molecule, wherein the
Fab light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)), and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In 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
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab heavy chain constant region of the second Fab molecule which in
turn shares a carboxy-terminal peptide bond with the Fab light
chain polypeptide of the first Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VL.sub.(1)-CL.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule
(VL.sub.(1)-CL.sub.(1)-VH.sub.(2)-CL.sub.(2)), as appropriate.
[0192] 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 Fab molecule 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 Fab molecule (VL.sub.(3)-CL.sub.(3). In
certain embodiments the polypeptides are covalently linked, e.g.,
by a disulfide bond.
[0193] In some embodiments, the first Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule. In certain such
embodiments, the T cell activating bispecific antigen binding
molecule does not comprise an Fc domain. In certain embodiments,
the T cell activating bispecific antigen binding molecule
essentially consists of the first and the second Fab molecule, and
optionally one or more peptide linkers, wherein the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule. Such
a configuration is schematically depicted in FIGS. 1O and 1S.
[0194] In other embodiments, the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule. In certain such embodiments,
the T cell activating bispecific antigen binding molecule does not
comprise an Fc domain. In certain embodiments, the T cell
activating bispecific antigen binding molecule essentially consists
of the first and the second Fab molecule, and optionally one or
more peptide linkers, wherein the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule. Such a configuration is
schematically depicted in FIGS. 1P and 1T. In some embodiments, the
first Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second Fab
molecule, and the T cell activating bispecific antigen binding
molecule further comprises a third Fab molecule, wherein said third
Fab molecule is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the Fab heavy chain of the first Fab molecule. In
particular such embodiments, said third Fab molecule is a
conventional Fab molecule. In other such embodiments, said third
Fab molecule is a crossover Fab molecule as described herein, i.e.
a Fab molecule wherein the variable domains VH and VL or the
constant domains CL and CH1 of the Fab heavy and light chains are
exchanged/replaced by each other. In certain such embodiments, the
T cell activating bispecific antigen binding molecule essentially
consists of the first, the second and the third Fab molecule, and
optionally one or more peptide linkers, wherein the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule, and
the third Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first Fab
molecule. Such a configuration is schematically depicted in FIGS.
1Q and 1U (particular embodiments, wherein the third Fab molecule
is a conventional Fab molecule and preferably identical to the
first Fab molecule).
[0195] In some embodiments, the first Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule, and the T cell activating
bispecific antigen binding molecule further comprises a third Fab
molecule, wherein said third Fab molecule is fused at the
N-terminus of the Fab heavy chain to the C-terminus of the Fab
heavy chain of the second Fab molecule. In particular such
embodiments, said third Fab molecule is a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CH1 and CL of the Fab heavy and
light chains are exchanged/replaced by each other. In other such
embodiments, said third Fab molecule is a conventional Fab
molecule. In certain such embodiments, the T cell activating
bispecific antigen binding molecule essentially consists of the
first, the second and the third Fab molecule, and optionally one or
more peptide linkers, wherein the first Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule, and the third Fab molecule
is fused at the N-terminus of the Fab heavy chain to the C-terminus
of the Fab heavy chain of the second Fab molecule. Such a
configuration is schematically depicted in FIGS. 1W and 1Y
(particular embodiments, wherein the third Fab molecule is a
crossover Fab molecule and preferably identical to the second Fab
molecule).
[0196] In some embodiments, the second Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the T cell activating
bispecific antigen binding molecule further comprises a third Fab
molecule, wherein said third Fab molecule is fused at the
N-terminus of the Fab heavy chain to the C-terminus of the Fab
heavy chain of the first Fab molecule. In particular such
embodiments, said third Fab molecule is a conventional Fab
molecule. In other such embodiments, said third Fab molecule is a
crossover Fab molecule as described herein, i.e. a Fab molecule
wherein the variable domains VH and VL or the constant domains CH1
and CL of the Fab heavy and light chains are exchanged/replaced by
each other. In certain such embodiments, the T cell activating
bispecific antigen binding molecule essentially consists of the
first, the second and the third Fab molecule, and optionally one or
more peptide linkers, wherein the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the third Fab molecule
is fused at the N-terminus of the Fab heavy chain to the C-terminus
of the Fab heavy chain of the first Fab molecule. Such a
configuration is schematically depicted in FIGS. 1R and 1V
(particular embodiments, wherein the third Fab molecule is a
conventional Fab molecule and preferably identical to the first Fab
molecule).
[0197] In some embodiments, the second Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the T cell activating
bispecific antigen binding molecule further comprises a third Fab
molecule, wherein said third Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule. In particular such
embodiments, said third Fab molecule is a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CH1 and CL of the Fab heavy and
light chains are exchanged/replaced by each other. In other such
embodiments, said third Fab molecule is a conventional Fab
molecule. In certain such embodiments, the T cell activating
bispecific antigen binding molecule essentially consists of the
first, the second and the third Fab molecule, and optionally one or
more peptide linkers, wherein the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the third Fab molecule
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the Fab heavy chain of the second Fab molecule. Such a
configuration is schematically depicted in FIGS. 1X and 1Z
(particular embodiments, wherein the third Fab molecule is a
crossover Fab molecule and preferably identical to the first Fab
molecule).
[0198] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
variable region of the second Fab molecule, which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
variable region is replaced by a light chain variable region)
(VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)). In some
embodiments the T cell activating bispecific antigen binding
molecule further comprises a polypeptide wherein the Fab heavy
chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)).
[0199] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab light chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab heavy chain constant region of the second Fab molecule (i.e.
the second Fab molecule 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 first Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)). In some
embodiments the T cell activating bispecific antigen binding
molecule further comprises a polypeptide wherein the Fab heavy
chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)).
[0200] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab light chain constant region of the second Fab molecule (i.e.
the second Fab molecule 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 first Fab molecule
(VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)). In some embodiments
the T cell activating bispecific antigen binding molecule further
comprises a polypeptide wherein the Fab light chain variable region
of the second Fab molecule shares a carboxy-terminal peptide bond
with the Fab heavy chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)).
[0201] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain of
the first Fab molecule, which in turn shares a carboxy-terminal
peptide bond with the Fab light chain variable region of the second
Fab molecule, which in turn shares a carboxy-terminal peptide bond
with the Fab heavy chain constant region of the second Fab molecule
(i.e. the second Fab molecule comprises a crossover Fab heavy
chain, wherein the heavy chain variable region is replaced by a
light chain variable region)
(VH.sub.(3)-CH1.sub.(3)-VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises the Fab light
chain polypeptide of a third Fab molecule
(VL.sub.(3)-CL.sub.(3).
[0202] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain of
the first Fab molecule, which in turn shares a carboxy-terminal
peptide bond with the Fab heavy chain variable region of the second
Fab molecule, which in turn shares a carboxy-terminal peptide bond
with the Fab light chain constant region of the second Fab molecule
(i.e. the second Fab molecule comprises a crossover Fab heavy
chain, wherein the heavy chain constant region is replaced by a
light chain constant region)
(VH.sub.(3)-CH1.sub.(3)-VH.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises the Fab light
chain polypeptide of a third Fab molecule
(VL.sub.(3)-CL.sub.(3).
[0203] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab light chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab heavy chain constant region of the second Fab molecule (i.e.
the second Fab molecule 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 first Fab molecule,
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain of a third Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)-VH.sub.(3)-CH1.sub.(3)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises the Fab light
chain polypeptide of a third Fab molecule
(VL.sub.(3)-CL.sub.(3).
[0204] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab light chain constant region of the second Fab molecule (i.e.
the second Fab molecule 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 first Fab molecule,
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain of a third Fab molecule
(VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)-VH.sub.(3)-CH1.sub.(3)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises the Fab light
chain polypeptide of a third Fab molecule
(VL.sub.(3)-CL.sub.(3)).
[0205] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
variable region of the second Fab molecule, which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
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
light chain variable region of a third Fab molecule, which in turn
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of a third Fab molecule (i.e. the third Fab
molecule comprises a crossover Fab heavy chain, wherein the heavy
chain variable region is replaced by a light chain variable region)
(VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)-VL.sub.(3)-CH1.sub.(3)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises a polypeptide
wherein the Fab heavy chain variable region of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of a third Fab molecule
(VH.sub.(3)-CL.sub.(3)).
[0206] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule, which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (i.e. the second Fab molecule
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 variable region of a third Fab molecule, which in turn
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of a third Fab molecule (i.e. the third Fab
molecule comprises a crossover Fab heavy chain, wherein the heavy
chain constant region is replaced by a light chain constant region)
(VH.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)-VH.sub.(3)-CL.sub.(3).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises a polypeptide
wherein the Fab light chain variable region of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of a third Fab molecule
(VL.sub.(3)-CH1.sub.(3)).
[0207] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab light chain variable region of a third
Fab molecule shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of a third Fab molecule (i.e. the third
Fab molecule 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 light chain variable region of the second Fab molecule,
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of the second Fab molecule (i.e. the
second Fab molecule 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 first Fab molecule
(VL.sub.(3)-CH1.sub.(3)-VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises a polypeptide
wherein the Fab heavy chain variable region of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of a third Fab molecule
(VH.sub.(3)-CL.sub.(3)).
[0208] In certain embodiments the T cell activating bispecific
antigen binding molecule according to the invention comprises a
polypeptide wherein the Fab heavy chain variable region of a third
Fab molecule shares a carboxy-terminal peptide bond with the Fab
light chain constant region of a third Fab molecule (i.e. the third
Fab molecule 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 variable region of the second Fab molecule,
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the second Fab molecule (i.e. the
second Fab molecule 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 first Fab molecule
(VH.sub.(3)-CL.sub.(3)-VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)).
In some embodiments the T cell activating bispecific antigen
binding molecule further comprises a polypeptide wherein the Fab
light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In some embodiments the T cell activating
bispecific antigen binding molecule further comprises a polypeptide
wherein the Fab light chain variable region of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of a third Fab molecule
(VL.sub.(3)-CH1.sub.(3)).
[0209] According to any of the above embodiments, components of the
T cell activating bispecific antigen binding molecule (e.g. Fab
molecules, 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 an integer from 1 to 10, typically from 2 to
4.
Fc Domain
[0210] 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 Fe 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.
[0211] 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 S228 (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: 13.
Fc Domain Modifications Promoting Heterodimerization
[0212] T cell activating bispecific antigen binding molecules
according to the invention comprise different Fab molecules, 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.
[0213] 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.
[0214] There exist several approaches for modifications in the CH3
domain of the Fc domain in order to enforce heterodimerization,
which are well described e.g. in WO 96/27011, WO 98/050431, EP
1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO
2013157954, WO 2013096291. Typically, in all such approaches the
CH3 domain of the first subunit of the Fc domain and the CH3 domain
of the second subunit of the Fc domain are both engineered in a
complementary manner so that each CH3 domain (or the heavy chain
comprising it) can no longer homodimerize with itself but is forced
to heterodimerize with the complementarily engineered other CH3
domain (so that the first and second CH3 domain heterodimerize and
no homdimers between the two first or the two second CH3 domains
are formed). These different approaches for improved heavy chain
heterodimerization are contemplated as different alternatives in
combination with the heavy-light chain modifications (VH and VL
exchange/replacement in one binding arm and the introduction of
substitutions of charged amino acids with opposite charges in the
CH1/CL interface) in the T cell activating bispecific antigen
binding molecule according to the invention which reduce light
chain mispairing and Bence Jones-type side products.
[0215] In a specific embodiment said modification promoting the
association of the first and the second subunit of the Fc domain 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.
[0216] 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). 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.
[0217] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), and tryptophan (W).
[0218] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), and valine (V).
[0219] 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.
[0220] In a specific embodiment, in the CH3 domain of the first
subunit of the Fc domain (the "knobs" subunit) 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 "hole" subunit) 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) (numberings according to Kabat EU index).
[0221] 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) or the glutamic acid residue at
position 356 is replaced with a cysteine residue (E356C), and in
the second subunit of the Fc domain additionally the tyrosine
residue at position 349 is replaced by a cysteine residue (Y349C)
(numberings according to Kabat EU index). 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)).
[0222] In a particular embodiment, the first subunit of the Fc
domain comprises amino acid substitutions S354C and T366W, and the
second subunit of the Fc domain comprises amino acid substitutions
Y349C, T366S, L368A and Y407V (numbering according to Kabat EU
index).
[0223] In a particular embodiment the Fab molecule which
specifically binds an activating T cell antigen is fused
(optionally via a Fab molecule which specifically binds 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 Fab molecule which specifically binds an activating T cell
antigen to the knob-containing subunit of the Fc domain will
(further) minimize the generation of antigen binding molecules
comprising two Fab molecules which bind to an activating T cell
antigen (steric clash of two knob-containing polypeptides).
[0224] Other techniques of CH3-modification for enforcing the
heterodimerization are contemplated as alternatives according to
the invention and are described e.g. in WO 96/27011, WO 98/050431,
EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO
2013/157954, WO 2013/096291.
[0225] In one embodiment the heterodimerization approach described
in EP 1870459 A1, is used alternatively. This approach is based on
the introduction of charged amino acids with opposite charges at
specific amino acid positions in the CH3/CH3 domain interface
between the two subunits of the Fc domain. One preferred embodiment
for the T cell activating bispecific antigen binding molecule of
the invention are amino acid mutations R409D; K370E in one of the
two CH3 domains (of the Fc domain) and amino acid mutations D399K;
E357K in the other one of the CH3 domains of the Fc domain
(numbering according to Kabat EU index).
[0226] In another embodiment the T cell activating bispecific
antigen binding molecule of the invention comprises amino acid
mutation T366W in the CH3 domain of the first subunit of the Fc
domain and amino acid mutations T366S, L368A, Y407V in the CH3
domain of the second subunit of the Fc domain, and additionally
amino acid mutations R409D; K370E in the CH3 domain of the first
subunit of the Fc domain and amino acid mutations D399K; E357K in
the CH3 domain of the second subunit of the Fc domain (numberings
according to Kabat EU index).
[0227] In another embodiment T cell activating bispecific antigen
binding molecule of the invention comprises amino acid mutations
S354C, T366W in the CH3 domain of the first subunit of the Fc
domain and amino acid mutations Y349C, T366S, L368A, Y407V in the
CH3 domain of the second subunit of the Fc domain, or said T cell
activating bispecific antigen binding molecule comprises amino acid
mutations Y349C, T366W in the CH3 domain of the first subunit of
the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V
in the CH3 domains of the second subunit of the Fc domain and
additionally amino acid mutations R409D; K370E in the CH3 domain of
the first subunit of the Fc domain and amino acid mutations D399K;
E357K in the CH3 domain of the second subunit of the Fc domain (all
numberings according to Kabat EU index).
[0228] In one embodiment the heterodimerization approach described
in WO 2013/157953 is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutation T366K and a second CH3
domain comprises amino acid mutation L351D (numberings according to
Kabat EU index). In a further embodiment the first CH3 domain
comprises further amino acid mutation L351K. In a further
embodiment the second CH3 domain comprises further an amino acid
mutation selected from Y349E, Y349D and L368E (preferably L368E)
(numberings according to Kabat EU index).
[0229] In one embodiment the heterodimerization approach described
in WO 2012/058768 is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutations L351Y, Y407A and a second
CH3 domain comprises amino acid mutations T366A, K409F. In a
further embodiment the second CH3 domain comprises a further amino
acid mutation at position T411, D399, S400, F405, N390, or K392,
e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or
T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or
S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R,
N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E
(numberings according to Kabat EU index). In a further embodiment a
first CH3 domain comprises amino acid mutations L351Y, Y407A and a
second CH3 domain comprises amino acid mutations T366V, K409F. In a
further embodiment a first CH3 domain comprises amino acid mutation
Y407A and a second CH3 domain comprises amino acid mutations T366A,
K409F. In a further embodiment the second CH3 domain further
comprises amino acid mutations K392E, T411E, D399R and S400R
(numberings according to Kabat EU index).
[0230] In one embodiment the heterodimerization approach described
in WO 2011/143545 is used alternatively, e.g. with the amino acid
modification at a position selected from the group consisting of
368 and 409 (numbering according to Kabat EU index).
[0231] In one embodiment the heterodimerization approach described
in WO 2011/090762, which also uses the knobs-into-holes technology
described above, is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutation T366W and a second CH3
domain comprises amino acid mutation Y407A. In one embodiment a
first CH3 domain comprises amino acid mutation T366Y and a second
CH3 domain comprises amino acid mutation Y407T (numberings
according to Kabat EU index).
[0232] In one embodiment the T cell activating bispecific antigen
binding molecule or its Fc domain is of IgG.sub.2 subclass and the
heterodimerization approach described in WO 2010/129304 is used
alternatively.
[0233] 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 no. 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. In
one such embodiment a first CH3 domain comprises amino acid
substitution of K392 or N392 with a negatively charged amino acid
(e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or
N392D) and a second CH3 domain comprises amino acid substitution of
D399, E356, D356, or E357 with a positively charged amino acid
(e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K,
or E357K, and more preferably D399K and E356K). In a further
embodiment the first CH3 domain further comprises amino acid
substitution of K409 or R409 with a negatively charged amino acid
(e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or
R409D). In a further embodiment the first CH3 domain further or
alternatively comprises amino acid substitution of K439 and/or K370
with a negatively charged amino acid (e.g. glutamic acid (E), or
aspartic acid (D)) (all numberings according to Kabat EU
index).
[0234] In yet a further embodiment the heterodimerization approach
described in WO 2007/147901 is used alternatively. In one
embodiment a first CH3 domain comprises amino acid mutations K253E,
D282K, and K322D and a second CH3 domain comprises amino acid
mutations D239K, E240K, and K292D (numberings according to Kabat EU
index).
[0235] In still another embodiment the heterodimerization approach
described in WO 2007/110205 can be used alternatively.
[0236] In one embodiment, the first subunit of the Fc domain
comprises amino acid substitutions K392D and K409D, and the second
subunit of the Fc domain comprises amino acid substitutions D356K
and D399K (numbering according to Kabat EU index).
Fc Domain Modifications Reducing Fc Receptor Binding and/or
Effector Function
[0237] 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.
[0238] 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
Fc.gamma. 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.
[0239] In certain embodiments the Fe 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
Fc.gamma. 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).
[0240] 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 (numberings
according to Kabat EU index). 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 (numberings according to
Kabat EU index). In some embodiments the Fc domain comprises the
amino acid substitutions L234A and L235A (numberings according to
Kabat EU index). 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 (numberings
according to Kabat EU index). 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 (numberings according to Kabat EU index). 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 (numberings according to Kabat EU
index). 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.
[0241] 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 S228, specifically the amino acid
substitution S228P (numberings according to Kabat EU index). 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 (numberings according to Kabat EU
index). In another embodiment, the IgG.sub.4 Fc domain comprises an
amino acid substitution at position P329, specifically the amino
acid substitution P329G (numberings according to Kabat EU index).
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 (numberings
according to Kabat EU index). Such IgG.sub.4 Fc domain mutants and
their Fc receptor binding properties are described in PCT
publication no. WO 2012/130831, incorporated herein by reference in
its entirety. 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 (numberings according to Kabat EU index).
[0242] 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) (numberings according to Kabat EU index).
[0243] 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) (numberings
according to Kabat EU index). 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).
[0244] Mutant Fe 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.
[0245] 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.RTM. 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.
[0246] 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 (PBMC) 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).
[0247] 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
[0248] 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 particular embodiments of 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 domain). 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 domains.
[0249] Preferably, at least one of the antigen binding moieties is
a crossover Fab molecule. Such modification reduces 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 variable domains of the Fab light chain and the Fab
heavy chain (VL and VH, respectively) are exchanged. Even with this
domain exchange, however, the preparation of the T cell activating
bispecific antigen binding molecule may comprise certain side
products due to a so-called Bence Jones-type interaction between
mispaired heavy and light chains (see Schaefer et al, PNAS, 108
(2011) 11187-11191). To further reduce mispairing of heavy and
light chains from different Fab molecules and thus increase the
purity and yield of the desired T cell activating bispecific
antigen binding molecule, according to the present invention
charged amino acids with opposite charges may be introduced at
specific amino acid positions in the CH1 and CL domains of either
the Fab molecule(s) specifically binding to a target cell antigen,
or the Fab molecule specifically binding to an activating T cell
antigen. Charge modifications are made either in the conventional
Fab molecule(s) comprised in the T cell activating bispecific
antigen binding molecule (such as shown e.g. in FIGS. 1 A-C, G-J),
or in the VH/VL crossover Fab molecule(s) comprised in the T cell
activating bispecific antigen binding molecule (such as shown e.g.
in FIG. 1 D-F, K-N) (but not in both). In particular embodiments,
the charge modifications are made in the conventional Fab
molecule(s) comprised in the T cell activating bispecific antigen
binding molecule (which in particular embodiments specifically
bind(s) to the target cell antigen).
[0250] 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 an activating T cell antigen, particularly 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 an activating T
cell antigen. 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 the
activating T cell antigen, particularly CD3, without simultaneous
binding to the target cell antigen does not result in T cell
activation.
[0251] 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.
[0252] 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.
Activating T Cell Antigen Binding Moiety
[0253] The T cell activating bispecific antigen binding molecule of
the invention comprises at least one antigen binding moiety,
particularly a Fab molecule, which specifically binds to an
activating T cell antigen (also referred to herein as an
"activating T cell antigen binding moiety, or activating T cell
antigen binding Fab molecule"). 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
an activating T cell antigen. In one embodiment the T cell
activating bispecific antigen binding molecule provides monovalent
binding to the activating T cell antigen.
[0254] In particular embodiments, the antigen binding moiety which
specifically binds an activating T cell antigen is a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other. In such embodiments, the antigen binding moiety(ies) which
specifically binds a target cell antigen is preferably a
conventional Fab molecule. In embodiments where there is more than
one antigen binding moiety, particularly Fab molecule, which
specifically binds to a target cell antigen comprised in the T cell
activating bispecific antigen binding molecule, the antigen binding
moiety which specifically binds to an activating T cell antigen
preferably is a crossover Fab molecule and the antigen binding
moieties which specifically bind to a target cell antigen are
conventional Fab molecules.
[0255] In alternative embodiments, the antigen binding moiety which
specifically binds an activating T cell antigen is a conventional
Fab molecule. In such embodiments, the antigen binding moiety(ies)
which specifically binds a target cell antigen is a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other.
[0256] In a particular embodiment the activating T cell antigen is
CD3, particularly human CD3 (SEQ ID NO: 1) or cynomolgus CD3 (SEQ
ID NO: 2), most particularly human CD3. In a particular embodiment
the activating T cell antigen binding moiety is cross-reactive for
(i.e. specifically binds to) human and cynomolgus CD3. In some
embodiments, the activating T cell antigen is the epsilon subunit
of CD3 (CD3 epsilon).
[0257] In some embodiments, the activating T cell antigen binding
moiety specifically binds to CD3, particularly CD3 epsilon, 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.
[0258] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising 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, and a
light chain variable region comprising 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.
[0259] In another embodiment the CD3 binding antigen binding
moiety, particularly Fab molecule, comprises a heavy chain variable
region comprising the heavy chain CDR1 of SEQ ID NO: 4, the heavy
chain CDR2 of SEQ ID NO: 46, the heavy chain CDR3 of SEQ ID NO: 6,
and a light chain variable region comprising the light chain CDR1
of SEQ ID NO: 47, the light chain CDR2 of SEQ ID NO: 9, and the
light chain CDR3 of SEQ ID NO: 10.
[0260] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, 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.
[0261] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, 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.
[0262] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises the heavy chain variable
region sequence of SEQ ID NO: 3 and the light chain variable region
sequence of SEQ ID NO: 7.
[0263] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 48 and a light chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 49.
[0264] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 48 and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 49.
[0265] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises the heavy chain variable
region sequence of SEQ ID NO: 48 and the light chain variable
region sequence of SEQ ID NO: 49.
Target Cell Antigen Binding Moiety
[0266] The T cell activating bispecific antigen binding molecule of
the invention comprises at least one antigen binding moiety,
particularly a Fab molecule, which specifically binds to CEA
(target cell antigen). In certain embodiments, the T cell
activating bispecific antigen binding molecule comprises two
antigen binding moieties, particularly Fab molecules, which
specifically bind to CEA.
[0267] 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, i.e. they comprise the same
amino acid sequences including the same amino acid substitutions in
the CH1 and CL domain as described herein (if any). In one
embodiment, the T cell activating bispecific antigen binding
molecule comprises an immunoglobulin molecule which specifically
binds to CEA. In one embodiment the T cell activating bispecific
antigen binding molecule comprises not more than two antigen
binding moieties, particularly Fab molecules, which specifically
bind to CEA.
[0268] In particular embodiments, the antigen binding moiety(ies)
which specifically bind to CEA is/are a conventional Fab molecule.
In such embodiments, the antigen binding moiety(ies) which
specifically binds an activating T cell antigen is a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other.
[0269] In alternative embodiments, the antigen binding moiety(ies)
which specifically bind to CEA is/are a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CH1 and CL of the Fab heavy and
light chains are exchanged/replaced by each other. In such
embodiments, the antigen binding moiety(ies) which specifically
binds an activating T cell antigen is a conventional Fab
molecule.
[0270] The CEA binding moiety 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 expresses
CEA.
[0271] In one embodiment, the antigen binding moiety, particularly
Fab molecule, which specifically binds to CEA comprises a heavy
chain variable region comprising the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 14, the heavy chain CDR 2
of SEQ ID NO: 15, and the heavy chain CDR 3 of SEQ ID NO: 16, and a
light chain variable region comprising the light chain CDR 1 of SEQ
ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and the light
chain CDR 3 of SEQ ID NO: 19. In a further embodiment, the antigen
binding moiety, particularly Fab molecule, which specifically binds
to CEA comprises a heavy chain variable region that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
22, and a light chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 23. In
still a further embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CEA
comprises the heavy chain variable region sequence of SEQ ID NO:
22, and the light chain variable region sequence of SEQ ID NO: 23.
In a particular embodiment, the T cell activating bispecific
antigen binding molecule comprises a polypeptide that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
34, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 36, a polypeptide that is
at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 37, and a polypeptide that is at least 95%, 96%, 97%,
98%, or 99% identical to the sequence of SEQ ID NO: 38. In a
further particular embodiment, the T cell activating bispecific
antigen binding molecule comprises a polypeptide sequence of SEQ ID
NO: 34, a polypeptide sequence of SEQ ID NO: 36, a polypeptide
sequence of SEQ ID NO: 37 and a polypeptide sequence of SEQ ID NO:
38. In another embodiment, the T cell activating bispecific antigen
binding molecule comprises a polypeptide that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34, a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 37, a polypeptide that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
38, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 39. In a further
embodiment, the the T cell activating bispecific antigen binding
molecule comprises a polypeptide sequence of SEQ ID NO: 34, a
polypeptide sequence of SEQ ID NO: 37, a polypeptide sequence of
SEQ ID NO: 38 and a polypeptide sequence of SEQ ID NO: 39. In still
another embodiment, the T cell activating bispecific antigen
binding molecule comprises a polypeptide that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34, a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 36, a polypeptide that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
38, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 40. In a further
embodiment, the the T cell activating bispecific antigen binding
molecule comprises a polypeptide sequence of SEQ ID NO: 34, a
polypeptide sequence of SEQ ID NO: 36, a polypeptide sequence of
SEQ ID NO: 38 and a polypeptide sequence of SEQ ID NO: 40. In a
further embodiment, the T cell activating bispecific antigen
binding molecule comprises a polypeptide that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34, a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 36, a polypeptide that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
38, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 41. In a further
embodiment, the the T cell activating bispecific antigen binding
molecule comprises a polypeptide sequence of SEQ ID NO: 34, a
polypeptide sequence of SEQ ID NO: 36, a polypeptide sequence of
SEQ ID NO: 38 and a polypeptide sequence of SEQ ID NO: 41. In yet
another embodiment, the T cell activating bispecific antigen
binding molecule comprises a polypeptide that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34, a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 50, a polypeptide that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
51, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 52. In a further
embodiment, the the T cell activating bispecific antigen binding
molecule comprises a polypeptide sequence of SEQ ID NO: 34, a
polypeptide sequence of SEQ ID NO: 50, a polypeptide sequence of
SEQ ID NO: 51 and a polypeptide sequence of SEQ ID NO: 52.
CEA Antibodies
[0272] In one aspect the invention provides an antibody,
particularly a humanized antibody, which specifically binds to CEA,
wherein said antibody comprises a heavy chain variable region
comprising the heavy chain complementarity determining region (CDR)
1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, and the
heavy chain CDR 3 of SEQ ID NO: 16, and a light chain variable
region comprising the light chain CDR 1 of SEQ ID NO: 17, the light
chain CDR 2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID
NO: 19. In one embodiment, the antibody comprises a heavy chain
variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 22, and a light chain
variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 23. In a further
embodiment, the antigen binding moiety, particularly Fab molecule,
which specifically binds to CEA comprises the heavy chain variable
region sequence of SEQ ID NO: 22, and the light chain variable
region sequence of SEQ ID NO: 23. In one embodiment, the antibody
is a Fab molecule.
Polynucleotides
[0273] 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.
[0274] 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 a Fab molecule 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
Fab molecule, an Fc domain subunit and optionally (part of) another
Fab molecule. When co-expressed, the heavy chain polypeptides will
associate with the light chain polypeptides to form the Fab
molecule. 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 Fab molecules
could be encoded by a separate polynucleotide from the portion of
the T cell activating bispecific antigen binding molecule
comprising the the other of the two Fc domain subunits and
optionally (part of) a Fab molecule. When co-expressed, the Fc
domain subunits will associate to form the Fc domain.
[0275] 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.
[0276] 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
[0277] 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).
[0278] 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.
[0279] 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 0-glucuronidase.
[0280] 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.
[0281] 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.RTM. 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)), MRC 5 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).
[0282] 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.
[0283] 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).
[0284] 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.
[0285] 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).
[0286] 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.
[0287] 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.RTM. 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.).
[0288] 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
[0289] 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
[0290] 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.RTM. 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.
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.
[0291] 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 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.
[0292] 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 is approximately 12000 R U. 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.
[0293] 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
[0294] 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
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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.
[0310] 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, gastric cancer, pancreatic cancer, ovarian cancer. In one
embodiment, the cancer is a solid tumor. 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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.
[0315] 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.
[0316] 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.
[0317] 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.
[0318] 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.
[0319] 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). 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
[0320] 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.
[0321] 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.
[0322] 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
[0323] 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
[0324] 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
[0325] 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
[0326] DNA sequences were determined by double strand
sequencing.
Gene Synthesis
[0327] 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.
Example 1
Binding of Different Humanized Variants of T84.66 IgG to Cells
[0328] Novel humanized variants of the murine antibody T84.66
(Wagener et al., J Immunol 130, 2308 (1983), Neumaier et al., J
Immunol 135, 3604 (1985)) were developed by grafting of the CDRs
onto human germline framework acceptor sequences.
[0329] In this example, the binding of different humanized variants
of T84.66 IgG was tested on CEA-expressing human gastric
adenocarcinoma cells (MKN45, DSMZ ACC 409).
[0330] Briefly, cells were harvested, counted, checked for
viability and re-suspended at 2.times.10.sup.6 cells/ml in FACS
buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of 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 IgG (4 ng/ml-60 g/ml), 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 Fcg 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). Binding curves and EC50 values were
obtained and calculated using GraphPadPrism5 (FIG. 2, binding to
MKN45 cells).
[0331] FIG. 2 shows the different binding pattern of selected
humanized variants of the T84.66 IgG to human CEA, expressed on
MKN45 cells. Based on the binding pattern and the calculated EC50
binding values (Table 1), the humanized variant 1 (SEQ ID NOs 22
and 23) was selected for further evaluation.
TABLE-US-00002 TABLE 1 Binding of different humanized variants of
T84.66 IgGs to cells (EC50 values, based on binding curves shown in
FIG. 2, calculated by Graph Pad Prism). EC50 (.mu.g/ml) Parental
chimeric T84.66 0.99 Humanized variant 1 1.5 Humanized variant 2
8.6 Humanized variant 3 1.4 Humanized variant 4 3.1 Humanized
variant 5 -- Humanized variant 6 --
Example 2
Preparation of Anti-CEA/Anti-CD3 T Cell Bispecific (TCB)
Molecules
[0332] The following molecules were prepared in this example;
schematic illustrations thereof are shown in FIG. 3: [0333] A. "2+1
IgG CrossFab, inverted" with charge modifications (VH/VL exchange
in CD3 binder, charge modification in CEA binder, parental murine
CEA binder (T84.66)) (FIG. 3A, SEQ ID NOs 32-35) [0334] B. "2+1 IgG
CrossFab, inverted" with charge modifications (VH/VL exchange in
CD3 binder, charge modification in CEA binder, humanized CEA
binder) (FIG. 3B, SEQ ID NOs 34, 36-38) [0335] C. "1+1 IgG
CrossFab, inverted" with charge modifications (VH/VL exchange in
CD3 binder, charge modification in CEA binder, humanized CEA
binder) (FIG. 3C, SEQ ID NOs 34, 37-39) [0336] D. "1+1 IgG
CrossMab" with charge modifications (VH/VL exchange in CD3 binder,
charge modification in CEA binder, humanized CEA binder) (FIG. 3D,
SEQ ID NOs 34, 36, 38, 40) [0337] E. "2+1 IgG CrossFab, inverted"
with charge modifications (VH/VL exchange in CD3 binder, charge
modification in CEA binder, humanized CEA binder, longer linker)
(FIG. 3E, SEQ ID NOs 34, 36, 38, 41). [0338] F. "2+1 IgG CrossFab,
inverted" without charge modifications (VH/VL exchange in CD3
binder, humanized CEA binder) (FIG. 3F, SEQ ID NOs 34, 50-52).
[0339] The DNA sequences encoding the variable heavy and light
chain regions of the CD3 and CEA binders were subcloned in frame
with the respective constant regions which are pre-inserted into
the respective recipient mammalian expression vector. Protein
expression is driven by an MPSV or a CMV promoter. Polyadenylation
is driven by a synthetic polyA signal sequence located at the 3'
end of the CDS. In addition each vector contains an EBV OriP
sequence for autosomal replication.
[0340] For production of the molecules, HEK293-EBNA cells growing
in suspension were co-transfected with the respective expression
vectors using polyethylenimine (PEI) as transfection reagent. The
cells were transfected with the corresponding expression vectors in
a 1:2:1:1 ratio (A, B, E and F: "vector heavy chain
(VH-CH1-VL-CH1-CH2-CH3)": "vector light chain (VL-CL)": "vector
heavy chain (VH-CH1-CH2-CH3)": "vector light chain (VH-CL)") or in
a 1:1:1:1 ratio (C: "vector heavy chain (VH-CH1-VL-CH1-CH2-CH3)":
"vector light chain (VL-CL)": "vector heavy chain (CH2-CH3)":
"vector light chain (VH-CL)", D: "vector heavy chain
(VL-CH1-CH2-CH3)": "vector light chain (VL-CL)": "vector heavy
chain (VH-CH1-CH2-CH3)": "vector light chain (VH-CL)").
[0341] For transfection, HEK293 EBNA cells were cultivated in
suspension serum free in Excell culture medium containing 6 mM
L-glutamine and 250 mg/l G418. For the production in 600 ml
tubespin flasks (max. working volume 400 ml) 600 million HEK293
EBNA cells were seeded 24 hours before transfection. For
transfection, cells were centrifuged for 5 min at 210.times.g, and
supernatant was replaced by 20 ml pre-warmed CD CHO medium.
Expression vectors are mixed in 20 ml CD CHO medium to a final
amount of 400 .mu.g DNA. After addition of 1080 .mu.l PEI solution
(2.7 .mu.g/ml) 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 600 ml tubespin
flask and incubated for 3 hours at 37.degree. C. in an incubator
with a humidified 5% CO.sub.2 atmosphere. After incubation, 360 ml
Excell medium containing 6 mM L-glutamine, 5 g/L Pepsoy and 1.0 mM
VPA was added and cells were cultivated for 24 hours. One day after
transfection 7% Feed 1 was added. After 7 days cultivation
supernatant was collected for purification by centrifugation for
20-30 min at 3600.times.g (Sigma 8K centrifuge), the solution was
sterile filtered (0.22 .mu.m filter) and sodium azide in a final
concentration of 0.01% w/v was added. The solution was kept at
4.degree. C.
[0342] The titer of the molecules in the culture medium was
determined by Protein A-HPLC (Table 2). Calculation of the titer is
based on a two-step process and includes binding of Fc-containing
molecules to Protein A at pH 8.0 and release in a step elution at
pH 2.5. Both buffers used for the analysis contained Tris (10 mM),
glycine (50 mM), and NaCl (100 mM) and were adjusted to the
respective pHs (8 and 2.5). The column body was an Upchurch
2.times.20 mm pre-column with an internal volume of .about.63 .mu.l
packed with POROS 20A. After initial calibration, 100 .mu.l of each
sample was injected with a flow rate of 0.5 ml/min. After 0.67
minutes the sample was eluted with a pH step to pH 2.5.
Quantitation was done by determination of 280 nm absorbance and
calculation using a standard curve with a concentration range of
human IgG1 from 16 to 166 mg/l.
[0343] The secreted proteins were purified from cell culture
supernatants by affinity chromatography using Protein A affinity
chromatography, followed by a size exclusion chromatographic
step.
[0344] For affinity chromatography supernatant was loaded on a
HITRAP.RTM. ProteinA HP (molecule A, B and F) or a MabSelectSure
(molecule C, D and E) column (CV=5 mL, GE Healthcare) equilibrated
with 25 ml 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5.
Unbound protein was removed by washing with at least 10 column
volumes 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5, and
target protein was eluted in 6 column volumes 20 mM sodium citrate,
100 mM sodium chloride, 100 mM glycine, pH 3.0. Protein solution
was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8.0.
For in-process analytics after Protein A chromatography, the purity
and molecular weight of the molecules in the single fractions were
analyzed by SDS-PAGE in the absence of a reducing agent and
staining with Coomassie (InstantBlue.TM., Expedeon). The
NuPAGE.RTM. Pre-Cast gel system (4-12% Bis-Tris, Invitrogen) was
used according to the manufacturer's instruction. Selected
fractions of target protein were concentrated and filtrated prior
to loading on a HILOAD.RTM. SUPERDEX.RTM. 200 column (GE
Healthcare) equilibrated with 20 mM histidine, 140 mM sodium
chloride, 0.01% Tween-20, pH 6.0.
[0345] 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.
[0346] The aggregate content of the molecules was analyzed using a
TSKGEL.RTM. 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.
[0347] Purity and molecular weight of molecules after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LABCHIP.RTM. GXII
system (Caliper lifescience) was used according to the
manufacturer's instruction (FIG. 5 and Table 3).
[0348] Mass spectrometry analysis of the molecules was performed on
an Agilent LC-MS system (Agilent Technologies, Santa Clara, Calif.,
USA). The chromatography system (Agilent 1260 Infinity) was coupled
on an Agilent 6224 TOF LC/MS ESI device. About 5 .mu.g of sample
were injected on a NUCLEOGEL.RTM. RP1000-8, 250 mm.times.4.6 mm
column (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany) at a flow
rate of 1 ml/min at 40.degree. C. The mobile phase was as follows
A: 5% acetonitrile, 0.05% formic acid, and B: 95% acetonitrile,
0.05% formic acid. To apply an elution gradient, 15% B was raised
to 60% B within 10 min, then to 100% B in 2.5 min.
[0349] The mass spectrometer was measuring in high resolution mode
4 GHz positive, and recorded a range from 500 to 3200 m/z. The m/z
spectra were deconvoluted manually with the MassAnalyzer 2.4.1 from
Roche (Hoffman-La Roche, Ltd).
[0350] All molecules were produced and purified essentially
following the same method. The final quality was very good for both
molecules A and B with almost 100% monomer content and 100% purity
on CE-SDS (Table 2 and 3, FIG. 4). LC-MS analysis was performed for
molecule B and revealed no mispairing of light chains. In contrast,
molecule F (without charge modifications) had a very low recovery
because side products had to be removed and LC-MS measurements
still showed around 10% mispairing. Molecule C could be purified
with a slightly better yield and quality than molecule D. The final
quality was also good for both molecules C and D with almost 100%
monomer content and >96% purity on CE-SDS (Table 2 and 3, FIG.
4).
TABLE-US-00003 TABLE 2 Summary of production and purification of
anti-CEA/anti-CD3 TCB molecules with and without charge
modifications. Analytical SEC (HMW/ Titer Recovery Yield
Monomer/LMW) Molecule [mg/l] [%] [mg/l] [%] A 10 40 4 0/100/0 B 1.5
61 0.9 0.8/99.2/0 C 20 36 7.3 0/100/0 D 10 17 1.7 0/99/1 E 16 27
4.3 3/97/0 F 15 3 0.46 0/100/0
TABLE-US-00004 TABLE 3 CE-SDS analyses (non-reduced) of
anti-CEA/anti-CD3 TCB molecules with and without charge
modifications. Molecule Peak # Size [kDa] Purity [%] A 1 218.6 100
B 1 199.6 100 C 1 169 100 D 1 98 4 2 166 96 E 1 190 2 2 200 94 3
210 4 F 1 172 2 2 197 2 3 220 2 4 230 94
Example 3
Comparison of Different Anti-CEA/Anti-CD3 T Cell Bispecific
Molecule Formats
Example 3A
Binding of Different Anti-CEA/Anti-CD3 T Cell Bispecific (CEA CD3
TCB) Molecules to Cells
[0351] The binding of different formats of anti-CEA/anti-CD3 T cell
bispecific (CEA CD3 TCB) molecules was tested on human gastric
adenocarcinoma cells (MKN45, DSMZ ACC 409, .about.513 000 CEA
binding sites), colon adenocarcinoma cells (LS174T, ATCC.RTM.
CL-188, .about.40 700 CEA binding sites), and colon adenocarcinoma
cells HT29 (DSMZ ACC 299, .about.10 000 CEA binding sites), as well
as on CD3-expressing immortalized T lymphocyte cells (Jurkat, DSMZ
ACC 282).
[0352] 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 of 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 CD3 TCB molecules (310 pM-500
nM), washed twice with cold PBS 0.1% BSA, re-incubated for further
30 min at 4.degree. C. with the Fluorescein (FITC)-AffiniPure
F(ab')2 Fragment Goat Anti-Human IgG, Fc.gamma. Fragment Specific
antibody (Jackson Immuno Research Lab #109-096-008), washed twice
with cold PBS 0.1% BSA.
[0353] Staining was fixed by incubation of cells with FACS buffer,
containing 2% PFA for 30 min at 4.degree. C. in the dark. For the
measurement, cells were re-suspended in 150 FACS buffer and
fluorescence was measured using Miltenyi MACSQUANT.RTM.
[0354] Results are shown in FIG. 5. Binding curves were obtained
using GraphPadPrism5 (upper row from left to right, MKN45 cells;
LS174T cells, binding to HT29 cells, lower row, binding to Jurkat
cells).
[0355] FIG. 5 shows higher fluorescence intensities for molecule D
and molecule C at high concentration on cell lines with medium
(LS174T) or low CEA expression level (HT29). This points to the
fact, that more of the monovalently binding constructs are able to
bind under these conditions compared to molecule B, that binds to
human CEA bivalently. The binding to human CD3 on Jurkat cells is
comparable for molecule C and molecule B, whereas molecule D shows
better binding. This might be due to better accessibility of the
CD3-targeting moiety in molecule D.
Example 3B
Tumor Cell Killing Induced by Different Anti-CEA/Anti-CD3 T Cell
Bispecific (CEA CD3 TCB) Molecules
[0356] T-cell mediated killing of different tumor cells by CEA CD3
TCB molecules was assessed using MKN45, BxPC-3 (ECACC 93120816, a
human primary pancreatic adenocarcinoma cell line) or HT-29 human
tumor cells as targets, and human PBMCs as effector cells. Lysis of
tumor cells was detected at 24 h and 48 h of incubation with the
indicated CEA CD3 TCB molecules.
[0357] 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 tumor cell lysis
assay, the CEA CD3 TCB molecules were added at the indicated
concentrations (range of 1 pM-20 nM in triplicates). PBMCs were
added to target cells at final E:T ratio of 10:1. Target cell
killing was assessed after 24 h and 48 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.
[0358] FIG. 6 shows that molecule D induced the strongest killing
of target cell lines, followed by molecule C and finally molecule
B. The best differentiation between the three different formats may
be seen on tumor cells lines with low CEA expression levels (FIGS.
6C and 6F). EC50 values of tumor cell lysis were calculated using
Graph Pad Prism5 and are given in Table 4 (24 h) and Table 5 (48
h).
TABLE-US-00005 TABLE 4 EC50 values (pM) for T-cell mediated killing
of CEA-expressing tumor cells induced by different CEA CD3 TCB
molecules after 24 h. CEA binding Cell line sites Molecule B
Molecule C Molecule D MKN45 513 000 78.46 72.96 13.58 BxPC-3 44 400
113.5 91.33 22.19 HT-29 10 000 214.5 142.0
TABLE-US-00006 TABLE 5 EC50 values (pM) for T-cell mediated killing
of CEA-expressing tumor cells induced by different CEA CD3 TCB
molecules after 48 h. CEA binding Cell line sites Molecule B
Molecule C Molecule D MKN45 513 000 41.24 47.64 11.60 BxPC-3 44 400
46.98 32.42 13.67 HT-29 10 000 31.72 63.80 62.44
Example 3C
CD25 Up-Regulation on CD4+ and CD8+ Effector Cells after Killing of
CEA-Expressing Tumor Cells Induced by Different CEA CD3 TCB
Molecules
[0359] Activation of CD4+ (FIG. 7 A-D) and CD8+ T cells (FIG. 7
E-H) after killing of CEA-expressing MKN45, BxPC3 or HT29 tumor
cells mediated by different CEA CD3 TCB molecules was assessed by
FACS analysis using antibodies recognizing the T cell activation
marker CD25 (late activation marker). In addition, CD25 was
analyzed on CD4 and CD8 T effector cells upon co-incubation of
effector cells with the different CEA CD3 TCB molecules in the
absence of target cells to check for antigen-unspecific T cell
activation.
[0360] The antibody and the killing assay conditions were
essentially as described above (Example 3B), using the same
antibody concentration range (1 pM-20 nM in triplicates), E:T ratio
10:1 and an incubation time of 48 h.
[0361] 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, BioLegend #344704), CD4 (PECy7 anti-human
CD4, BioLegend #344612) and CD25 (APC anti-human CD25 BioLegend
#302610) was performed according to the suppliers' indications.
Cells were washed twice with 150 .mu.l/well PBS containing 0.1% BSA
and fixed for 30 min at 4.degree. C. using 150 .mu.l/well of FACS
buffer, containing 2% PFA. After centrifugation, the samples were
re-suspended in 200 .mu.l/well PBS 0.1% BSA and analyzed using a BD
FACS Fortessa.
[0362] FIG. 7 shows that molecule D induced the strongest T cell
activation, as measured by percentage of CD25-positive T cells.
However, this molecule also induced activation of T cells in the
absence of target cells at the highest 2-3 concentrations and was
therefore not selected as preferred format. Molecule C is the
second most potent molecule to induce T cell activation in the
presence of CEA-expressing target cells, which is especially
apparent in settings with target cells that express rather low
levels of CEA (FIG. 7C). Also molecule B is able to induce strong
and concentration-dependent T cell activation in the presence of
CEA-expressing target cells. In this example, both molecules B and
C do not induce significant T cell activation in the absence of
target cells.
[0363] To further elaborate this important safety point, additional
assays were performed.
Example 3D
CD69 Up-Regulation on CD4+ and CD8+ Effector Cells Upon
Co-Incubation with Different CEA
[0364] CD3 TCB molecules and CEA-expressing tumor or primary
epithelial cells Activation of CD4+ (FIG. 8 A-E) and CD8+ T cells
(FIG. 8 F-J) was assessed after co-incubation of CEA-expressing
MKN45, LS174T (ECACC 87060401, a human colon adenocarcinoma cell
line with approximately 40 700 CEA binding sites), HT29 or CCD 841
CoN (ATCC.RTM. CRL-1790.TM., a primary epithelial cell line from
human colon, expressing <2000 CEA binding sites) with human
PBMCs and different CEA CD3 TCB molecules for 48 h. To check for
antigen-unspecific T cell activation, CD69 was analyzed on CD4 and
CD8 T effector cells upon co-incubation of effector cells with the
different CEA CD3 TCB molecules in the absence of target cells as
well.
[0365] The antibody and assay conditions were essentially as
described above (Example 3B and 3C), using the same antibody
concentration range (1 pM-20 nM in triplicates) and an E:T ratio of
10:1, as well as the FACS staining protocol as described above (PE
anti-human CD69, BioLegend #310906). FIG. 8 shows that again
molecule D induced the strongest T cell activation, as measured by
percentage of CD69-positive T cells in the presence of different
CEA-expressing tumor cells. However, this molecule also induced
activation of T cells in the presence of primary epithelial cells
(CCD841, see FIGS. 8D and 8I), as well as in the absence of target
cells (FIGS. 8E and 8J). These findings confirm the
antigen-independent activation of T cells and in addition point to
a potential safety issue in the presence of primary epithelial
cells with very low CEA expression levels.
[0366] However, with these reactive PBMC effector cells,
antigen-independent T cell activation was observed with the second
most potent molecule as well, molecule C. Consequently, the only
format that showed strong and concentration-dependent killing of
various CEA-expressing tumor cells, but no significant killing of
primary epithelial cells, nor antigen-independent T cell activation
in the presence of primary epithelial cells or in the absence of
target cells is molecule B. Therefore, the "2+1 IgG CrossFab,
inverted" format was selected as the preferred format.
Example 4
Comparison of Anti-CEA/Anti-CD3 T Cell Bispecific Molecules
Comprising Parental or Humanized CEA Binders
Example 4A
T Cell Activation and Tumor Cell Killing Induced by Different CEA
CD3 TCB Molecules (Chimeric T84.66 Versus Humanized Variant 1)
[0367] The impact of the CEA binder (parental chimeric T84.66
versus humanized variant 1) on the final potency of the CEA CD3 TCB
to induce T cell activation or tumor cell lysis was assessed with a
classical tumor cell lysis assay with subsequent staining of T cell
activation markers as described above (Example 3B and Example 3C).
To further evaluate the safety window, both molecules were
co-incubated with effector cells only as well.
[0368] Briefly, target cells included in this assay were MKN45 and
CCD841 CoN cells. The CEA CD3 TCB molecules A and B were added at
the indicated concentration range of 1 pM-20 nM (in triplicates).
PBMCs were added to target cells at a final E:T ratio of 10:1.
Lysis of tumor or primary epithelial cells was detected at 48 h of
incubation with the indicated CEA CD3 TCB molecules. PBMCs of this
assay were harvested and stained for CD8+ and the early activation
marker CD69 as described above (Example 3C).
[0369] FIG. 9 shows that the CEA CD3 TCB molecule containing the
parental chimeric T84.66 CEA binder (molecule A) induced both T
cell activation (FIG. 9 A, B) and cell lysis (FIG. 9 C, D) not only
in the presence of CEA high expressing tumor cells (MKN45), but
also in the presence of primary epithelial cells CCD841 with very
low CEA expression levels or in the absence of target cells.
[0370] In contrast, the CEA CD3 TCB molecule containing the
humanized binder (molecule B) induced T cell activation only in the
presence of the tumor cell line MKN45, but not in the presence of
the primary epithelial cell line. The same is true for cell lysis.
In addition, there was no sign of significant T cell activation in
the absence of target cells.
Example 4B
[0371] To further evaluate the safety window of the CEA CD3 TCB
molecules, containing either the parental, chimeric T84.66 CEA
binder (molecule A) or the humanized variant 1 (molecule B), a
sensitive Jurkat-NFAT reporter assay was conducted. In principle,
the simultaneous binding of the TCB molecule to human CEA on
antigen-expressing cells and to human CD3 on Jurkat-NFAT reporter
cells (a human acute lymphatic leukemia reporter cell line with a
NFAT promoter-regulated luciferase expression, GloResponse Jurkat
NFAT-RE-luc2P, Promega # CS176501) the NFAT promoter is activated
and leads to expression of active firefly luciferase. The intensity
of the luminescence signal (obtained upon addition of luciferase
substrate) is proportional to the intensity of CD3 activation and
signaling.
[0372] For the assay, target cells were harvested with trypsin/EDTA
and viability was determined using ViCell. 20 000 cells/well were
plated in a flat-bottom, white-walled 96-well-plate (#655098,
greiner bio-one) and diluted antibodies or medium (for controls)
was added at the indicated concentration range (0.4 pM-100 nM).
Subsequently, Jurkat-NFAT reporter cells were harvested and
viability assessed using ViCell. Cells were resuspended in cell
culture medium and added to tumor cells to obtain a final E:T of
2.5:1 and a final volume of 100 .mu.l per well. Cells were
incubated for 4 h at 37.degree. C. in a humidified incubator. At
the end of the incubation time, 100 .mu.l/well of ONE-Glo solution
(1:1 ONE-Glo and assay medium volume per well) were added to wells
and incubated for 10 min at room temperature in the dark.
Luminescence was detected using WALLAC Victor3 ELISA reader
(PerkinElmer2030), 1 sec/well as detection time.
[0373] FIG. 10 shows that the CEA CD3 TCB molecule containing the
parental chimeric T84.66 CEA binder (molecule A) induced Jurkat T
cell activation not only in the presence of CEA high and medium
expressing tumor cells (MKN45 and LS174T respectively), but also in
the presence of primary epithelial cells CCD841 with very low CEA
expression levels or in the absence of target cells (FIG. 10A).
[0374] In contrast, the CEA CD3 TCB molecule containing the
humanized binder (molecule B) induced Jurkat T cell activation only
in the presence of the tumor cell lines MKN45 and LS174T, but not
in the presence of the primary epithelial cell line or in the
absence of targets (FIG. 10B). These results surprisingly show that
the molecule comprising the humanized CEA binder is better in terms
of safety.
Example 5
Comparison of Anti-CEA/Anti-CD3 T Cell Bispecific Molecules
Comprising Different Humanized CEA Binders
Example 5A
T Cell Activation and Tumor Cell Killing Induced by CEA CD3 TCB
Molecules Comprising Different Humanized CEA Binders
[0375] The impact of the CEA binder (humanized variant 1 (SEQ ID
NOs 22 and 23) versus a different humanized CEA binder (SEQ ID NOs
30 and 31, not based on T84.66)) on the final potency of the CEA
TCB to induce tumor cell lysis was assessed with a classical tumor
cell lysis assay as described above (Example 3B).
[0376] Briefly, target cells included in this assay were BxPC-3,
NCI-H2122 (ATCC.RTM. CRL-5985, a human non-small cell lung cancer
cell line, .about.13 300 CEA binding sites), COR-L105
(Sigma-Aldrich #92031918, a human lung adenocarcinoma cell line,
.about.1200 CEA binding sites) and HBEpiC (Chemie Brunschwig AG
#3210, human bronchial epithelial cells, <500 CEA binding
sites). The CEA CD3 TCB molecule comprising the humanized variant 1
CEA binder (molecule B) was added at the indicated concentration
range of 1 pM-20 nM (in triplicates), the CEA CD3 TCB molecule
comprising the different humanized CEA binder (i.e. a CEA CD3 TCB
molecule of similar structure as molecule B, but comprising a
different CEA binder, see SEQ ID NOs 42-45) was added at the
indicated concentration range of 6 pM-100 nM. PBMCs were added to
target cells at a final E:T ratio of 10:1. Lysis of tumor or
primary epithelial cells was detected at 47 h of incubation with
the indicated CEA CD3 TCB molecules.
[0377] Subsequently, PBMCs of this assay were harvested and stained
for the early activation marker CD69 on human CD8+ T cells as
described above.
[0378] FIG. 11 A-D shows that the CEA CD3 TCB based on humanized
variant 1 (molecule B of Example 2) induced stronger T cell
activation upon simultaneous binding to T effector and CEA-positive
target cells compared to the TCB molecule based on the different
humanized CEA binder (referred to in the following as "molecule X",
SEQ ID NOs 42-45).
[0379] This is in line with the tumor cell lysis data depicted in
FIG. 11 E-H, where stronger killing of CEA-expressing tumor cells
was observed with the molecule based on humanized variant 1.
[0380] Remarkably, none of the CEA CD3 TCB molecules induced lysis
of CEA-low primary epithelial HBEpiC cells.
[0381] Taken together, the CEA CD3 TCB molecule based on humanized
variant 1 (molecule B) is able to kill tumor cells with much lower
CEA levels as compared to the CEA CD3 TCB based on a different
humanized CEA binder (molecule X), while maintaining the safety
window.
Example 5B
T Cell Proliferation Induced by CEA CD3 TCB Molecules Comprising
Different Humanized CEA Binders
[0382] As an alternative read-out, the TCB molecules used in
Example 5A were analyzed for their capability to induce T cell
proliferation upon cross-linkage in the presence of the respective
tumor target cells (MKN45, LS174T, HT29). As a control, primary
epithelial cells CCD841 CoN with very low CEA expression levels
were included as alternative target cells as well.
[0383] Briefly, freshly isolated human PBMCs were adjusted to 1
million cells per ml in warm PBS and stained with 0.1 .mu.M CFSE in
a humidified incubator at 37.degree. C. for 15 minutes. The
staining was stopped by addition of 1/10 volume of FCS, that was
incubated for 1 min at room temperature. Subsequently, the cells
were centrifuged, re-suspended in pre-warmed medium and incubated
for another 30 min in a humidified incubator at 37.degree. C. to
remove remaining CFSE. After the incubation the cells were washed
once with warm medium, counted and re-suspended in medium at 2 mio
cells per ml.
[0384] 0.02 million target cells were plated per well of a
flat-bottom 96-well plate and the different TCB molecules were
added at the indicated concentrations. CFSE-labeled PBMCs were
added to obtain a final E:T ratio of 10:1 and the assay plates were
incubated for five days in a humidified incubator at 37.degree.
C.
[0385] On day five, the effector cells were harvested, washed twice
with FACS buffer (PBS, 0.1% BSA) and stained for surface expression
of CD4 and CD8. Proliferation of the different T cell
subpopulations was analyzed using a BD FACS Fortessa, equipped with
PD FACS Diva Software. Proliferation curves were analyzed by
GraphPadPrism5.
[0386] FIG. 12 shows that the CEA CD3 TCB based on humanized
variant 1 (molecule B) induced stronger T cell activation and
subsequent T cell proliferation in the presence of CEA-positive
tumor target cells compared to the CEA CD3 TCB based on the
different humanized CEA binder (molecule X). Proliferation of CD8+
T cells is shown in FIG. 12 A-D, proliferation of CD4+ T cells in
FIG. 12 E-H.
[0387] Notably, even after 5 days of incubation no sign of
significant T cell activation and subsequent T cell proliferation
could be observed with either one of the molecules in the presence
of primary epithelial cells (FIG. 12, CCD841 CoN cells).
[0388] This further confirms the favorable potency and safety
window of the CEA CD3 TCB based on humanized variant 1 (molecule
B), even as compared to the CEA CD3 TCB based on a different
humanized CEA binder (molecule X).
Example 5C
Binding of Different Anti-CEA/Anti-CD3 T Cell Bispecific (CEA CD3
TCB) Molecules to Cells
[0389] The binding of the TCB molecules used in Example 5A and B
was tested on different CEA-expressing tumor and CD3-expressing
Jurkat (DSMZ ACC 282) cells.
[0390] Briefly, cells were harvested, counted, checked for
viability and re-suspended at 2.times.10.sup.6 cells/ml in FACS
buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of 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 IgG (31 pM-500 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 Fcg Fragment Specific secondary antibody
(Jackson Immuno Research Lab FITC #109-096-008, 1:40 pre-diluted in
PBS 0.1% BSA), washed twice with cold PBS 0.1% BSA and fixed using
150 .mu.l PBS 0.1% BSA, containing 2% PFA and incubation at
4.degree. C. for 20 min. Thereafter, cells were washed once for 8
min at 400.times.g, 4.degree. C. and finally re-suspended in 150
.mu.l FACS buffer for the FACS measurement. Fluorescence was
measured using Miltenyi MACSQuant.
[0391] Binding curves and EC50 values were obtained and calculated
using GraphPadPrism6 (FIG. 16 A, binding to MKN45 cells, FIG. 16 B,
binding to LS-174T cells, FIG. 16 C, binding to HT-29 cells, FIG.
16 D, Table 6).
[0392] FIG. 16 shows, that the CEA CD3 molecule based on humanized
variant 1 (molecule B) displays better binding to CEA-expressing
tumor cells than the CEA CD3 TCB based on a different humanized CEA
binder (molecule X) (better EC50 values and maximal binding,
particularly on medium and low CEA-expressing target cells). Both
TCB molecules show concentration-dependent binding to human CD3 on
Jurkat cells.
TABLE-US-00007 TABLE 6 Binding of CEA CD3 TCB molecule B and CEA
CD3 TCB molecule X to cells (EC50 values, based on binding curves
shown in FIG. 16, calculated by Graph Pad Prism). CEA Molecule B
Molecule X binding EC50 binding EC50 binding Cell Line Vendor sites
(nM) (nM) MKN45 DSMZ ~513 300 26.61 27.85 #ACC409 LS-174T ATCC
.RTM. ~40 700 4.256 11.69 #CL-188 HT-29 DSMZ ~10 000 18.66 n.c.
#ACC299
[0393] CEA binding sites were determined by a FACS-based Qifikit
analysis, according to the manufacturers' instructions, using 10
.mu.g/ml anti-human CEA antibody (Santa Cruz Biotechnology,
sc-23928).
Example 5D
Tumor Cell Lysis Induced by CEA CD3 TCB Molecules Comprising
Different Humanized CEA Binders
[0394] T-cell mediated lysis of different tumor cells by the CEA
CD3 TCB molecules used in Examples 5A-C was assessed using human
tumor cells as target cells, and human PBMCs as effector cells.
Lysis of tumor cells was detected at 48 h of incubation with the
indicated CEA CD3 TCB molecules. Briefly, target cells were
harvested with Trypsin/EDTA, washed, and plated at density of 25
000-000 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 a cell incubator until further use (no longer than 24
h). For the tumor cell lysis assay, the CEA CD3 TCB molecules were
added at the indicated concentrations (range of 0.26 pM-20 nM for
CEA CD3 TCB based on humanized variant 1 (molecule B), respective
1.28 pM-100 nM for CEA CD3 TCB based on different humanized CEA
binder (molecule X), in triplicates). PBMCs were added to target
cells at final E:T ratio of 10:1. Target cell killing was assessed
after 48 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.
[0395] FIG. 17 shows that the CEA CD3 TCB molecule based on
humanized variant 1 (molecule B) induced significant and
concentration-dependent lysis of all shown tumor cell lines,
whereas the CEA CD3 TCB based on the different humanized CEA binder
(molecule X) induced tumor lysis of KatoTI and to a lesser extent
of NCT-H2122 only. This clearly demonstrates the higher potency of
molecule B as compared to molecule X, especially for tumor cell
lines with rather low CEA expression levels.
[0396] EC50 values of tumor cell lysis were calculated using Graph
Pad Prism6 and are given in Table 7 (48 h).
TABLE-US-00008 TABLE 7 EC50 values (pM) for T-cell mediated lysis
of low CEA-expressing tumor cells induced by different CEA CD3 TCB
molecules after 48 h. CEA Molecule Molecule Tumor binding B EC50 X
EC50 Cell Line Indication Reference sites lysis lysis Kato III
Gastric ECACC ~22 700 55.4 8233 Cancer #86093004 HCC1954 Breast ATC
~15 750 421.7 -- Cancer #CRL-2338 NCI-H2122 Lung ATCC ~13 300 27.9
-- Cancer #CRL-598 CX-1 Colon DSMZ ~4750 430.4 -- Cancer #ACC 129
NCI-H596 Lung ATCC ~1 900 18.5 -- Cancer #HTB-178
[0397] CEA binding sites were determined by a FACS-based Qifikit
analysis, according to the manufacturers' instructions, using 10
.mu.g/ml anti-human CEA antibody (Santa Cruz Biotechnology,
sc-23928).
Example 6
Comparison of Anti-CEA/Anti-CD3 T Cell Bispecific Molecules
Comprising Different Linkers Between CEA and CD3 Binders
Example 6A
Binding of CEA CD3 TCB Molecules Comprising Different Linkers
Between CEA and CD3 Binders to Cells
[0398] The binding of a variant of molecule B with a longer linker
between the CD3 Fab and the CEA Fab (molecule E) was compared to
molecule B for its binding to cells.
[0399] The binding to human CEA was tested on MKN45, LS174T or
HT29, the binding to human CD3 was tested on Jurkat cells. The
assay set-up and conditions were as described above (Example
3A).
[0400] Results are shown in FIG. 13. Binding curves were obtained
using GraphPadPrism5 (A, binding to MKN45 cells; B, binding to
LS174T cells, C, binding to HT29 cells, D, binding to Jurkat
cells).
[0401] FIG. 13 shows comparable binding of both molecules to human
CEA, as well as to human CD3 on cells.
Example 6B
Lysis of Various Tumor Cells by CEA CD3 TCB Molecules Comprising
Different Linkers Between CEA and CD3 Binders
[0402] To further evaluate the impact of the linker length on the
potency of the molecule to induce T cell-mediated lysis of
CEA-expressing tumor cells, a classical tumor cell lysis assay was
performed, as described above (e.g. in Example 3B). The CEA CD3 TCB
molecules of Example 6A (molecule B and a corresponding molecule
with a longer linker between the CEA and CD3 binders, molecule E)
were added at the indicated concentrations (range of 1 pM-20 nM in
triplicates) and tumor cell lysis was assessed after 24 h (FIG. 14
A-D) and 48 h (FIG. 14 E-H). EC50 values were calculated by
GraphPadPrism5 and are given in Table 8.
[0403] FIG. 14 shows comparable lysis of tumor cells expressing
high (MKN45) or medium levels of CEA (LS174T) and no killing of
primary epithelial cells CCD841.
[0404] However, target cells with low CEA expression levels (HT29)
showed higher overall killing with the molecule comprising the
longer linker.
TABLE-US-00009 TABLE 8 EC50 values (pM) for T-cell mediated killing
of low CEA-expressing HT29 tumor cells induced by different CEA CD3
TCB molecules after 24 h and 48 h. EC50 (pM) Molecule B Molecule E
24 h 512 679 48 h 338 342
Example 7
Tumor Cell Lysis and T Cell Activation Induced by CEA CD3 TCB
Molecule
Example 7A
[0405] Lysis of Cell Lines with Different CEA Expression Levels by
CEA CD3 TCB Molecule B
[0406] In another experiment (FIG. 18A), the CEA CD3 TCB molecule B
was characterized in the presence of the low CEA-expressing primary
epithelial cell line HBEpiC versus different tumor cell lines to
assess its safety. The assay set-up and antibody range was as
described in Example 5D for molecule B. EC50 values of tumor cell
lysis were calculated using Graph Pad Prism6 and are given in Table
9 (48 h).
[0407] As depicted in FIG. 18A and Table 9, primary epithelial
cells were not killed by the CEA CD3 TCB molecule, whereas tumor
cell lines with varying CEA expression levels could be lysed by the
CEA CD3 TCB molecule in a concentration-dependent manner.
TABLE-US-00010 TABLE 9 EC50 values (pM) for T-cell mediated lysis
of CEA-expressing tumor cells induced by CEA CD3 TCB molecule B
after 48 h. CEA binding Cell Line Reference sites Molecule B BxPC-3
ECACC ~44 000 49.63 #93120816 NCI-H2122 ATCC ~13 300 237.5 #CRL-598
COR-L105 Sigma-Aldrich ~1 200 n.c.* #92031918 HBEpiC ScienCell
#3210 <600 -- *The EC50 for COR-L105 could not be calculated
properly because the curve did not reach saturation at high
concentrations.
[0408] CEA binding sites were determined by a FACS-based Qifikit
analysis, according to the manufacturers' instructions, using 10
.mu.g/ml anti-human CEA antibody (Santa Cruz Biotechnology,
sc-23928.
[0409] In another experiment (FIG. 19A), the CEA CD3 TCB molecule B
was characterized in the presence of another low CEA-expressing
primary epithelial cell line (CCD841CoN) versus different tumor
cell lines to assess its safety, using another PBMC donor. The
assay set-up and antibody range was as described in Example 5D for
the CEA CD3 TCB molecule B. EC50 values of tumor cell lysis were
calculated using Graph Pad Prism6 and are given in Table 10 (48
h).
TABLE-US-00011 TABLE 10 EC50 values (pM) for T-cell mediated lysis
of CEA-expressing tumor cells induced by CEA CD3 TCB molecule B
after 48 h. CEA binding Cell Line Reference sites Molecule B MKN45
DSMZ ~513 300 41.24 #ACC409 BxPC-3 ECACC ~44 000 46.98 #93120816
HT-29 DSMZ ~10 000 562.7 #ACC299 CCD- ATCC <600 -- 841CoN
#CRL-1790
[0410] CEA binding sites were determined by a FACS-based Qifikit
analysis, according to the manufacturers' instructions, using 10
.mu.g/ml anti-human CEA antibody (Santa Cruz Biotechnology,
sc-23928).
[0411] As depicted in FIG. 19A and Table 10, primary epithelial
cells were not killed by CEA CD3 TCB molecule B, whereas tumor cell
lines with varying CEA expression levels could be lysed by the CEA
CD3 TCB molecule in a concentration-dependent manner.
Example 7B
[0412] CD69 Up-Regulation on CD4+ Effector Cells after Killing of
CEA-Expressing Tumor Cells Induced by CEA CD3 TCB Molecule B
[0413] Activation of CD4+ and CD8+ effector cells after lysis of
CEA-expressing tumor cells mediated by the CEA CD3 TCB molecule B
was assessed by FACS analysis using antibodies recognizing the T
cell activation marker CD69 (early activation marker).
[0414] The antibody and the killing assay conditions were
essentially as described above (Example 5D), using the same
antibody concentration range (0.26 pM-20 nM in triplicates), E:T
ratio 10:1 and an incubation time of 48 h.
[0415] 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 Biosciences #555634), CD4 (PECy7
anti-human CD4, BD Biosciences #557852) and CD69 (PE anti-human
CD69 BioLegend #310906) was performed according to the suppliers'
indications. Cells were washed twice with 150 .mu.l/well PBS
containing 0.1% BSA and fixed for 30 min at 4.degree. C. using 150
.mu.l/well of FACS buffer, containing 2% PFA. After centrifugation,
the samples were re-suspended in 200 .mu.l/well PBS 0.1% BSA and
analyzed using a BD FACS Fortessa.
[0416] FIG. 18B shows that the CEA CD3 TCB molecule B induced
concentration-dependent T cell activation in the presence of
different CEA-expressing tumor cell lines, as measured by
percentage of CD69-positive CD4+ T cells. In contrast, no T cell
activation occurred in the presence of low CEA-expressing primary
epithelial cells. Similar results were obtained for CD8+ cells
(data not shown). The data suggests that therapeutic administration
of CEA CD3 TCB molecule B should not lead to adverse effects on
primary epithelial cells with low CEA expression levels.
[0417] EC50 values of T-cell activation were calculated using Graph
Pad Prism6 and are given in Table 11.
TABLE-US-00012 TABLE 11 EC50 values (pM) for T-cell activation upon
simultaneous binding of CEA CD3 TCB molecule B to CEA-expressing
cells and CD3-expressing T cells after 48 h CEA binding Cell Line
Reference sites Molecule B BxPC-3 ECACC ~44 000 84.74 #93120816
NCI-H2122 ATCC ~13 300 149.1 #CRL-598 COR-L105 Sigma-Aldrich ~1 200
n.c.* #92031918 HBEpiC ScienCell #3210 <600 -- *The EC50 for
COR-L105 could not be calculated properly because the curve did not
reach saturation at high concentrations.
[0418] CEA binding sites were determined by a FACS-based Qifikit
analysis, according to the manufacturers' instructions, using 10
.mu.g/ml anti-human CEA antibody (Santa Cruz Biotechnology,
sc-23928).
Example 7C
[0419] CD25 Up-Regulation on CD4+ Effector Cells after Killing of
CEA-Expressing Tumor Cells Induced by CEA CD3 TCB Molecule B
[0420] Activation of CD4+ and CD8+ after lysis of CEA-expressing
tumor cells mediated by the CEA CD3 TCB molecule B was assessed by
FACS analysis using antibodies recognizing the T cell activation
marker CD25 (late activation marker).
[0421] The antibody and the killing assay conditions were
essentially as described above (Example 5D), using the same
antibody concentration range (0.26 pM-20 nM in triplicates), E:T
ratio 10:1 and an incubation time of 48 h.
[0422] After the incubation, PBMCs were transferred to a
round-bottom 96-well plate, centrifuged at 350.times.15 g for 5 min
and washed twice with PBS containing 0.1% BSA. Surface staining for
CD8 (FITC anti-human CD8, BioLegend #344704), CD4 (PECy7 anti-human
CD4, BioLegend #344612) and CD25 (APC anti-human CD25 BioLegend
#302610) was performed according to the suppliers' indications.
Cells were washed twice with 150 .mu.l/well PBS containing 0.1% BSA
and fixed for 30 min at 4.degree. C. using 150 .mu.l/well of FACS
buffer, containing 2% PFA. After centrifugation, the samples were
re-suspended in 200 .mu.l/well PBS 0.1% BSA and analyzed using a BD
FACS Fortessa.
[0423] FIG. 19B shows that the CEA CD3 TCB molecule B induced
concentration-dependent T cell activation in the presence of
CEA-expressing tumor cells, as measured by percentage of
CD25-positive T cells. In contrast, no T cell activation occurred
in the presence of low CEA-expressing primary epithelial cells.
Similar results were obtained for CD8+ cells (data not shown). The
data suggests that therapeutic administration of CEA CD3 TCB
molecule B should not lead to adverse effects on primary epithelial
cells with low CEA expression levels.
Example 8
Specific Binding of CEA CD3 Molecule B to Human CEACAM5
[0424] To show the specific binding of the CEA CD3 TCB molecule B
to human CEACAM5, but not to the other closest family members
CEACAM1 and CEACAM6, binding of CEA CD3 TCB molecule B to transient
HEK293T transfected cells, expressing either human CEACAM5, CEACAM1
or CEACAM6, was evaluated.
[0425] Briefly, cells were harvested, counted, checked for
viability and re-suspended at 1.times.10.sup.6 cells/ml in FACS
buffer (100 .mu.l PBS 0.1% BSA). 100 .mu.l of the cell suspension
(containing 0.1.times.10.sup.6 cells) were plated iton round-bottom
96-well plates and washed twice with 150 .mu.l of cold PBS. Cells
were stained for 30 min at 4.degree. C., using a 1:5000 pre-diluted
suspension of the fixable viability dye eFluor660 (eBioscience,
#65-0864-14) in PBS. Thereafter, the cells were washed twice with
PBS, once with FACS buffer and stained for 30 min at 4.degree. C.
with increasing concentrations of the CEA CD3 TCB molecule B. The
antibody concentration range was 30.5 pM-500 nM. Cells were 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 Fcg Fragment Specific secondary antibody
(Jackson Immuno Research Lab FITC #109-096-098, 1:50 pre-diluted in
PBS 0.1% BSA), washed twice with cold PBS 0.1% BSA and fixed using
150 .mu.l PBS 0.1% BSA, containing 2% PFA and incubation at
4.degree. C. for 20 min. Thereafter, cells were washed once for 8
min at 400.times.g, 4.degree. C. and finally re-suspended in 150
.mu.l FACS buffer for the FACS measurement. Fluorescence was
measured using BD FACS CantoII. Binding curves were obtained using
GraphPadPrism6 (FIG. 20A).
[0426] To determine the transfection efficacy, all transfectants
were stained for 30 min at 4.degree. C., using a commercially
available anti-human CD66 antibody (FITC mouse anti-human CD66, BD
Biosciences #551479, 20 .mu.l per sample) (FIG. 20B). As shown in
FIG. 20B, the highest expression level was detected for human
CEACAM1, followed by CEACAM5 and CEACAM6.
[0427] FIG. 20A clearly shows that the CEA CD3 TCB molecule B shows
concentration-dependent binding to transient transfectants
expressing human CEACAM5, but not to any of the other
transfectants, expressing human CEACAM1 or CEACAM6, demonstrating
the specificity of the binding to CEA.
Example 9
Single Dose PK of CEA CD3 TCB Molecules Comprising Different
Humanized CEA Binders in Healthy NOG Mice
[0428] Single dose pharmacokinetic studies (SDPK) were performed in
healthy NOG mice to evaluate exposure of CEA CD3 TCB molecule B and
CEA CD3 TCB molecule X (FIG. 15). An intravenous (i.v.) bolus
injection of 0.5 mg/kg was administered to NOG mice and blood
samples were taken at selected time points for pharmacokinetic
evaluation. A generic immunoassay was used for measuring total
concentrations of CEA CD3 TCB molecules. The calibration range of
the standard curve for both TCB molecules was 0.078 to 5 ng/ml,
where 1.5 ng/ml is the lower limit of quantification (LLOQ).
[0429] A biphasic decline was observed with a half-life of 10 days
(non-compartmental analysis) for CEA CD3 TCB molecule B and 6.5
days for CEA CD3 TCB molecule X. A clearance (CL) of 8.1 ml/d/kg
was detected for CEA CD3 TCB molecule B and 19 ml/d/kg for CEA CD3
TCB molecule X, respectively (Table 12). Overall, CEA CD3 TCB
molecule B showed longer half-life and lower CL in NOG mice
compared to CEA CD3 TCB molecule X.
[0430] The Phoenix v6.4 from Pharsight Ltd was used for PK
analysis, modelling and simulation.
TABLE-US-00013 TABLE 12 Pharmacokinetic parameters of a 0.5 mg/kg
iv bolus administration of CEA CD3 TCB molecules in NOG mice.
Half-life CL Construct (d) (mL/d/kg) Molecule B 10 8.1 Molecule X
6.5 19
Example 10
Anti-Tumor Activity of CEA CD3 TCB Molecules Comprising Different
Humanized CEA Binders in the MKN45 Model
[0431] Anti-tumor activity of CEA CD3 TCB molecule B and CEA CD3
TCB molecule X was tested in fully humanized
NOD/Shi-scid/IL-2R.gamma..sup.null (NOG) mice bearing the gastric
carcinoma cell line MKN45.
[0432] Fully humanized NOG mice at 14 weeks of age, bearing
physiological levels of circulating human B- and T-cells (Hayakawa
et al., Stem Cells 27 (2009) 175-182), were injected sub-cutaneous
(s.c.) with 1.times.10.sup.6 MKN45 cells (originally obtained from
the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH). When average tumor volume reached 100 mm.sup.3, mice
received CEA CD3 TCB molecule X or CEA CD3 TCB molecule B i.v. at
the dose of 2.5 mg/kg administered either twice or once a week, and
at the dose of 0.5 mg/kg administered once a week (FIG. 21). CEA
CD3 TCB molecule B showed significantly stronger anti-tumor
activity at all tested doses and schedules. Importantly, only CEA
CD3 TCB molecule B could mediate tumor regression with tumor-free
mice detected in the 2.5 mg/kg and 0.5 mg/kg treated groups (FIG.
21, Table 13).
TABLE-US-00014 TABLE 13 Number of tumor-free mice per group at
study termination (day 70). Tumor-free mice at termination
Treatment (study day 70) Vehicle 0/9 Molecule X 2.5 mg/kg
twice/week 0/9 Molecule B 2.5 mg/kg twice/week 3/9 Molecule X 2.5
mg/kg once/week 0/9 Molecule B 2.5 mg/kg once/week 1/9 Molecule X
0.5 mg/kg once/week 0/9 Molecule B 0.5 mg/kg once/week 1/8
Example 11
[0433] Based on the SDPK data shown in Example 9, a 2-compartmental
model was compiled to describe the PK of CEA CD3 TCB molecule B and
CEA CD3 TCB molecule X in NOG mice (FIG. 22). The different dose
levels and schedules selected for the dose-range efficacy study
described in the next example were simulated. As shown in FIG. 22,
to compensate for the lower clearance of CEA5 CD3 TCB molecule B
the design of the study was adapted. The frequency of
administration for CEA CD3 TCB molecule X was increased to a twice
weekly schedule and also higher dose levels up to 12.5 mg/kg were
used. The simulated profiles show that at a bi-weekly dose of 12.5
mg of CEA CD3 TCB molecule X the exposure is substantially higher
as compared to 0.5 mg/kg CEA CD3 TCB molecule B.
Example 12
Dose-Range Efficacy Study with CEA CD3 TCB Molecules Comprising
Different Humanized CEA Binders in the MKN45 Model
[0434] To more specifically assess the in vivo fold difference in
anti-tumor activity between the two molecules, CEA CD3 TCB molecule
B and CEA CD3 TCB molecule X were tested at a larger range of doses
in fully humanized NSG mice bearing the gastric carcinoma cell line
MKN45 (FIG. 23).
[0435] Fully humanized NSG mice were injected s.c. with
1.times.10.sup.6 MKN45 cells. When average tumor volume reached 180
mm.sup.3, mice received CEA CD3 TCB molecule B or CEA CD3 TCB
molecule X i.v. at the different doses and schedules depicted in
FIG. 23. The doses and schedules were selected based on the
analysis described in Example 11. The efficacy data obtained
clearly show that CEA CD3 TCB molecule B could mediate stronger
anti-tumor activity when compared to CEA CD3 TCB molecule X with a
significant fold difference of at least 25 times (as highlighted by
the star in the graph on FIG. 23).
Example 13
Anti-Tumor Activity of CEA CD3 TCB Molecules Comprising Different
Humanized CEA Binders in the HPAF-II Model
[0436] Anti-tumor activity of CEA CD3 TCB molecule B and CEA CD3
TCB molecule X was tested in NOD.Cg-Prkdc.sup.scid
Il2rg.sup.tmlWjl/SzJ (NSG) mice bearing the human pancreatic
carcinoma cell line HPAF-II and transferred with human peripheral
mononuclear cells (PBMC). Briefly, female NOG mice were injected
sub-cutaneously (s.c.) with 1.times.10.sup.6 HPAF-II cells
(originally obtained from the American Type Culture Collection
(ATCC)). When average tumor volume reached 150 mm.sup.3, mice
received i.v. injection of human PBMC (10.times.10.sup.6 cells per
mouse) as source of human T-cells. Three days later, mice received
CEA CD3 TCB molecule B or CEA CD3 TCB molecule X i.v. at a dose of
2.5 mg/kg, administered once a week. As depicted in FIG. 24, after
3 weeks treatment, both TCB molecules show potent anti-tumor
activity, with only molecule B able to mediate tumor regression (at
day 32, study day termination) (FIG. 24).
Example 14
Single Dose PK of CEA CD3 TCB Molecules Comprising Different
Humanized CEA Binders in Cynomolgus Monkeys
[0437] Single dose pharmacokinetic studies (SDPK) were performed in
cynomolgus monkeys to assess the exposure of CEA CD3 TCB molecule B
and CEA CD3 TCB molecule B, respectively (FIG. 25). An IV bolus
administration of 0.01 mg/kg was administered and blood samples
were taken at selected time points for pharmacokinetic evaluation.
Specific immunoassays were used measuring binding competent
concentrations of CEA CD3 TCB molecule B and CEA CD3 TCB molecule
X. For CEA CD3 TCB molecule X the lower limit of quantification
(LLOQ) was 0.1 ng/ml and for CEA CD3 TCB molecule B 0.44 ng/ml.
[0438] A biphasic decline was observed with a half-life of
184.+-.40 hours (non-compartmental analysis) for CEA CD3 TCB
molecule B versus 32.+-.11 hours for CEA CD3 TCB molecule X. A
clearance (CL) of 7.+-.0.9 ml/d/kg was detected for CEA CD3 TCB
molecule B and 25.+-.6 ml/d/kg for CEA CD3 TCB molecule X. Overall,
CEA CD3 TCB molecule B showed IgG-like properties and displayed a
longer half-life and a slower clearance in cynomolgus monkeys as
compared to CEA CD3 TCB molecule X.
TABLE-US-00015 TABLE 14 Summary of pharmacokinetic parameters of
CEA CD3 TCB molecule B in serum after a single intravenous (bolus)
administration of 0.01 mg/kg to cynomolgus monkeys (N = 3) Half
Life CL Cmax AUC.sub.INF Vc ID (h) (ml/clay/kg) (ng/ml) (h*ng/ml)
(ml/kg) Mean 184 7 257 33351 39 SD 40 0.9 18 4259 2 CV % 22 13 7 13
6
TABLE-US-00016 TABLE 15 Summary of pharmacokinetic parameters of
CEA CD3 TCB molecule X in serum after a single intravenous (bolus)
administration of 0.01 mg/kg to cynomolgus monkeys (N = 5). Half
Life CL Cmax AUC.sub.INF Vc ID (h) (ml/clay/kg) (ng/ml) (h*ng/ml)
(ml/kg) Mean 32 25 321 9991 31 SD 11 6 24 2133 2 CV % 34 23 7 21
7
[0439] The Phoenix v6.4 from Pharsight Ltd was used for PK
assessment.
[0440] 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
521207PRTHomo sapiens 1Met 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 2052198PRTMacaca
fascicularis 2Met 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
1953125PRTArtificial SequenceCD3 VH 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 SequenceCD3 HCDR1 4Thr Tyr Ala Met Asn1
5519PRTArtificial SequenceCD3 HCDR2 5Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys
Gly614PRTArtificial SequenceCD3 HCDR3 6His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr1 5 107109PRTArtificial SequenceCD3 VL
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 SequenceCD3 LCDR1 8Gly Ser
Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5
1097PRTArtificial SequenceCD3 LCDR2 9Gly Thr Asn Lys Arg Ala Pro1
5109PRTArtificial SequenceCD3 LCDR3 10Ala Leu Trp Tyr Ser Asn Leu
Trp Val1 51110PRTArtificial Sequencelinker 11Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser1 5 101211PRTArtificial Sequencelinker 12Asp Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 1013225PRTHomo sapiens 13Asp
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 220Pro225145PRTMus musculus 14Asp Thr Tyr
Met His1 51517PRTMus musculus 15Arg Ile Asp Pro Ala Asn Gly Asn Ser
Lys Tyr Val Pro Lys Phe Gln1 5 10 15Gly1612PRTMus musculus 16Phe
Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr1 5 101715PRTMus
musculus 17Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu
His1 5 10 15187PRTMus musculus 18Arg Ala Ser Asn Arg Ala Thr1
5199PRTMus musculus 19Gln Gln Thr Asn Glu Asp Pro Tyr Thr1
520121PRTMus musculus 20Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Glu Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr
Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Thr Ser
Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly
Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly
Thr Ser Val Thr Val Ser Ser 115 12021111PRTMus musculus 21Asp Ile
Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln
Arg Ala Thr Met Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25
30Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro
Val 50 55 60Arg Phe Ser Gly Thr Gly Ser Arg Thr Asp Phe Thr Leu Ile
Ile Asp65 70 75 80Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys
Gln Gln Thr Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 11022121PRTArtificial SequenceHumanized CEA
binder VH 22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile
Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys
Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val
Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ser 115 12023111PRTArtificial SequenceHumanized CEA
binder VL 23Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val
Asp Ile Phe 20 25 30Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Thr Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110245PRTArtificial
SequenceCEA HCDR1 24Glu Phe Gly Met Asn1 52517PRTArtificial
SequenceCEA HCDR2 25Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val
Glu Glu Phe Lys1 5 10 15Gly2612PRTArtificial SequenceCEA HCDR3
26Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr1 5
102711PRTArtificial SequenceCEA LCDR1 27Lys Ala Ser Ala Ala Val Gly
Thr Tyr Val Ala1 5 10287PRTArtificial SequenceCEA LCDR2 28Ser Ala
Ser Tyr Arg Lys Arg1 52910PRTArtificial SequenceCEA LCDR3 29His Gln
Tyr Tyr Thr Tyr Pro Leu Phe Thr1 5 1030121PRTArtificial SequenceCEA
VH 30Gln 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 12031108PRTArtificial SequenceCEA VL 31Asp 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 10532449PRTArtificial Sequenceparental CEA VH-CH1(EE)-Fc (hole,
P329G LALA) 32Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Glu
Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn
Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln
Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser
Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr Ala Asp
Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Thr Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr
Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Ser
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 Glu 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 Glu 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 445Pro33674PRTArtificial
Sequenceparental CEA VH-CH1(EE)-CD3 VL-CH1-Fc (knob, P329G LALA)
33Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Glu Pro Gly Ala1
5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu
Trp Ile 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val
Pro Lys Phe 50 55 60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser
Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Thr Ser Leu Thr Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp
Tyr Ala Met Ala Tyr Trp Gly
100 105 110Gln Gly Thr Ser 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val
Thr225 230 235 240Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr
Val Thr Leu Thr 245 250 255Cys Gly Ser Ser Thr Gly Ala Val Thr Thr
Ser Asn Tyr Ala Asn Trp 260 265 270Val Gln Glu Lys Pro Gly Gln Ala
Phe Arg Gly Leu Ile Gly Gly Thr 275 280 285Asn Lys Arg Ala Pro Gly
Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 290 295 300Gly Gly Lys Ala
Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu305 310 315 320Ala
Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 325 330
335Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro
340 345 350Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr 355 360 365Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr 370 375 380Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro385 390 395 400Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410 415Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 420 425 430His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 435 440 445Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 450 455
460Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu465 470 475 480Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser 485 490 495His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu 500 505 510Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr 515 520 525Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro545 550 555 560Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570
575Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
580 585 590Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 595 600 605Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 610 615 620Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr625 630 635 640Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 645 650 655Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670Ser
Pro34232PRTArtificial SequenceCD3 VH-CL 34Glu 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 Val 115 120 125Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys 130 135 140Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg145 150 155 160Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200
205Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
210 215 220Lys Ser Phe Asn Arg Gly Glu Cys225 23035218PRTArtificial
Sequenceparental CEA VL-CL(RK) 35Asp Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Met Ser Cys
Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30Gly Val Gly Phe Leu His
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Val 50 55 60Arg Phe Ser Gly
Thr Gly Ser Arg Thr Asp Phe Thr Leu Ile Ile Asp65 70 75 80Pro Val
Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 21536449PRTArtificial
Sequencehumanized CEA VH-CH1(EE)-Fc (hole, P329G LALA) 36Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala
Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu 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 Glu 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 Glu 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 445Pro37674PRTArtificial Sequencehumanized CEA
VH-CH1(EE)-CD3 VL-CH1-Fc (knob, P329G LALA) 37Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp
Gly 100 105 110Gln Gly Thr Leu 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val
Val Thr225 230 235 240Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
Thr Val Thr Leu Thr 245 250 255Cys Gly Ser Ser Thr Gly Ala Val Thr
Thr Ser Asn Tyr Ala Asn Trp 260 265 270Val Gln Glu Lys Pro Gly Gln
Ala Phe Arg Gly Leu Ile Gly Gly Thr 275 280 285Asn Lys Arg Ala Pro
Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 290 295 300Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu305 310 315
320Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly
325 330 335Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys
Gly Pro 340 345 350Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 355 360 365Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr 370 375 380Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro385 390 395 400Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410 415Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 420 425 430His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 435 440
445Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
450 455 460Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu465 470 475 480Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 485 490 495His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 500 505 510Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 515 520 525Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro545 550 555
560Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val 580 585 590Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 595 600 605Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 610 615 620Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr625 630 635 640Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 645 650 655Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670Ser
Pro38218PRTArtificial Sequencehumanized CEA VL-CL(RK) 38Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30Gly
Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40
45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Thr Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Arg Lys 115 120 125Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185
190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21539225PRTArtificial SequenceFc (hole, P329G LALA) 39Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly Ala
Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys Ala 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 Val 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 220Pro22540439PRTArtificial SequenceCD3 VL-CH1-Fc (knob,
P329G LALA) 40Gln 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
Asp Lys Thr His Thr Cys Pro Pro Cys Pro 210 215 220Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys225 230 235 240Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250
255Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
260 265 270Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 275 280 285Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys305 310 315 320Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu 340 345 350Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375
380Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu385 390 395 400Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val 405 410 415Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln 420 425 430Lys Ser Leu Ser Leu Ser Pro
43541684PRTArtificial Sequencehumanized CEA VH-CH1(EE)-CD3
VL-CH1-Fc (knob, P329G LALA) (longer linker) 41Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp
Gly 100 105 110Gln Gly Thr Leu 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser225 230 235 240Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln
Glu Pro Ser Leu Thr 245 250 255Val Ser Pro Gly Gly Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly 260 265 270Ala Val Thr Thr Ser Asn Tyr
Ala Asn Trp Val Gln Glu Lys Pro Gly 275 280 285Gln Ala Phe Arg Gly
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly 290 295 300Thr Pro Ala
Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu305 310 315
320Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala
325 330 335Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr 340 345 350Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala 355 360 365Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu 370 375 380Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly385 390 395 400Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 405 410 415Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 420 425 430Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 435 440
445Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
450 455 460Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val Phe465 470 475 480Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 485 490 495Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 500 505 510Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 515 520 525Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 530 535 540Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys545 550 555
560Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
565 570 575Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 580 585 590Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Trp Cys Leu Val 595 600 605Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 610 615 620Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp625 630 635 640Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 645 650 655Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 660 665 670Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 675
68042694PRTArtificial SequenceCEA VH-CH1-CD3 VH-CL-Fc (knob, P329G
LALA) 42Gln 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
69043451PRTArtificial SequenceCEA VH-CH1-Fc (hole, P329G LALA)
43Gln 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 45044214PRTArtificial
SequenceCD3 VL-CH1 44Gln 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
21045215PRTArtificial SequenceCEA VL-CL 45Asp 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 2154619PRTArtificial SequenceCD3
HCDR2 46Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
Ser1 5 10 15Val Lys Asp4714PRTArtificial SequenceCD3 LCDR1 47Arg
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5
1048125PRTArtificial SequenceCD3 VH 48Glu 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
Asp 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 12549109PRTArtificial SequenceCD3 VL 49Gln Ala Val Val Thr Gln
Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr
Cys Arg 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
10550674PRTArtificial Sequencehumanized CEA VH-CH1-CD3 VL-CH1-Fc
(knob, P329G LALA) 50Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly
Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly
Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly
Thr Leu 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 Gln Ala Val Val Thr225 230 235
240Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr
245 250 255Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala
Asn Trp 260 265 270Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu
Ile Gly Gly Thr 275 280 285Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg
Phe Ser Gly Ser Leu Leu 290 295 300Gly Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala Gln Pro Glu Asp Glu305 310 315 320Ala Glu Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 325 330 335Gly Gly Thr
Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro 340 345 350Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 355 360
365Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
370 375 380Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro385 390 395 400Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr 405 410 415Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn 420 425 430His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser 435 440 445Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 450 455 460Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu465 470 475
480Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
485 490 495His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 500 505 510Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 515 520 525Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 530 535 540Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro545 550 555 560Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575Val Tyr Thr
Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val 580 585 590Ser
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600
605Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
610 615 620Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr625 630 635 640Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 645 650 655Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 660 665 670Ser Pro51449PRTArtificial
Sequencehumanized CEA VH-CH1-Fc (hole, P329G LALA) 51Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met
Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu 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 445Pro52218PRTArtificial Sequencehumanized CEA VL-CL 52Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe
20 25 30Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro 35 40 45Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Thr Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170
175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215
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