U.S. patent application number 15/434921 was filed with the patent office on 2017-08-17 for bcma antibodies and use of same to treat cancer and immunological disorders.
This patent application is currently assigned to SEATTLE GENETICS, INC.. The applicant listed for this patent is SEATTLE GENETICS, INC.. Invention is credited to Michael Feldhaus, Maureen Ryan, Django Sussman, Lori Westendorf.
Application Number | 20170233484 15/434921 |
Document ID | / |
Family ID | 59562012 |
Filed Date | 2017-08-17 |
United States Patent
Application |
20170233484 |
Kind Code |
A1 |
Sussman; Django ; et
al. |
August 17, 2017 |
BCMA ANTIBODIES AND USE OF SAME TO TREAT CANCER AND IMMUNOLOGICAL
DISORDERS
Abstract
The invention provides humanized antibodies that specifically
bind to BCMA. The antibodies are useful for treatment and diagnoses
of various cancers and immune disorders as well as detecting
BCMA.
Inventors: |
Sussman; Django; (Seattle,
WA) ; Ryan; Maureen; (Bellevue, WA) ;
Westendorf; Lori; (Snohomish, WA) ; Feldhaus;
Michael; (Lebanon, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEATTLE GENETICS, INC. |
Bothell |
WA |
US |
|
|
Assignee: |
SEATTLE GENETICS, INC.
Bothell
WA
|
Family ID: |
59562012 |
Appl. No.: |
15/434921 |
Filed: |
February 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62396084 |
Sep 16, 2016 |
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62296594 |
Feb 17, 2016 |
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Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/522 20130101; A61K 38/05 20130101; C07K 2317/14 20130101;
C07K 2317/734 20130101; C07K 16/2878 20130101; C07K 2317/24
20130101; C07K 2317/76 20130101; A61K 47/6803 20170801; A61P 35/00
20180101; C07K 2317/72 20130101; A61K 31/40 20130101; A61K 47/6851
20170801; C07K 2317/71 20130101; A61K 47/6849 20170801; C07K
2317/732 20130101; C07K 2317/41 20130101; C07K 2317/524 20130101;
A61K 31/5517 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; A61K 38/05 20060101
A61K038/05 |
Claims
1. A humanized, chimeric or veneered antibody, which is a
humanized, chimeric or veneered form of an antibody deposited as
ATCC PTC-6937.
2. The antibody of claim 1, comprising a mature heavy chain
variable region having at least 90% sequence identity to hSG16.17
VH3 (SEQ ID NO: 13) and a mature light chain variable region having
at least 90% sequence identity to hSG16.17 VK2 (SEQ ID NO: 19).
3. The antibody of claim 2, comprising a mature heavy chain
variable region having at least 95% sequence identity to hSG16.17
VH3 (SEQ ID NO: 13) and a mature light chain variable region having
at least 95% sequence identity to hSG16.17 VK2 (SEQ ID NO: 19).
4. The antibody of claim 2, comprising the three Kabat CDRs (SEQ ID
NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three Kabat CDRs
(SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19) provided that
position H58 can be occupied by N or K, position H60 can be
occupied by A or N, position H61 can be occupied by Q or E,
position H62 can be occupied by K or N, position H64 can be
occupied by Q or K, position H65 can be occupied by G or T,
position L24 can be occupied by R or L, and position L53 can be
occupied by S or R.
5. The antibody of claim 2 comprising the three Kabat CDRs (SEQ ID
NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three Kabat CDRs
(SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19).
6. The antibody of claim 2, wherein positions H20, H48, H69, H71,
H73, H76, H80, H88, H91 and H93 are occupied by L, I, M, A, K, N,
V, A, F, and T respectively, and positions L46, L48 and L87 are
occupied by V, V and F respectively.
7. The antibody of claim 1, wherein the mature heavy chain variable
has the sequence of hSG16.17 VH3 (SEQ ID NO: 13) and the mature
light chain variable region has the sequence of hSG16.17 VK2 (SEQ
ID NO: 19).
8. The antibody of claim 2, wherein the mature heavy chain variable
region is fused to a heavy chain constant region and the mature
light chain variable region is fused to a light chain constant
region.
9. The antibody of claim 6, wherein the heavy chain constant region
is a mutant form of natural human constant region which has reduced
binding to an Fc.gamma. receptor relative to the natural human
constant region.
10. The antibody of claim 8, wherein the heavy chain constant
region is of IgG1 isotype.
11. The antibody of claim 8, wherein the heavy chain constant
region has an amino acid sequence comprising SEQ ID NO: 5 and the
light chain constant region has an amino acid sequence comprising
SEQ ID NO: 3.
12. The antibody of claim 8, wherein the heavy chain constant
region has an amino acid sequence comprising SEQ ID NO:7 (S239C)
and the light chain constant region has an amino acid sequence
comprising SEQ ID NO:3.
13. The antibody of claim 2, which is a naked antibody.
14. The antibody of any of claim 2, wherein the antibody is
conjugated to a cytotoxic or cytostatic agent.
15. The antibody of claim 14, wherein the antibody is conjugated to
a cytotoxic agent.
16. The antibody of claim 15, wherein the cytotoxic agent is
conjugated to the antibody via an enzyme cleavable linker.
17. The antibody of claim 15, wherein the cytotoxic agent is a DNA
minor groove binder.
18. The antibody of claim 17, wherein the cytotoxic agent has the
formula ##STR00008##
19. The antibody of claim 15, wherein the cytotoxic agent is MMAE
or MMAF.
20. A pharmaceutical composition comprising an antibody of claim 2
and a pharmaceutically acceptable carrier.
21. A method of treating a patient having or at risk of having a
cancer that expresses BCMA comprising administering to the patient
an effective regime of an antibody of any claim 2.
22. The method of claim 20, wherein the cancer is a hematological
cancer.
23. The method of claim 22, wherein the hematological cancer is a
myeloma, leukemia or a lymphoma.
24. The method of claim 22, wherein the hematological cancer is
multiple myeloma.
25. The method of claim 22, wherein the hematological cancer is
non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma.
26. The method of claim 22, wherein the hematological cancer is
myelodysplastic syndromes (MDS), myeloproliferative syndromes
(MPS), Waldenstrom's macroglobulinemia or Burkett's lymphoma.
27. A method of treating a patient having or at risk of having an
immune disorder mediated by immune cells expressing BCMA comprising
administering to the patient an effective regime of a humanized
antibody of claim 2.
28. The method of claim 27, which is a B cell mediated
disorder.
29. The method of claim 27, wherein the immune disorder is
rheumatoid arthritis, systemic lupus E (SLE), Type I diabetes,
asthma, atopic dermitus, allergic rhinitis, thrombocytopenic
purpura, multiple sclerosis, psoriasis, Sjorgren's syndrome,
Hashimoto's thyroiditis, Grave's disease, primary biliary
cirrhosis, Wegener's granulomatosis, tuberculosis, and graft versus
host disease.
30. A humanized, chimeric or veneered antibody, which is a
humanized, chimeric or veneered form of the rat SG16.45 antibody
having a mature heavy chain variable region of SEQ ID NO: 23 and a
mature light chain variable region of SEQ ID NO: 33.
31. The antibody of claim 30, comprising a mature heavy chain
variable region having at least 90% sequence identity to hSG16.45
VH5 (SEQ ID NO: 31) and a mature light chain variable region having
at least 90% sequence identity to hSG16.45 VK2 (SEQ ID NO: 36).
32. The antibody of claim 31, comprising a mature heavy chain
variable region having at least 95% sequence identity to hSG16.45
VH5 (SEQ ID NO: 31) and a mature light chain variable region having
at least 95% sequence identity to hSG16.45 VK2 (SEQ ID NO: 36).
33. The antibody of any of claim 31, comprising the three Kabat
CDRs (SEQ ID NOs: 152-154) of hSG16.45 VH5 (SEQ ID NO: 31) and
three Kabat CDRs (SEQ ID NOs: 179-181) of hSG16.45 VK2 (SEQ ID NO:
36) provided that position H50 can be occupied by A or S, position
L24 can be occupied by R or L and position L26 can be occupied by S
or T.
34. The antibody of any of claim 31 comprising the three Kabat CDRs
(SEQ ID NOs: 152-154) of hSG16.45 VH5 (SEQ ID NO: 31) and three
Kabat CDRs (SEQ ID NOs: 179-181) of hSG16.45 VK2 (SEQ ID NO:
36).
35. The antibody of any one of claim 31, wherein positions H30, H93
and H94 are occupied by N, T and S respectively.
36. The antibody of claim 30, wherein the mature heavy chain
variable region has the sequence of hSG16.45 VH5 (SEQ ID NO: 31)
and the mature light chain variable region has the sequence of
hSG16.45 VK2 (SEQ ID NO: 36) or the mature heavy chain variable
region has the sequence of hSG16.45 VH1 (SEQ ID NO: 27) and the
mature light chain variable region has the sequence of hSG16.45 VK1
(SEQ ID NO: 35) or the mature heavy chain variable region has the
sequence of hSG16.45 VH1 (SEQ ID NO: 27) and the mature light chain
variable region has the sequence of hSG16.45 VK3 (SEQ ID NO:
37).
37. The antibody of any one of claim 31, wherein the mature heavy
chain variable region is fused to a heavy chain constant region and
the mature light chain variable region is fused to a light chain
constant region.
38. The antibody of claim 37, wherein the heavy chain constant
region is a mutant form of natural human constant region which has
reduced binding to an Fc.gamma. receptor relative to the natural
human constant region.
39. The antibody of claim 37, wherein the heavy chain constant
region is of IgG1 isotype.
40. The antibody of claim 37, wherein the heavy chain constant
region has an amino acid sequence comprising SEQ ID NO: 5 and the
light chain constant region has an amino acid sequence comprising
SEQ ID NO: 3.
41. The antibody of claim 37, wherein the heavy chain constant
region has an amino acid sequence comprising SEQ ID NO:7 (S239C)
and the light chain constant region has an amino acid sequence
comprising SEQ ID NO:3.
42. The antibody of any one of claim 31, which is a naked
antibody.
43. The antibody of any one of claim 31, wherein the antibody is
conjugated to a cytotoxic or cytostatic agent.
44. The antibody of claim 43, wherein the antibody is conjugated to
a cytotoxic agent.
45. The antibody of claim 44, wherein the cytotoxic agent is
conjugated to the antibody via an enzyme cleavable linker.
46. The antibody of claim 43, wherein the cytotoxic agent is a DNA
minor groove binder.
47. The antibody of claim 46, wherein the cytotoxic agent has the
formula ##STR00009##
48. The antibody of claim 44, wherein the cytotoxic agent is MMAE
or MMAF.
49. The antibody of claim 2 or 31, wherein less than 5% of
N-glycoside linked sugar chains at an asn residue at EU position
297 of heavy chain constant region include fucose or an analog
thereof in which cells expressing the antibody were cultured to
reduce fucosylation of the antibody.
50. A pharmaceutical composition comprising an antibody of any one
of claim 31 and a pharmaceutically acceptable carrier.
51. A method of treating a patient having or at risk of having a
cancer that expresses BCMA comprising administering to the patient
an effective regime of a humanized antibody of any one of claim
31.
52. The method of claim 51, wherein the cancer is a hematological
cancer.
53. The method of claim 52, wherein the hematological cancer is a
myeloma, leukemia or a lymphoma.
54. The method of claim 52, wherein the hematological cancer is
multiple myeloma.
55. The method of claim 52, wherein the hematological cancer is
non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma.
56. The method of claim 52, wherein the hematological cancer is
myelodysplastic syndromes (MDS), myeloproliferative syndromes
(MPS), Waldenstrom's macroglobulinemia or Burkett's lymphoma.
57. A method of treating a patient having or at risk of having an
immune disorder mediated by immune cells expressing BCMA comprising
administering to the patient an effective regime of an antibody of
claim 31.
58. The method of claim 56, which is a B cell mediated
disorder.
59. The method of claim 56, wherein the immune disorder is
rheumatoid arthritis, systemic lupus E (SLE), Type I diabetes,
asthma, atopic dermitus, allergic rhinitis, thrombocytopenic
purpura, multiple sclerosis, psoriasis, Sjorgren's syndrome,
Hashimoto's thyroiditis, Grave's disease, primary biliary
cirrhosis, Wegener's granulomatosis, tuberculosis, and graft versus
host disease.
60. A humanized antibody the specifically binds to the human BCMA
protein, the antibody comprising a mature heavy chain variable
region having at least 90% sequence identity to hSG16.17 VH3 (SEQ
ID NO: 13) and a mature light chain variable region having at least
90% sequence identity to hSG16.17 VK2 (SEQ ID NO: 19).
61. The antibody of claim 60, comprising a mature heavy chain
variable region having at least 95% sequence identity to hSG16.17
VH3 (SEQ ID NO: 13) and a mature light chain variable region having
at least 95% sequence identity to hSG16.17 VK2 (SEQ ID NO: 19).
62. The antibody of claim 60, comprising the three Kabat CDRs (SEQ
ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three Kabat CDRs
(SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19) provided that
position H58 can be occupied by N or K, position H60 can be
occupied by A or N, position H61 can be occupied by Q or E,
position H62 can be occupied by K or N, position H64 can be
occupied by Q or K, position H65 can be occupied by G or T,
position L24 can be occupied by R or L, and position L53 can be
occupied by S or R.
63. The antibody of claim 60 comprising the three Kabat CDRs (SEQ
ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three Kabat CDRs
(SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19).
64. The antibody of claim 60, wherein positions H20, H48, H69, H71,
H73, H76, H80, H88, H91 and H93 are occupied by L, I, M, A, K, N,
V, A, F, and T respectively, and positions L46, L48 and L87 are
occupied by V, V and F respectively.
65. The antibody of claim 60, wherein the mature heavy chain
variable has the sequence of hSG16.17 VH3 (SEQ ID NO: 13) and the
mature light chain variable region has the sequence of hSG16.17 VK2
(SEQ ID NO: 19).
66. The antibody of claim 60, wherein the mature heavy chain
variable region is fused to a heavy chain constant region and the
mature light chain variable region is fused to a light chain
constant region.
67. The antibody of claim 65, wherein the mature heavy chain
variable region is fused to a heavy chain constant region and the
mature light chain variable region is fused to a light chain
constant region.
68. A pharmaceutical composition comprising an antibody of claim 67
and a pharmaceutically acceptable carrier.
69. The pharmaceutical composition of claim 68, wherein less than
about 10% of the antibodies have core fucosylation by fucose or a
fucose analogue.
70. The pharmaceutical composition of claim 68, wherein less than
about 5% of the antibodies have core fucosylation by fucose or a
fucose analogue.
71. The pharmaceutical composition of claim 69, wherein about 2% of
the antibodies have core fucosylation by fucose or a fucose
analogue.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
application no. U.S. 62/296,594 filed Feb. 17, 2016 and U.S.
provisional application No. 62/396,084 filed Sep. 16, 2016, both of
which are incorporated herein by reference in their entirety for
all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] Sequences disclosed in the application are contained in a
sequence listing filed herewith.
BACKGROUND
[0003] B-cell maturation antigen (BCMA, CD269) is a member of the
TNF receptor superfamily. Expression of BCMA is restricted to the
B-cell lineage where it is predominantly expressed in the
interfollicular region of germinal centers and on differentiated
plasma cells and plasma blasts. BCMA binds to two distinct ligands,
a proliferation inducing ligand (APRIL) and B-cell activating
factor (BAFF, also known as BlyS, TALL-1, and THANK). The ligands
for BCMA bind two additional TNF receptors, transmembrane activator
and calcium modulator and cyclophilin ligand interactor (TACI) and
BAFF receptor (BAFF-R also called BR3). TACI binds APRIL and BAFF,
whereas BAFF-R shows restricted but high-affinity binding to BAFF.
Together, BCMA, TACI, BAFF-R, and their corresponding ligands
regulate different aspects of humoral immunity, B-cell development,
and homeostasis.
[0004] BCMA is virtually absent on naive and memory B cells (Novak
et al., Blood 103, 689-94 (2004)) but it is selectively induced
during plasma cell differentiation where it may support humoral
immunity by promoting the survival of normal plasma cells and
plasma blasts (O'Conner et al., J. Exp Med. 199, 91-98 (2004)).
BCMA has been reported to be expressed in primary multiple myeloma
(MM) samples. BCMA has also been detected on the Reed-Sternberg
cells (CD30.sup.+) from patients with Hodgkin's disease. It has
been reported based on knockdown experiments that that BCMA
contributed to both proliferation and survival of a Hodgkin's
disease cell line (Chiu et al., Blood 109, 729-39 (2007)).
SUMMARY OF THE CLAIMED INVENTION
[0005] The invention provides a humanized, chimeric or veneered
antibody, which is a humanized or chimeric form of an antibody
deposited as ATCC PTC-6937. Optionally the antibody comprises a
mature heavy chain variable region having at least 90% sequence
identity to hSG16.17 VH3 (SEQ ID NO: 13) and a mature light chain
variable region having at least 90% sequence identity to hSG16.17
VK2 (SEQ ID NO: 19). Optionally, the antibody comprises a mature
heavy chain variable region having at least 95% sequence identity
to hSG16.17 VH3 (SEQ ID NO: 13) and a mature light chain variable
region having at least 95% sequence identity to hSG16.17 VK2 (SEQ
ID NO: 19). Optionally, the antibody comprising the three Kabat
CDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three
Kabat CDRs (SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19)
provided that position H58 can be occupied by N or K, position H60
can be occupied by A or N, position H61 can be occupied by Q or E,
position H62 can be occupied by K or N, position H64 can be
occupied by Q or K, position H65 can be occupied by G or T,
position L24 can be occupied by R or L and position L53 can be
occupied by S or R. Optionally, the antibody comprises the three
Kabat CDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and
three Kabat CDRs (SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO:
19). Optionally, positions H58, H60, H61, H62, H64 and H65 are
occupied by N, A, Q K, Q and G respectively and L24 and L53 are
occupied by R and S respectively. Optionally, positions H20, H48,
H69, H71, H73, H76, H80, H88, H91 and H93 are occupied by L, I, M,
A, K, N, V, A, F, and T respectively, and positions L46, L48 and
L87 are occupied by V, V and F respectively. Optionally, the mature
heavy chain variable has the sequence of hSG16.17 VH3 (SEQ ID NO:
13) and the mature light chain variable region has the sequence of
hSG16.17 VK2 (SEQ ID NO: 19).
[0006] The invention further provides a humanized, chimeric or
veneered antibody, which is a humanized, chimeric or veneered form
of the rat SG16.45 antibody having the VH (SEQ ID NO: 23) and VK
(SEQ ID NO: 33) sequences. Optionally, the antibody comprises a
heavy chain mature variable region having at least 90% sequence
identity to hSG16.45 VH5 (SEQ ID NO: 31) and a mature light chain
variable region having at least 90% sequence identity to hSG16.45
VK2 (SEQ ID NO: 36). Optionally, the antibody comprises a mature
heavy chain variable region having at least 95% sequence identity
to hSG16.45 VH5 (SEQ ID NO: 31) and a mature light chain variable
region having at least 95% sequence identity to hSG16.45 VK2 (SEQ
ID NO: 36). Optionally, the comprises the three Kabat CDRs (SEQ ID
NOs: 152-154) of hSG16.45 VH5 (SEQ ID NO: 31) and three Kabat CDRs
(SEQ ID NOs: 179-181) of hSG16.45 VK2 (SEQ ID NO: 36) provided that
positions H50 can be occupied by A or S and position L24 can be
occupied by R or L and position L26 can be occupied by S or T.
Optionally, the antibody comprises the three Kabat CDRs (SEQ ID
NOs: 152-154) of hSG16.45 VH5 (SEQ ID NO: 31) and three Kabat CDRs
(SEQ ID NOs: 179-181) of hSG16.45 VK2 (SEQ ID NO: 36). Optionally
positions H30, H93 and H94 are occupied by N, T and S respectively.
Optionally, the mature heavy chain variable region has the sequence
of hSG16.45 VH5 (SEQ ID NO: 31) and the mature light chain variable
region has the sequence of hSG16.45 VK2 (SEQ ID NO: 36) or the
mature heavy chain variable region has the sequence of hSG16.45 VH1
(SEQ ID NO: 27) and the mature light chain variable region has the
sequence of hSG16.45 VK1 (SEQ ID NO: 35) or the mature heavy chain
variable region has the sequence of hSG16.45 VH1 (SEQ ID NO: 27)
and the mature light chain variable region has the sequence of
hSG16.45 VK3 (SEQ ID NO: 37).
[0007] In any of the above antibodies, the mature heavy chain
variable region can be fused to a heavy chain constant region and
the mature light chain variable region can be fused to a light
chain constant region. Optionally, the heavy chain constant region
is a mutant form of natural human constant region which has reduced
binding to an Fc.gamma. receptor relative to the natural human
constant region. Optionally, the heavy chain constant region is of
IgG1 isotype. Optionally, the heavy chain constant region has an
amino acid sequence comprising SEQ ID NO: 5 and the light chain
constant region has an amino acid sequence comprising SEQ ID NO: 3.
Optionally, the heavy chain constant region has an amino acid
sequence comprising SEQ ID NO:7 (S239C) and the light chain
constant region has an amino acid sequence comprising SEQ ID NO:3.
Optionally, the antibody is a naked antibody. Optionally, the
antibody is conjugated to a cytotoxic or cytostatic agent.
Optionally, the antibody is conjugated to a cytotoxic agent.
Optionally, the cytotoxic agent is conjugated to the via an enzyme
cleavable linker. Optionally, the cytotoxic agent is a DNA minor
groove binder, e.g., the cytotoxic agent having the formula
##STR00001##
Optionally, the cytotoxic agent is MMAE or MMAF.
[0008] The invention further provides pharmaceutical compositions
comprising any of the antibodies described above and a
pharmaceutically acceptable carrier.
[0009] In one embodiment, the invention provides an antibody
comprising the three Kabat CDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3
(SEQ ID NO: 13) and three Kabat CDRs (SEQ ID NOs: 90-92) of
hSG16.17 VK2 (SEQ ID NO: 19). In a further embodiment, the
invention provides an antibody having a mature heavy chain variable
with the sequence of hSG16.17 VH3 (SEQ ID NO: 13) and a mature
light chain variable region with the sequence of hSG16.17 VK2 (SEQ
ID NO: 19). In another embodiment, the mature heavy chain variable
region is fused to a heavy chain constant region and the mature
light chain variable region is fused to a light chain constant
region. The antibody can be, e.g., an IgG1 antibody. In another
embodiment, the antibody lacks core fucosylation by fucose or a
fucose analogue. The antibodies can by formulated into a
pharmaceutical composition, e.g., with addition of a
pharmaceutically acceptable carrier.
[0010] In a further embodiment, the pharmaceutical composition has
a plurality of antibodies having a mature heavy chain variable with
the sequence of hSG16.17 VH3 (SEQ ID NO: 13) and a mature light
chain variable region with the sequence of hSG16.17 VK2 (SEQ ID NO:
19). The variable regions of these antibodies are preferably fused
to appropriate heavy and light chain constant regions. In another
embodiment the antibodies are IgG1 antibodies. In a further
embodiment, the plurality of antibodies has less than about 5% of
the antibodies have core fucosylation by fucose or a fucose
analogue. In a further embodiment, the plurality of antibodies has
less than about 10% of the antibodies have core fucosylation by
fucose or a fucose analogue. In another embodiment, the plurality
of antibodies includes about 2% antibodies with core fucosylation
by fucose or a fucose analogue. In another embodiment, the
plurality of antibodies includes 2% antibodies with core
fucosylation by fucose or a fucose analogue.
[0011] The invention further provides a method of treating a
patient having or at risk of having a cancer that expresses BCMA
comprising administering to the patient an effective regime of an
antibody as described above. Optionally the cancer is a
hematological cancer. Optionally, the hematological cancer is a
myeloma, leukemia or a lymphoma. Optionally, the hematological
cancer is multiple myeloma. Optionally the hematological cancer is
non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma. Optionally, the
hematological cancer is myelodysplastic syndromes (MDS),
myeloproliferative syndromes (MPS), Waldenstrom's macroglobulinemia
or Burkett's lymphoma.
[0012] The invention further provides a method of treating a
patient having or at risk of having an immune disorder mediated by
immune cells expressing BCMA comprising administering to the
patient an effective regime of any of the above described
antibodies. Optionally, the disorder is a B cell mediated disorder.
Optionally, the immune disorder is rheumatoid arthritis, systemic
lupus E (SLE), Type I diabetes, asthma, atopic dermitus, allergic
rhinitis, thrombocytopenic purpura, multiple sclerosis, psoriasis,
Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease,
primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis,
and graft versus host disease.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1A shows the structure of BCMA.
[0014] FIG. 1B shows the structural interaction of the
extracellular domain of BCMA with BAFF.
[0015] FIG. 2 shows an antibody selection procedure.
[0016] FIG. 3 shows cell binding data and ligand blockade activity
for uncloned hybridoma wells.
[0017] FIG. 4 shows blocking activity/percent inihibition of of
anti-BCMA antibodies.
[0018] FIG. 5 shows inhibiton of APRIL blocking titrated with
anti-BCMA antibodies.
[0019] FIG. 6 shows a titration of BAFF blocking using anti-BCMA
antibodies.
[0020] FIG. 7 shows alignment of hSG16.17 heavy chain variants with
human VH acceptorsequence, HV1-2/HJ3. It shows rat SG16.17 vH (SEQ
ID NO: 8) with Kabat CDRs (SEQ ID Nos: 39-41) and IMGT CDRs (SEQ ID
NOs: 42 and 43); Hu HV1-2/HJ3 (SEQ ID NO: 9) with Kabat CDRs (SEQ
ID NOs: 44 and 45) and IMGT CDRs (SEQ ID NO: 46 and "AR"); hSG16.17
vH1 (SEQ ID NO: 11) with Kabat CDRs (SEQ ID NOs: 50-52) and IMGT
CDRs (SEQ ID NOs: 53 and 54); hSG16.17 vH2 (SEQ ID NO: 12) with
Kabat CDRs (SEQ ID NOs: 55-57) and IMGT CDRs (SEQ ID NOs: 58 and
59); hSG16.17 vH3 (SEQ ID NO: 13) with Kabat CDRs (SEQ ID NOs:
60-62) and IMGT CDRs (SEQ ID NOs: 63 and 64); and hSG16.17 vH4 (SEQ
ID NO: 14) with Kabat CDRs (SEQ ID NOs: 65-67) and IMGT CDRs (SEQ
ID NOs: 68 and 69).
[0021] FIG. 8 shows alignment of hSG16.17 heavy chain variants with
human VH acceptor sequence; HV1-46/HJ3. It shows the sequences of
rat SG16.17 vH (SEQ ID NO: 8) with Kabat CDRs (SEQ ID NOs: 39-41)
and IMGT CDRs (SEQ ID NOs: 42 and 43); Hu HV1-46/HJ3 (SEQ ID NO:
10) with Kabat CDRs (SEQ ID NOs: 47 and 48) and IMGT CDRs (SEQ ID
NO: 49 and "AR"); hSG16.17 vH5 (SEQ ID NO: 15) with Kabat CDRs (SEQ
ID NOs: 70-72) and IMGT CDRs (SEQ ID NOs: 73 and 74); and hSG16.17
vH6 (SEQ ID NO: 16) with Kabat CDRs (SEQ ID NOs: 75-77) and IMGT
CDRs (SEQ ID NOs: 78 and 79).
[0022] FIG. 9 shows alignment of hSG16.17 heavy chain variants. It
shows the sequences of hSG16.17 vH1-6 (SEQ ID NOs: 11-16).
[0023] FIG. 10 shows alignment of hSG16.17 light chain variants
with human VK acceptor sequence; KV1-12/KJS. It shows the sequences
of rat SG16.17 vK (SEQ ID NO: 17) with Kabat CDRs (SEQ ID NOs:
80-82) and IMGT CDRs (SEQ ID NO: 83, "TTS", and SEQ ID NO: 84,
respectively); Hu KV1-12/KJ5 (SEQ ID NO: 18) with Kabat CDRs (SEQ
ID NOs: 85-87) and IMGT CDRs (SEQ ID NO: 88, "AAS", and SEQ ID NO:
89, respectively); hSG16.17 vK2 (SEQ ID NO: 19) with Kabat CDRs
(SEQ ID NOs: 90-92) and IMGT CDRs (SEQ ID NO: 93, "TTS", and SEQ ID
NO: 94, respectively); hSG16.17 vK3 (SEQ ID NO: 20) with Kabat CDRs
(SEQ ID NOs: 95-97) and IMGT CDRs (SEQ ID NO: 98, "TTS", and SEQ ID
NO: 99, respectively); hSG16.17 vK4 (SEQ ID NO: 21) with Kabat CDRs
(SEQ ID NOs. 100-102) and IMGT CDRs (SEQ ID NO: 103, "TTS", and SEQ
ID NO: 104, respectively); and hSG16.17 vK5(SEQ ID NO: 22) with
Kabat CDRs (SEQ ID NOs: 105-107) and IMGT CDRs (SEQ ID NO: 108,
"TTS", and SEQ ID NO: 109, respectively).
[0024] FIG. 11 shows alignment of hSG16.17 light chain variants. It
shows the sequences of hSG16.17 vK2, vK3, vK4, vK5 (SEQ ID NOs:
19-22).
[0025] FIG. 12 shows competition binding assay showing binding of
chimeric SG16.17 to human FcRIIIa.
[0026] FIG. 13: shows chimeric SG16.17 induces signallying through
Fc.gamma.RIIIA.
[0027] FIG. 14 shows alignment of hSG16.45 heavy chain variants
with human HV acceptor sequence HV3-23/HJ3. It shows the sequences
of Rat SG16.45 vH (SEQ ID NO: 23) with Kabat CDRs (SEQ ID NOs:
110-112) and IMGT CDRs (SEQ ID NOs: 113-115); Hu HV3-23/HJ3 (SEQ ID
NO: 24) with Kabat CDRs (SEQ ID NOs: 116 and 117) and IMGT CDRs
(SEQ ID NOs: 118 and 119, and "AK", respectively); hSG16.45 vH1
(SEQ ID NO: 27) with Kabat CDRs (SEQ ID NOs: 128-130) and IMGT CDRs
(SEQ ID NOs: 131-133); hSG16.45 vH2 (SEQ ID NO: 28) with Kabat CDRs
(SEQ ID NOs: 134-136) and IMGT CDRs (SEQ ID NOs: 137-139); hSG16.45
vH3 (SEQ ID NO: 29) with Kabat CDRs (SEQ ID NOs: 140-142) and IMGT
CDRs (SEQ ID NOs: 143-145); and hSG16.45 vH4 (SEQ ID NO: 30) with
Kabat CDRs (SEQ ID NOs: 146-148) and IMGT CDRs (SEQ ID NOs:
149-151).
[0028] FIG. 15 shows alignment of hSG16.45 heavy chain variants
with human HV acceptor sequence HV3-74/HJ3. It shows the sequences
of Rat SG16.45 vH (SEQ ID NO: 23) with Kabat CDRs (SEQ ID NOs:
110-112) and IMGT CDRs (SEQ ID NOs: 113-115); Hu HV3-74/HJ3 (SEQ ID
NO: 25) with Kabat CDRs (SEQ ID NOs: 120 and 121) and IMGT CDRs
(SEQ ID NOs: 122 and 123, and "AR", respectively); hSG16.45 vH5
(SEQ ID NO: 31) with Kabat CDRs (SEQ ID NOs: 152-154) and IMGT CDRs
(SEQ ID NOs: 155-157).
[0029] FIG. 16 shows alignment of hSG16.45 heavy chain variants
with human HV acceptor sequence HV3-9/HJ3. It shows the sequences
of Rat SG16.45 vH (SEQ ID NO: 23) with Kabat CDRs (SEQ ID NOs:
110-112) and IMGT CDRs (SEQ ID NOs: 113-115); Hu HV3-9/HJ3 (SEQ ID
NO: 26) with Kabat CDRs (SEQ ID NOs: 124 and 125) and IMGT CDRs
(SEQ ID NOs: 126 and 127, and "AR", respectively); hSG16.45 vH6
(SEQ ID NO: 32) with Kabat CDRs (SEQ ID NOs: 158-160) and IMGT CDRs
(SEQ ID NOs: 161-163).
[0030] FIG. 17 shows alignment of hSG16.45 heavy chain variants. It
shows the sequences of hSG16.45 vH1-6 (SEQ ID NOs: 27-32).
[0031] FIG. 18 shows alignment of hSG16.45 light chain variants
with human KV acceptor sequence KV3-20/KJ2. It shows the sequences
of Rat SG16.45 vK (SEQ ID NO: 33) with Kabat CDRs (SEQ ID NOs:
164-166) and IMGT CDRs (SEQ ID NO: 167, "STS", and SEQ ID NO: 168,
respectively); Hu KV3-20/KJ2 (SEQ ID NO: 34) with Kabat CDRs (SEQ
ID NOs: 169-171) and IMGT CDRs (SEQ ID NO: 172, "STS", and SEQ ID
NO: 173, respectively); hSG16.45 vK1 (SEQ ID NO: 35) with Kabat
CDRs (SEQ ID NOs: 174-176) and IMGT CDRs (SEQ ID NO: 177, "STS",
and SEQ ID NO: 178, respectively); hSG16.45 vK2 (SEQ ID NO: 36)
with Kabat CDRs (SEQ ID NOs: 179-181) and IMGT CDRs (SEQ ID NO:
182, "STS", and SEQ ID NO: 183, respectively); hSG16.45 vK3 (SEQ ID
NO: 37) with Kabat CDRs (SEQ ID NOs: 184-186) and IMGT CDRs (SEQ ID
NO: 187, "STS", and SEQ ID NO: 188, respectively); and hSG16.45 vK5
(SEQ ID NO: 38) with Kabat CDRs (SEQ ID NOs: 189-191) and IMGT CDRs
(SEQ ID NO: 192, "STS", and SEQ ID NO: 193, respectively).
[0032] FIG. 19. shows alignment of hSG16.45 light chain variants.
It shows the sequences of hSG16.45 vK1, vK2, vK3, vK5 (SEQ ID NOs:
35-38).
[0033] FIGS. 20A-C show in vivo activity of multi dosed
hSG16.17-SEA in MM1S disseminated tumor model in SCID mice.
[0034] FIGS. 21A-C show in vivo activity of single dosed
hSG16.17-SEA in EJM disseminated tumor model in NSG mice.
[0035] FIG. 22 show in vivo activity of multi dosed hSG16.17-SEA in
NCI-H929-luciferase disseminated tumor model in NSG mice.
[0036] FIGS. 23A-B show in vivo activity of single dosed
hSG16.17-SEA in NCI-H929-luciferase disseminated tumor model in NSG
mice.
[0037] FIG. 24 provides in vivo activity of single dosed
hSG16.17-SEA in MOLP-8-luciferase disseminated tumor model in SCID
mice.
[0038] FIG. 25 provides ADCC activity of the SG16.17 SEA antibody
on MM1R target cells.
DEFINITIONS
[0039] An "isolated" antibody refers to an antibody that has been
identified and separated and/or recovered from components of its
natural environment and/or an antibody that is recombinantly
produced. A "purified antibody" is an antibody that is typically at
least 50% w/w pure of interfering proteins and other contaminants
arising from its production or purification but does not exclude
the possibility that the monoclonal antibody is combined with an
excess of pharmaceutical acceptable carrier(s) or other vehicle
intended to facilitate its use. Interfering proteins and other
contaminants can include, for example, cellular components of the
cells from which an antibody is isolated or recombinantly produced.
Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95
or 99% w/w pure of interfering proteins and contaminants from
production or purification. The antibodies described herein,
including rat, chimeric, veneered and humanized antibodies can be
provided in isolated and/or purified form.
[0040] A "monoclonal antibody" refers to an antibody obtained from
a population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler et al. (1975) Nature 256:495, or may be made by
recombinant DNA methods (see, for example, U.S. Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al. (1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol.
Biol., 222:581-597, for example or may be made by other methods.
The antibodies described herein are monoclonal antibodies.
[0041] Specific binding of a monoclonal antibody to its target
antigen means an affinity of at least 10.sup.6, 10.sup.7, 10.sup.8,
10.sup.9, or 10.sup.10 M.sup.-1. Specific binding is detectably
higher in magnitude and distinguishable from non-specific binding
occurring to at least one unrelated target. Specific binding can be
the result of formation of bonds between particular functional
groups or particular spatial fit (e.g., lock and key type) whereas
nonspecific binding is usually the result of van der Waals
forces.
[0042] The basic antibody structural unit is a tetramer of
subunits. Each tetramer includes two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. This variable region
is initially expressed linked to a cleavable signal peptide. The
variable region without the signal peptide is sometimes referred to
as a mature variable region. Thus, for example, a light chain
mature variable region, means a light chain variable region without
the light chain signal peptide. The carboxy-terminal portion of
each chain defines a constant region primarily responsible for
effector function.
[0043] Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, and
define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 or more amino acids. (See generally, Fundamental
Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7,
incorporated by reference in its entirety for all purposes).
[0044] The mature variable regions of each light/heavy chain pair
form the antibody binding site. Thus, an intact antibody has two
binding sites. Except in bifunctional or bispecific antibodies, the
two binding sites are the same. The chains all exhibit the same
general structure of relatively conserved framework regions (FR)
joined by three hypervariable regions, also called complementarity
determining regions or CDRs. The CDRs from the two chains of each
pair are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4. The assignment of amino acids to each domain is in
accordance with the definitions of Kabat, Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.
196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989), or a
composite of Kabat and Chothia, or IMGT, AbM or Contact or other
conventional definition of CDRs. Kabat also provides a widely used
numbering convention (Kabat numbering) in which corresponding
residues between different heavy chains or between different light
chains are assigned the same number. Unless otherwise apparent from
the context, Kabat numbering is used to designate the position of
amino acids in the variable regions. Unless otherwise apparent from
the context EU numbering is used to designated positions in
constant regions.
[0045] The term "antibody" includes intact antibodies and binding
fragments thereof. Typically, antibody fragments compete with the
intact antibody from which they were derived for specific binding
to the target including separate heavy chains, light chains Fab,
Fab', F(ab').sub.2, F(ab)c, diabodies, Dabs, nanobodies, and Fv.
Fragments can be produced by recombinant DNA techniques, or by
enzymatic or chemical separation of intact immunoglobulins. The
term "antibody" also includes a diabody (homodimeric Fv fragment)
or a minibody (V.sub.L--V.sub.H--C.sub.H3), a bispecific antibody
or the like. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites (see, e.g., Songsivilai and Lachmann,
Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J.
Immunol., 148:1547-53 (1992)).
[0046] The term "antibody" includes an antibody by itself (naked
antibody) or an antibody conjugated to a cytotoxic or cytostatic
drug.
[0047] A chimeric antibody is an antibody in which the mature
variable regions of light and heavy chains of a non-human antibody
(e.g., a mouse) are combined with human light and heavy chain
constant regions. Such antibodies substantially or entirely retain
the binding specificity of the mouse antibody, and are about
two-thirds human sequence.
[0048] A veneered antibody is a type of humanized antibody that
retains some and usually all of the CDRs and some of the non-human
variable region framework residues of a non-human antibody but
replaces other variable region framework residues that may
contribute to B- or T-cell epitopes, for example exposed residues
(Padlan, Mol. Immunol. 28:489, 1991) with residues from the
corresponding positions of a human antibody sequence. The result is
an antibody in which the CDRs are entirely or substantially from a
non-human antibody and the variable region frameworks of the
non-human antibody are made more human-like by the
substitutions.
[0049] The term "epitope" refers to a site on an antigen to which
an antibody binds. An epitope can be formed from contiguous amino
acids or noncontiguous amino acids juxtaposed by tertiary folding
of one or more proteins. Epitopes formed from contiguous amino
acids are typically retained on exposure to denaturing solvents
whereas epitopes formed by tertiary folding are typically lost on
treatment with denaturing solvents. An epitope typically includes
at least 3, and more usually, at least 5 or 8-10 amino acids in a
unique spatial conformation. Methods of determining spatial
conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols, in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed. (1996).
[0050] Antibodies that recognize the same or overlapping epitopes
can be identified in a simple immunoassay showing the ability of
one antibody to compete with the binding of another antibody to a
target antigen. The epitope of an antibody can also be defined by
X-ray crystallography of the antibody bound to its antigen to
identify contact residues. Alternatively, two antibodies have the
same epitope if all amino acid mutations in the antigen that reduce
or eliminate binding of one antibody reduce or eliminate binding of
the other. Two antibodies have overlapping epitopes if some amino
acid mutations that reduce or eliminate binding of one antibody
reduce or eliminate binding of the other.
[0051] Competition between antibodies is determined by an assay in
which an antibody under test inhibits specific binding of a
reference antibody to a common antigen (see, e.g., Junghans et al.,
Cancer Res. 50:1495, 1990). A test antibody competes with a
reference antibody if an excess of a test antibody (e.g., at least
2.times., 5.times., 10.times., 20.times. or 100.times.) inhibits
binding of the reference antibody by at least 50% but preferably
75%, 90% or 99% as measured in a competitive binding assay.
Antibodies identified by competition assay (competing antibodies)
include antibodies binding to the same epitope as the reference
antibody and antibodies binding to an adjacent epitope sufficiently
proximal to the epitope bound by the reference antibody for steric
hindrance to occur. Antibodies that compete with the h2H12 antibody
for binding to the human BCMA protein are included in this
disclosure.
[0052] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0053] For purposes of classifying amino acids substitutions as
conservative or nonconservative, amino acids are grouped as
follows: Group I (hydrophobic side chains): met, ala, val, leu,
ile; Group II (neutral hydrophilic side chains): cys, ser, thr;
Group III (acidic side chains): asp, glu; Group IV (basic side
chains): asn, gln, his, lys, arg; Group V (residues influencing
chain orientation): gly, pro; and Group VI (aromatic side chains):
trp, tyr, phe. Conservative substitutions involve substitutions
between amino acids in the same class. Nonconservative
substitutions constitute exchanging a member of one of these
classes for a member of another.
[0054] Percentage sequence identities are determined with antibody
sequences maximally aligned by the Kabat numbering convention.
After alignment, if a subject antibody region (e.g., the entire
mature variable region of a heavy or light chain) is being compared
with the same region of a reference antibody, the percentage
sequence identity between the subject and reference antibody
regions is the number of positions occupied by the same amino acid
in both the subject and reference antibody region divided by the
total number of aligned positions of the two regions, with gaps not
counted, multiplied by 100 to convert to percentage.
[0055] Compositions or methods "comprising" one or more recited
elements may include other elements not specifically recited. For
example, a composition that comprises antibody may contain the
antibody alone or in combination with other ingredients.
[0056] Designation of a range of values includes all integers
within or defining the range.
[0057] An antibody effector function refers to a function
contributed by an Fc domain(s) of an Ig. Such functions can be, for
example, antibody-dependent cellular cytotoxicity,
antibody-dependent cellular phagocytosis or complement-dependent
cytotoxicity. Such function can be effected by, for example,
binding of an Fc effector domain(s) to an Fc receptor on an immune
cell with phagocytic or lytic activity or by binding of an Fc
effector domain(s) to components of the complement system.
Typically, the effect(s) mediated by the Fc-binding cells or
complement components result in inhibition and/or depletion of the
BCMA targeted cell. Fc regions of antibodies can recruit Fc
receptor (FcR)-expressing cells and juxtapose them with
antibody-coated target cells. Cells expressing surface FcR for IgGs
including Fc.gamma.RIII (CD16), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD64) can act as effector cells for the destruction
of IgG-coated cells. Such effector cells include monocytes,
macrophages, natural killer (NK) cells, neutrophils and
eosinophils. Engagement of Fc.gamma.R by IgG activates
antibody-dependent cellular cytotoxicity (ADCC) or
antibody-dependent cellular phagocytosis (ADCP). ADCC is mediated
by CD16.sup.+ effector cells through the secretion of membrane
pore-forming proteins and proteases, while phagocytosis is mediated
by CD32.sup.+ and CD64.sup.+ effector cells (see Fundamental
Immunology, 4.sup.th ed., Paul ed., Lippincott-Raven, N.Y., 1997,
Chapters 3, 17 and 30; Uchida et al., 2004, J. Exp. Med.
199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65;
Watanabe et al., 1999, Breast Cancer Res. Treat. 53:199-207). In
addition to ADCC and ADCP, Fc regions of cell-bound antibodies can
also activate the complement classical pathway to elicit
complement-dependent cytotoxicity (CDC). C1q of the complement
system binds to the Fc regions of antibodies when they are
complexed with antigens. Binding of C1q to cell-bound antibodies
can initiate a cascade of events involving the proteolytic
activation of C4 and C2 to generate the C3 convertase. Cleavage of
C3 to C3b by C3 convertase enables the activation of terminal
complement components including C5b, C6, C7, C8 and C9.
Collectively, these proteins form membrane-attack complex pores on
the antibody-coated cells. These pores disrupt the cell membrane
integrity, killing the target cell (see Immunobiology, 6.sup.th
ed., Janeway et al., Garland Science, N. Y., 2005, Chapter 2).
[0058] The term "antibody-dependent cellular cytotoxicity", or
ADCC, is a mechanism for inducing cell death that depends upon the
interaction of antibody-coated target cells with immune cells
possessing lytic activity (also referred to as effector cells).
Such effector cells include natural killer cells,
monocytes/macrophages and neutrophils. The effector cells attach to
an Fc effector domain(s) of Ig bound to target cells via their
antigen-combining sites. Death of the antibody-coated target cell
occurs as a result of effector cell activity.
[0059] The term "antibody-dependent cellular phagocytosis", or
ADCP, refers to the process by which antibody-coated cells are
internalized, either in whole or in part, by phagocytic immune
cells (e.g., macrophages, neutrophils and dendritic cells) that
bind to an Fc effector domain(s) of Ig.
[0060] The term "complement-dependent cytotoxicity", or CDC, refers
to a mechanism for inducing cell death in which an Fc effector
domain(s) of a target-bound antibody activates a series of
enzymatic reactions culminating in the formation of holes in the
target cell membrane. Typically, antigen-antibody complexes such as
those on antibody-coated target cells bind and activate complement
component C1q which in turn activates the complement cascade
leading to target cell death. Activation of complement may also
result in deposition of complement components on the target cell
surface that facilitate ADCC by binding complement receptors (e.g.,
CR3) on leukocytes.
[0061] A "cytotoxic effect" refers to the depletion, elimination
and/or the killing of a target cell. A "cytotoxic agent" refers to
an agent that has a cytotoxic effect on a cell.
[0062] Cytotoxic agents can be conjugated to an antibody or
administered in combination with an antibody.
[0063] A "cytostatic effect" refers to the inhibition of cell
proliferation. A "cytostatic agent" refers to an agent that has a
cytostatic effect on a cell, thereby inhibiting the growth and/or
expansion of a specific subset of cells. Cytostatic agents can be
conjugated to an antibody or administered in combination with an
antibody.
[0064] The term "pharmaceutically acceptable" means approved or
approvable by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. The term "pharmaceutically compatible ingredient" refers
to a pharmaceutically acceptable diluent, adjuvant, excipient, or
vehicle with which an anti-BCMA antibody is administered to a
subject.
[0065] The phrase "pharmaceutically acceptable salt," refers to
pharmaceutically acceptable organic or inorganic salts of an
anti-BCMA-1 antibody or conjugate thereof or agent administered
with an anti-BCMA-1 antibody. Exemplary salts include sulfate,
citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,
bisulfate, phosphate, acid phosphate, isonicotinate, lactate,
salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,
bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,
gluconate, glucuronate, saccharate, formate, benzoate, glutamate,
methanesulfonate, ethanesulfonate, benzenesulfonate, p
toluenesulfonate, and pamoate (i.e., 1,1' methylene bis-(2 hydroxy
3 naphthoate)) salts. A pharmaceutically acceptable salt may
involve the inclusion of another molecule such as an acetate ion, a
succinate ion or other counterion. The counterion may be any
organic or inorganic moiety that stabilizes the charge on the
parent compound. Furthermore, a pharmaceutically acceptable salt
may have more than one charged atom in its structure. Instances
where multiple charged atoms are part of the pharmaceutically
acceptable salt can have multiple counter ions. Hence, a
pharmaceutically acceptable salt can have one or more charged atoms
and/or one or more counterion.
[0066] Unless otherwise apparent from the context, the term "about"
encompasses insubstantial variation having no significant effect on
functional properties (e.g., within a margin of error or
experimental measurement).
DETAILED DESCRIPTION
I. General
[0067] The invention provides monoclonal antibodies that
specifically bind to BCMA. The antibodies are useful for treatment
and diagnoses of various cancers and immunological disorders as
well as detecting BCMA.
II. Target Molecules
[0068] Unless otherwise indicated, BCMA means a human BCMA.
Exemplary human nucleic acid and amino acid sequences are provided
by SEQ ID NOS:1 and 2. Unless otherwise apparent from the context
reference to BMCA means at least an extracellular domain of the
protein (approximately residues 1-54 of SEQ ID NO: 2) and sometimes
the complete protein. Likewise, unless otherwise apparent from the
context reference to BAFF and APRIL and their receptors other than
BCMA refers to wild type human sequences e.g., as provided in the
Swiss Prot Database.
III. Antibodies of the Invention
[0069] A. Binding Specificity and Functional Properties
[0070] The SG16.17 antibody is a rat monoclonal antibody that
specifically binds to human BCMA as described in the examples. An
ATCC deposit was made on Aug. 15, 2005 under the Budapest Treaty.
The ATCC is located at 10801 University Boulevard, Manassas, Va.
20110-2209, USA. The ATCC deposit was assigned accession number of
PTA-6937. The SG16.17 antibody inhibits binding of BCMA to both of
its ligands, APRIL and BAFF. The SG16.17 antibody when linked to a
human IgG1 elicits ADCC, binds to and elicits signaling through
Fc.gamma. receptors. The SG16.17 antibody can also be incorporated
into an antibody drug conjugate to deliver a linked drug into the
interior of cells expressing BCMA. The SG16.45 antibody is another
rat monoclonal antibody that specifically binds to human BCMA,
inhibits its binding to its ligands and can deliver a linked drug
to the interior of cells expressing BCMA.
[0071] The invention provides humanized, chimeric and veneered
forms of the SG16.17 antibody (designated hSG16.17, cSG16.17 or
vSG16.17) and SG16.45 (analogously designated). Such antibodies
typically retain some or all of the properties for SG16.17 or
SG16.45 noted above. For any given property, humanized, chimeric or
veneered antibodies may exhibit the property to the same extent
within experimental error or more or less than rat SG16.17 or
SG16.45. The affinity of humanized, chimeric or veneered forms of
the rat SG16.17 antibody (i.e., Ka) can be greater than that of the
rat SG16.17 antibody, or within a factor of five or a factor of two
(i.e., more than or less than) than that of the rat SG16.17
antibody for human BCMA. Preferred humanized, chimeric or veneered
SG16.17 antibodies bind to the same epitope and/or compete with rat
SG16.17 antibodies for binding to human BCMA. The affinity of
humanized, chimeric or veneered forms of the rat SG16.45 antibody
(i.e., Ka) can be greater than that of the rat SG16.45 antibody, or
within a factor of five or a factor of two (i.e., more than or less
than) than that of the rat SG16.45 antibody for human BCMA.
Preferred humanized, chimeric or veneered SG16.45 antibodies bind
to the same epitope and/or compete with rat SG16.45 antibodies for
binding to human BCMA.
[0072] Preferred humanized, chimeric and veneered antibodies
inhibit cancer (e.g., growth of cells, metastasis and/or lethality
to the organisms) or B-cell mediated immune disorders as shown in
vitro, in an animal model or clinical trial.
[0073] B. Antibodies
[0074] A humanized antibody is a genetically engineered antibody in
which CDRs from a non-human "donor" antibody are grafted into human
"acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos.
5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter,
U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote,
U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be,
for example, a mature human antibody sequence, a composite of such
sequences, a consensus sequence of human antibody sequences, or a
germline region sequence. For humanization of SG16.17, a preferred
acceptor sequence for the heavy chain is the germline V.sub.H exon
V.sub.H1-2 and for the J exon (J.sub.H), exon J.sub.H-3. For the
light chain, a preferred acceptor sequence is exon V.sub.L1-12 and
J exon J.sub.K5. For humanization of SG16.45, a preferred heavy
chain acceptor sequence is HV3-23/HJ3 (SEQ ID NO: 24) and a
preferred light chain acceptor sequence is KV3-20/KJ2 (SEQ ID NO:
34).
[0075] Thus, a humanized antibody is an antibody having at least
four CDRs entirely or substantially from a non-human donor antibody
and variable region framework sequences and constant regions, if
present, entirely or substantially from human antibody sequences.
Similarly a humanized heavy chain has at least two and usually all
three CDRs entirely or substantially from a donor antibody heavy
chain, and a heavy chain variable region framework sequence and
heavy chain constant region, if present, substantially from human
heavy chain variable region framework and constant region
sequences. Similarly a humanized light chain has at least two and
usually all three CDRs entirely or substantially from a donor
antibody light chain, and a light chain variable region framework
sequence and light chain constant region, if present, substantially
from human light chain variable region framework and constant
region sequences. Other than nanobodies and dAbs, a humanized
antibody comprises a humanized heavy chain and a humanized light
chain. A CDR in a humanized or human antibody is substantially from
or substantially identical to a corresponding CDR in a non-human
antibody when at least 60%, 85%, 90%, 95% or 100% of corresponding
residues (as defined by Kabat) are identical between the respective
CDRs. The variable region framework sequences of an antibody chain
or the constant region of an antibody chain are substantially from
a human variable region framework sequence or human constant region
respectively when at least 70%, 80%, 85%, 90%, 95% or 100% of
corresponding residues defined by Kabat are identical.
[0076] Although humanized antibodies often incorporate all six CDRs
(preferably as defined by Kabat, but alternatively as defined by
IMGT, Chothia, composite Kabat-Chothia, AbM or Contact or other
conventional definition) from a mouse antibody, they can also be
made with less than all CDRs (e.g., at least 4, or 5) CDRs from a
mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002;
Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002;
lwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al,
Journal of Immunology, 164:1432-1441, 2000).
[0077] Certain amino acids from the human variable region framework
residues can be selected for substitution based on their possible
influence on CDR conformation and/or binding to antigen.
Investigation of such possible influences is by modeling,
examination of the characteristics of the amino acids at particular
locations, or empirical observation of the effects of substitution
or mutagenesis of particular amino acids.
[0078] For example, when an amino acid differs between a murine
variable region framework residue and a selected human variable
region framework residue, the human framework amino acid can be
substituted by the equivalent framework amino acid from the mouse
antibody when it is reasonably expected that the amino acid: [0079]
(1) noncovalently binds antigen directly, [0080] (2) is adjacent to
a CDR region, [0081] (3) otherwise interacts with a CDR region
(e.g. is within about 6 .ANG. of a CDR region); or [0082] (4)
mediates interaction between the heavy and light chains.
[0083] The invention provides humanized forms of the rat SG16.17
antibody including six exemplified humanized heavy chain mature
variable regions (hSG16.17 VH1-6) (SEQ ID Nos: 11-16) and four
exemplified humanized light chain mature variable regions (hSG16.17
VK2-5) (SEQ ID NOs: 19-22). The heavy and light chains can be
combined in any permutations, with permutations including any of
hSG16.17 VH1, VH3 or VH5 being preferred. The permutation having
the best combination of binding affinity, percentage sequence
identity to human germline, expression and percentage of monomeric
content was hSG16.17 VH3 VK2. This antibody shows similar affinity
within experimental error as the rat SG16.17, greater than 85%
sequence identity with human germline in both heavy and light chain
variable regions (thus, qualifying for "humanized" designation
under the new INN guideliness), high expression in CHO cells, and
high proportion of monomers. Compared with most other humanized
antibodies hSG16.17 VH3 VK2 is unusual in having a large number of
variable region framework mutations in which human acceptor
residues are changed to the corresponding rat residue (13) but also
having a large number of "forward" CDR mutations, in which a rat
residue in the Kabat CDRs is changed to the corresponding residue
in the human acceptor sequence, such that overall the antibody has
sufficient sequence identity to human germline sequences to be
classified as humanized under INN guidelines. Most previous
humanized antibodies have had Kabat CDR entirely from the donor
antibody.
[0084] The invention provides antibodies in which the heavy chain
variable region shows at least 90% identity to hSG16.17 VH3 (SEQ ID
NO: 13) and a light chain variable region at least 90% identical to
hSG16.17 VK2 (SEQ ID NO: 19). Some antibodies show at least 95%,
96%, 97%, 98% or 99% sequence identity to HV3 and at least 95%,
96%, 97%, 98% or 99% sequence identity to VK2. Some such antibodies
include the the three Kabat CDRs (SEQ ID NOs: 60-62) of hSG16.17
VH3 (SEQ ID NO: 13) and three Kabat CDRs (SEQ ID NOs: 90-92) of
hSG16.17 VK2 (SEQ ID NO: 19). Some such antibodies include the the
three Kabat CDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO:
13) and three Kabat CDRs (SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ
ID NO: 19) provided that position H58 can be occupied by N or K,
position H60 can be occupied by A or N, position H61 can be
occupied by Q or E, position H62 can be occupied by K or N,
position H64 can be occupied by Q or K, position H65 can be
occupied by G or T, position L24 can be occupied by R or L and
position L53 can be occupied by S or R. Preferably positions H58,
H60, H61, H62, H64 and H65 are occupied by N, A, Q, K, Q and G
respectively and L24 and L53 are occupied by R and S respectively.
These recited residues represent amino acids from a human acceptor
sequence occupying positions within the Kabat CDRs. Some antibodies
have at least 1, 2, 3, 4, 5, 6, 7 or 8 rat residues in the human
Kabat CDRs replaced with corresponding residues from a human
acceptor sequence. In some antibodies positions H58, H60, H61, H62,
H64 and H65 are occupied by N, A, Q, K, Q and G respectively and
L24 and L53 are occupied by R and S respectively. Some antibodies
include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14
backmutations representing replacement of variable region human
acceptor sequence residues with corresponding rat residues.
[0085] In some antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or 11 of positions H20, H48, H69, H71, H73, H76, H80, H88, H91 and
H93 are occupied by L, I, M, A, K, N, V, A, F, and T respectively.
In some antibodies at least 1, 2 or 3 of positions L46, L48 and L87
are occupied by V, V and F respectively. In some antibodies, each
of positions H20, H48, H69, H71, H73, H76, H80, H88, H91 and H93
are occupied by L, I, M, A, K, N, V, A, F, and T respectively and
each of L46, L48 and L87 are occupied by V, V and F
respectively.
[0086] Insofar as humanized antibodies show any variation from the
exemplified hSG16.17 VH3 VK2 humanized antibody, one possibility
for such additional variation is additional backmutations in the
variable region frameworks. Any or all of the positions backmutated
in other exemplified humanized heavy or light chain mature variable
regions can also be made (i.e., 1, 2, 3, 4, 5 or all 6) of H8
occupied by R, H67 occupied by A and H78 occupied by A, L40
occupied by S, L78 occupied by M and L85 occupied by D, or all 5 of
H38 occupied by N, H40 occupied by R, H73 occupied by K, H82A
occupied by S, and H83 occupied by T in the heavy chain and 1 or
both of L3 occupied by K, and L20 occupied by I in the light chain.
However, such additional backmutations are not preferred because
they in general do not improve affinity and introducing more mouse
residues may give increased risk of immunogenicity.
[0087] Another possible variation is to substitute more or fewer
residues in the CDRs of the mouse antibody with corresponding
residues from human CDRs sequences, typically from the CDRs of the
human acceptor sequences used in designing the exemplified
humanized antibodies. In some antibodies only part of the CDRs,
namely the subset of CDR residues required for binding, termed the
SDRs, are needed to retain binding in a humanized antibody. CDR
residues not contacting antigen and not in the SDRs can be
identified based on regions of Kabat CDRs lying outside CDRs
according to other definitions, such as Chothia hypervariable loops
(Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling
and/or empirically, or as described in Gonzales et al., Mol.
Immunol. 41: 863 (2004). In such humanized antibodies at positions
in which one or more donor CDR residues is absent or in which an
entire donor CDR is omitted, the amino acid occupying the position
can be an amino acid occupying the corresponding position (by Kabat
numbering) in the acceptor antibody sequence. The number of such
substitutions of acceptor for donor amino acids in the CDRs to
include reflects a balance of competing considerations. Such
substitutions are potentially advantageous in decreasing the number
of mouse amino acids in a humanized antibody and consequently
decreasing potential immunogenicity. However, substitutions can
also cause changes of affinity, and significant reductions in
affinity are preferably avoided. Positions for substitution within
CDRs and amino acids to substitute can also be selected
empirically.
[0088] Although not preferred other amino acid substitutions can be
made, for example, in framework residues not in contact with the
CDRs, or even some potential CDR-contact residues amino acids
within the CDRs. Often the replacements made in the variant
humanized sequences are conservative with respect to the replaced
hSG16.17 VH3 VK2 amino. Preferably, replacements relative to
hSG16.17 VH3 VK2 (whether or not conservative) have no substantial
effect on the binding affinity or potency of the humanized mAb,
that is, its ability to bind human BCMA and inhibit growth of
cancer cells.
[0089] Variants typically differ from the heavy and light chain
mature variable region sequences of hSG16.17 VH3 VK2 by a small
number (e.g., typically no more than 1, 2, 3, 5 or 10 in either the
light chain or heavy chain mature variable region, or both) of
replacements, deletions or insertions.
[0090] Other preferred combinations of humanized heavy and light
chains include any of hSG16.17 VH1 VK2, VH1 VK3, VH1 VK4, VH1 VK4,
VH3 VK2, VH3 VK3, VH3 VK4, and VH3 VK5, and VH5 VK2, VH5 VK3, VH5
VK4, VH5 VK5, as well as humanized antibodies in which the heavy
and light chain variable regions show at least 90, 95, 96, 97, 98,
or 99% identity with the heavy and light chain variable regions of
any of these antibodies.
[0091] The invention provides humanized forms of the rat SG16.45
antibody including six exemplified humanized heavy chain mature
variable regions (hSG16.45 VH1-6) (SEQ ID NOs: 27-32) and four
exemplified humanized light chain mature variable regions (hSG16.45
VK1, 2, 3, and 5) (SEQ ID NOs: 35-38). The heavy and light chains
can be combined in any permutations, with permuations hSG16.45 VH5
VK2, VH1 VK1 and VH1 VK5 being preferred. hSG16.45 HV5 VK2 shows
greater than 85% sequence identity with human germline in both
heavy and light chain variable regions (thus, qualifying for
"humanized" designation under the new INN guideliness), high
expression in CHO cells, a high proportion of monomers and adequate
binding albeit slightly less than that of rat or chimeric SG16.45.
hSG16.45 VH5 VK2 has 3 variable region backmutations (all in the
heavy chain) and 3 Kabat CDR forward mutations, in which a rat
residue in the Kabat CDRs is changed to the corresponding residue
in the human acceptor sequence, such that overall the antibody has
sufficient sequence identity to human germline sequences to be
classified as humanized under INN guidelines.
[0092] The invention provides antibodies in which the heavy chain
variable region shows at least 90% identity to hSG16.45 VH5 (SEQ ID
NO: 31) and a light chain variable region at least 90% identical to
hSG16.45 VK2. Some antibodies show at least 95%, 96%, 97%, 98% or
99% sequence identity to hSG16.45 VH5 and at least 95%, 96%, 97%,
98% or 99% sequence identity to VK2. Some such antibodies include
the the three Kabat CDRs (SEQ ID NOs: 152-154) of hSG16.45 VH5 (SEQ
ID NO: 31) and three Kabat CDRs (SEQ ID NOs: 179-181) of hSG16.45
VK2 (SEQ ID NO: 36). Some such antibodies include the the three
Kabat CDRs (SEQ ID NOs: 152-154) of hSG16.45 VH5 (SEQ ID NO: 31)
and three Kabat CDRs (SEQ ID NOs: 179-181) of hSG16.45 VK2 (SEQ ID
NO: 36) provided that position H50 can be occupied by A or S and
position L24 can be occupied by R or L and position L26 can be
occupied by S or T. Preferably positions H50 is occupied by A and
positions L24 and L26 are occupied by R and S. These recited
residues represent amino acids from a human acceptor sequence
occupying positions within the Kabat CDRs. Some antibodies have at
least 1, 2, or 3 rat residues in the human Kabat CDRs replaced with
corresponding residues from a human acceptor sequence. In some
antibodies positions H50, L24 and L26 are occupied by A, R and S
respectively. Some antibodies include at least 1, 2, or 3
backmutations representing replacement of variable region human
acceptor sequence residues with corrsponding rat residues.
[0093] In some antibodies at least 1, 2, or 3, of positions H30,
H93 and H94 are occupied by N, T and S respectively. In some
antibodies, each of positions H30, H93 and H94 are occupied by N, T
and S respectively
[0094] Insofar as humanized antibodies show any variation from the
exemplified hSG16.45 VH5 VK2 humanized antibody, one possibility
for such additional variation is additional backmutations in the
variable region frameworks. Any or all of the positions backmutated
in other exemplified humanized heavy or light chain mature variable
regions can also be made (i.e., 1, 2, 3, or 4) of H37, H48, H76,
H107 occupied by I, I, N, and V respectively and/or 1, 2, 3, 4, 5,
6 or 7 of L14, L19, L21, L38, L58, L71 and L78 occuiied by A, V, I,
H, V, Y, and M respectively. However, such additional backmutations
are not preferred because they in general do not improve affinity
and introducing more mouse residues may give increased risk of
immunogenicity.
[0095] Another possible variation is to substitute more or fewer
residues in the CDRs of the mouse antibody with corresponding
residues from human CDRs sequences, typically from the CDRs of the
human acceptor sequences used in designing the exemplified
humanized antibodies. In some antibodies only part of the CDRs,
namely the subset of CDR residues required for binding, termed the
SDRs, are needed to retain binding in a humanized antibody. CDR
residues not contacting antigen and not in the SDRs can be
identified based on regions of Kabat CDRs lying outside CDRs
according to other definitions, such as Chothia hypervariable loops
(Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling
and/or empirically, or as described in Gonzales et al., Mol.
Immunol. 41: 863 (2004). In such humanized antibodies at positions
in which one or more donor CDR residues is absent or in which an
entire donor CDR is omitted, the amino acid occupying the position
can be an amino acid occupying the corresponding position (by Kabat
numbering) in the acceptor antibody sequence. The number of such
substitutions of acceptor for donor amino acids in the CDRs to
include reflects a balance of competing considerations. Such
substitutions are potentially advantageous in decreasing the number
of mouse amino acids in a humanized antibody and consequently
decreasing potential immunogenicity. However, substitutions can
also cause changes of affinity, and significant reductions in
affinity are preferably avoided. Positions for substitution within
CDRs and amino acids to substitute can also be selected
empirically.
[0096] Although not preferred other amino acid substitutions can be
made, for example, in framework residues not in contact with the
CDRs, or even some potential CDR-contact residues amino acids
within the CDRs. Often the replacements made in the variant
humanized sequences are conservative with respect to the replaced
hSG16.45 VH3 VK2. Preferably, replacements relative to hSG16.45 VH5
VK2 (whether or not conservative) have no substantial effect on the
binding affinity or potency of the humanized mAb, that is, its
ability to bind human BCMA and inhibit growth of cancer cells.
[0097] Variants typically differ from the heavy and light chain
mature variable region sequences of SG16.45 VH5 VK2 by a small
number (e.g., typically no more than 1, 2, 3, 5 or 10 in either the
light chain or heavy chain mature variable region, or both) of
replacements, deletions or insertions.
[0098] Other preferred combinations of humanized heavy and light
chains include any of hSG16.45 VH1 VK1 and VH1 VK5, as well as
humanized antibodies in which the heavy and light chain variable
regions show at least 90, 95, 96, 97, 98, or 99% identity with the
heavy and light chain variable regions of any of these
antibodies.
[0099] C. Selection of Constant Region
[0100] Heavy and light chain variable regions of humanized
antibodies can be linked to at least a portion of a human constant
region. The choice of constant region depends, in part, whether
antibody-dependent cell-mediated cytotoxicity, antibody dependent
cellular phagocytosis and/or complement dependent cytotoxicity are
desired. For example, human isotopes IgG1 and IgG3 have strong
complement-dependent cytotoxicity, human isotype IgG2 weak
complement-dependent cytotoxicity and human IgG4 lacks
complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce
stronger cell mediated effector functions than human IgG2 and IgG4.
Light chain constant regions can be lambda or kappa. Antibodies can
be expressed as tetramers containing two light and two heavy
chains, as separate heavy chains, light chains, as Fab, Fab',
F(ab')2, and Fv, or as single chain antibodies in which heavy and
light chain variable domains are linked through a spacer.
[0101] Human constant regions show allotypic variation and
isoallotypic variation between different individuals, that is, the
constant regions can differ in different individuals at one or more
polymorphic positions. lsoallotypes differ from allotypes in that
sera recognizing an isoallotype binds to a non-polymorphic region
of a one or more other isotypes. Exemplary wild type human kappa
and IgG1 constant region sequences (the latter with or without the
C-terminal lysine) are provide in SEQ ID NOS: 3-5.
[0102] One or several amino acids at the amino or carboxy terminus
of the light and/or heavy chain, such as the C-terminal lysine of
the heavy chain, may be missing or derivatized in a proportion or
all of the molecules. Substitutions can be made in the constant
regions to reduce or increase effector function such as
complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al.,
U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and
Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to
prolong half-life in humans (see, e.g., Hinton et al., J. Biol.
Chem. 279:6213, 2004).
[0103] Exemplary substitution include the amino acid substitution
of the native amino acid to a cysteine residue is introduced at
amino acid position 234, 235, 237, 239, 267, 298, 299, 326, 330, or
332, preferably an S239C mutation in a human IgG1 isotype
(numbering is according to the EU index (Kabat, Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987 and 1991); see US 20100158909, which is herein
incorporated reference). Sequences of a heavy chain constant
regions with S239C with and without a C-terminal lysine are
provided by SEQ ID NOS: 6 and 7. The presence of an additional
cysteine residue allows interchain disulfide bond formation. Such
interchain disulfide bond formation can cause steric hindrance,
thereby reducing the affinity of the Fc region-Fc.gamma.R binding
interaction. The cysteine residue(s) introduced in or in proximity
to the Fc region of an IgG constant region can also serve as sites
for conjugation to therapeutic agents (i.e., coupling cytotoxic
drugs using thiol specific reagents such as maleimide derivatives
of drugs. The presence of a therapeutic agent causes steric
hindrance, thereby further reducing the affinity of the Fc
region-Fc.gamma.R binding interaction. Other substitutions at any
of positions 234, 235, 236 and/or 237 reduce affinity for Fc.gamma.
receptors, particularly Fc.gamma.RI receptor (see, e.g., U.S. Pat.
No. 6,624,821, U.S. Pat. No. 5,624,821.) A preferred combination of
mutations is S239D, A330L and I332E, which increases the affinity
of the Fc domain for Fc.gamma.RIIIA and consequently increases
ADCC.
[0104] The in vivo half-life of an antibody can also impact its
effector functions. The half-life of an antibody can be increased
or decreased to modify its therapeutic activities. FcRn is a
receptor that is structurally similar to MHC Class I antigen that
non-covalently associates with .beta.2-microglobulin. FcRn
regulates the catabolism of IgGs and their transcytosis across
tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766;
Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The IgG-FcRn
interaction takes place at pH 6.0 (pH of intracellular vesicles)
but not at pH 7.4 (pH of blood); this interaction enables IgGs to
be recycled back to the circulation (Ghetie and Ward, 2000, Ann.
Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res.
25:97-113). The region on human IgG1 involved in FcRn binding has
been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604).
Alanine substitutions at positions Pro238, Thr256, Thr307, GIn311,
Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn
binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1
molecules harboring these substitutions have longer serum
half-lives. Consequently, these modified IgG1 molecules may be able
to carry out their effector functions, and hence exert their
therapeutic efficacies, over a longer period of time compared to
unmodified IgG1. Other exemplary substitutions for increasing
binding to FcRn include a Gln at position 250 and/or a Leu at
position 428. EU numbering is used for all positions in the
constant region.
[0105] Oligosaccharides covalently attached to the conserved Asn297
are involved in the ability of the Fc region of an IgG to bind
Fc.gamma.R (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and
Morrison, 1997, Trends Biotechnol. 15:26-31). Engineering of this
glycoform on IgG can significantly improve IgG-mediated ADCC.
Addition of bisecting N-acetylglucosamine modifications (Umana et
al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001,
Biotech. Bioeng. 74:288-94) to this glycoform or removal of fucose
(Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawa et
al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer
Res. 64:2127-33) from this glycoform are two examples of IgG Fc
engineering that improves the binding between IgG Fc and
Fc.gamma.R, thereby enhancing Ig-mediated ADCC activity.
[0106] A systemic substitution of solvent-exposed amino acids of
human IgG1 Fc region has generated IgG variants with altered
Fc.gamma.R binding affinities (Shields et al., 2001, J. Biol. Chem.
276:6591-604). When compared to parental IgG1, a subset of these
variants involving substitutions at Thr256/Ser298, Ser298/Glu333,
Ser298/Lys334, or Ser298/Glu333/Lys334 to Ala demonstrate increased
in both binding affinity toward Fc.gamma.R and ADCC activity
(Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al.,
2004, J. Mol. Biol. 336:1239-49).
[0107] Complement fixation activity of antibodies (both C1q binding
and CDC activity) can be improved by substitutions at Lys326 and
Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575). The same
substitutions on a human IgG2 backbone can convert an antibody
isotype that binds poorly to C1q and is severely deficient in
complement activation activity to one that can both bind C1q and
mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75).
Several other methods have also been applied to improve complement
fixation activity of antibodies. For example, the grafting of an
18-amino acid carboxyl-terminal tail piece of IgM to the
carboxyl-termini of IgG greatly enhances their CDC activity. This
is observed even with IgG4, which normally has no detectable CDC
activity (Smith et al., 1995, J. Immunol. 154:2226-36). Also,
substituting Ser444 located close to the carboxy-terminal of IgG1
heavy chain with Cys induced tail-to-tail dimerization of IgG1 with
a 200-fold increase of CDC activity over monomeric IgG1 (Shopes et
al., 1992, J. Immunol. 148:2918-22). In addition, a bispecific
diabody construct with specificity for C1q also confers CDC
activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).
[0108] Complement activity can be reduced by mutating at least one
of the amino acid residues 318, 320, and 322 of the heavy chain to
a residue having a different side chain, such as Ala. Other
alkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or
Val, or such aromatic non-polar residues as Phe, Tyr, Trp and Pro
in place of any one of the three residues also reduce or abolish
C1q binding. Ser, Thr, Cys, and Met can be used at residues 320 and
322, but not 318, to reduce or abolish C1q binding activity.
Replacement of the 318 (Glu) residue by a polar residue may modify
but not abolish C1q binding activity. Replacing residue 297 (Asn)
with Ala results in removal of lytic activity but only slightly
reduces (about three fold weaker) affinity for C1q. This alteration
destroys the glycosylation site and the presence of carbohydrate
that is required for complement activation. Any other substitution
at this site also destroys the glycosylation site. The following
mutations and any combination thereof also reduce C1q binding:
D270A, K322A, P329A, and P311S (see WO 06/036291).
[0109] Reference to a human constant region includes a constant
region with any natural allotype or any permutation of residues
occupying polymorphic positions in natural allotypes. Also, up to
1, 2, 5, or 10 mutations may be present relative to a natural human
constant region, such as those indicated above to reduce Fc.gamma.
receptor binding or increase binding to FcRN.
[0110] D. Expression of Recombinant Antibodies
[0111] Humanized, chimeric or veneered antibodies are typically
produced by recombinant expression. Recombinant polynucleotide
constructs typically include an expression control sequence
operably linked to the coding sequences of antibody chains,
including naturally-associated or heterologous promoter regions.
Preferably, the expression control sequences are eukaryotic
promoter systems in vectors capable of transforming or transfecting
eukaryotic host cells. Once the vector has been incorporated into
the appropriate host, the host is maintained under conditions
suitable for high level expression of the nucleotide sequences, and
the collection and purification of the crossreacting
antibodies.
[0112] Mammalian cells are a preferred host for expressing
nucleotide segments encoding immunoglobulins or fragments thereof.
See Winnacker, From Genes to Clones, (VCH Publishers, N Y, 1987). A
number of suitable host cell lines capable of secreting intact
heterologous proteins have been developed in the art, and include
CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells,
HEK293 cells, L cells, and non-antibody-producing myelomas
including Sp2/0 and NS0. Preferably, the cells are nonhuman.
Expression vectors for these cells can include expression control
sequences, such as an origin of replication, a promoter, an
enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites, and transcriptional terminator
sequences. Preferred expression control sequences are promoters
derived from endogenous genes, cytomegalovirus, SV40, adenovirus,
bovine papillomavirus, and the like. See Co et al., J. Immunol.
148:1149 (1992).
[0113] Once expressed, antibodies can be purified according to
standard procedures of the art, including HPLC purification, column
chromatography, gel electrophoresis and the like (see generally,
Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
[0114] E. Glycosylation Variants
[0115] Antibodies may be glycosylated at conserved positions in
their constant regions (Jefferis and Lund, (1997) Chem. Immunol.
65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32). The
oligosaccharide side chains of the immunoglobulins affect the
protein's function (Boyd et al., (1996) Mol. Immunol. 32:1311-1318;
Wittwe and Howard, (1990) Biochem. 29:4175-4180), and the
intramolecular interaction between portions of the glycoprotein
which can affect the conformation and presented three-dimensional
surface of the glycoprotein (Hefferis and Lund, supra; Wyss and
Wagner, (1996) Current Opin. Biotech. 7:409-416). Oligosaccharides
may also serve to target a given glycoprotein to certain molecules
based upon specific recognition structures. For example, it has
been reported that in agalactosylated IgG, the oligosaccharide
moiety `flips` out of the inter-CH2 space and terminal
N-acetylglucosamine residues become available to bind mannose
binding protein (Malhotra et al., (1995) Nature Med. 1:237-243).
Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H
(a recombinant humanized murine monoclonal IgG1 antibody which
recognizes the CDw52 antigen of human lymphocytes) produced in
Chinese Hamster Ovary (CHO) cells resulted in a complete reduction
in complement mediated lysis (CMCL) (Boyd et al., (1996) Mol.
Immunol. 32:1311-1318), while selective removal of sialic acid
residues using neuraminidase resulted in no loss of DMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al. (1999) Mature
Biotech. 17:176-180).
[0116] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0117] Glycosylation variants of antibodies are variants in which
the glycosylation pattern of an antibody is altered. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, adding one or more carbohydrate moieties to the antibody,
changing the composition of glycosylation (glycosylation pattern),
the extent of glycosylation, etc.
[0118] Addition of glycosylation sites to the antibody can be
accomplished by altering the amino acid sequence such that it
contains one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites). The alteration may also be made
by the addition of, or substitution by, one or more serine or
threonine residues to the sequence of the original antibody (for
O-linked glycosylation sites). Similarly, removal of glycosylation
sites can be accomplished by amino acid alteration within the
native glycosylation sites of the antibody.
[0119] The amino acid sequence is usually altered by altering the
underlying nucleic acid sequence. These methods include isolation
from a natural source (in the case of naturally-occurring amino
acid sequence variants) or preparation by oligonucleotide-mediated
(or site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0120] The glycosylation (including glycosylation pattern) of
antibodies may also be altered without altering the amino acid
sequence or the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g., antibodies, as potential therapeutics is
rarely the native cell, significant variations in the glycosylation
pattern of the antibodies can be expected. See, e.g., Hse et al.,
(1997) J. Biol. Chem. 272:9062-9070. In addition to the choice of
host cells, factors which affect glycosylation during recombinant
production of antibodies include growth mode, media formulation,
culture density, oxygenation, pH, purification schemes and the
like. Various methods have been proposed to alter the glycosylation
pattern achieved in a particular host organism including
introducing or overexpressing certain enzymes involved in
oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261;
5,278,299). Glycosylation, or certain types of glycosylation, can
be enzymatically removed from the glycoprotein, for example using
endoglycosidase H (Endo H). In addition, the recombinant host cell
can be genetically engineered, e.g., make defective in processing
certain types of polysaccharides. These and similar techniques are
well known in the art.
[0121] The glycosylation structure of antibodies can be readily
analyzed by conventional techniques of carbohydrate analysis,
including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides
[0122] A preferred form of modification of glycosylation of
antibodies is reduced core fucosylation. "Core fucosylation" refers
to addition of fucose ("fucosylation") to N-acetylglucosamine
("GlcNAc") at the reducing terminal of an N-linked glycan.
[0123] A "complex N-glycoside-linked sugar chain" is typically
bound to asparagine 297 (according to the number of Kabat). As used
herein, the complex N-glycoside-linked sugar chain has a
biantennary composite sugar chain, mainly having the following
structure:
##STR00002##
where .+-. indicates the sugar molecule can be present or absent,
and the numbers indicate the position of linkages between the sugar
molecules. In the above structure, the sugar chain terminal which
binds to asparagine is called a reducing terminal (at right), and
the opposite side is called a non-reducing terminal. Fucose is
usually bound to N-acetylglucosamine ("GlcNAc") of the reducing
terminal, typically by an .alpha.1,6 bond (the 6-position of GlcNAc
is linked to the 1-position of fucose). "Gal" refers to galactose,
and "Man" refers to mannose.
[0124] A "complex N-glycoside-linked sugar chain" includes 1) a
complex type, in which the non-reducing terminal side of the core
structure has one or more branches of galactose-N-acetylglucosamine
(also referred to as "gal-GlcNAc") and the non-reducing terminal
side of Gal-GlcNAc optionally has a sialic acid, bisecting
N-acetylglucosamine or the like; or 2) a hybrid type, in which the
non-reducing terminal side of the core structure has both branches
of a high mannose N-glycoside-linked sugar chain and complex
N-glycoside-linked sugar chain.
[0125] In some embodiments, the "complex N-glycoside-linked sugar
chain" includes a complex type in which the non-reducing terminal
side of the core structure has zero, one or more branches of
galactose-N-acetylglucosamine (also referred to as "gal-GlcNAc")
and the non-reducing terminal side of Gal-GlcNAc optionally further
has a structure such as a sialic acid, bisecting
N-acetylglucosamine or the like.
[0126] According to the present methods, typically only a minor
amount of fucose is incorporated into the complex
N-glycoside-linked sugar chain(s) of humanized, chimeric or
veneered SG16.17 or SG16.45 antibodies. For example, in various
embodiments, less than about 60%, less than about 50%, less than
about 40%, less than about 30%, less than about 20%, less than
about 15%, less than about 10%, less than about 5%, or less than
about 3% of the molecules of an antibody have core fucosylation by
fucose. In some embodiments, about 2% of the molecules of the
antibody has core fucosylation by fucose.
[0127] In certain embodiments, only a minor amount of a fucose
analog (or a metabolite or product of the fucose analog) is
incorporated into the complex N-glycoside-linked sugar chain(s).
For example, in various embodiments, less than about 60%, less than
about 50%, less than about 40%, less than about 30%, less than
about 20%, less than about 15%, less than about 10%, less than
about 5%, or less than about 3% of humanized, chimeric or veneered
SG16.17 or SG16.45 antibodies have core fucosylation by a fucose
analog or a metabolite or product of the fucose analog. In some
embodiments, about 2% of humanized, chimeric or veneered SG16.17
antibodies have core fucosylation by a fucose analog or a
metabolite or product of the fucose analog.
[0128] Methods of making non-fucosylated antibodies by incubating
antibody-producing cells with a fucose analogue are described,
e.g., in WO2009/135181. Briefly, cells that have been engineered to
express humanized, chimeric or veneered SG16.17 antibodies antibody
are incubated in the presence of a fucose analogue or an
intracellular metabolite or product of the fucose analog. An
intracellular metabolite can be, for example, a GDP-modified analog
or a fully or partially de-esterified analog. A product can be, for
example, a fully or partially de-esterified analog. In some
embodiments, a fucose analogue can inhibit an enzyme(s) in the
fucose salvage pathway. For example, a fucose analog (or an
intracellular metabolite or product of the fucose analog) can
inhibit the activity of fucokinase, or
GDP-fucose-pyrophosphorylase. In some embodiments, a fucose analog
(or an intracellular metabolite or product of the fucose analog)
inhibits fucosyltransferase (preferably a 1,6-fucosyltransferase,
e.g., the FUT8 protein). In some embodiments, a fucose analog (or
an intracellular metabolite or product of the fucose analog) can
inhibit the activity of an enzyme in the de novo synthetic pathway
for fucose. For example, a fucose analog (or an intracellular
metabolite or product of the fucose analog) can inhibit the
activity of GDP-mannose 4,6-dehydratase or/or GDP-fucose
synthetase. In some embodiments, the fucose analog (or an
intracellular metabolite or product of the fucose analog) can
inhibit a fucose transporter (e.g., GDP-fucose transporter).
[0129] In one embodiment, the fucose analogue is 2-flurofucose.
Methods of using fucose analogues in growth medium and other fucose
analogues are disclosed, e.g., in WO/2009/135181, which is herein
incorporated by reference.
[0130] Other methods for engineering cell lines to reduce core
fucosylation included gene knock-outs, gene knock-ins and RNA
interference (RNAi). In gene knock-outs, the gene encoding FUT8
(alpha 1,6-fucosyltransferase enzyme) is inactivated. FUT8
catalyzes the transfer of a fucosyl residue from GDP-fucose to
position 6 of Asn-linked (N-linked) GlcNac of an N-glycan. FUT8 is
reported to be the only enzyme responsible for adding fucose to the
N-linked biantennary carbohydrate at Asn297. Gene knock-ins add
genes encoding enzymes such as GNTIII or a golgi alpha mannosidase
II. An increase in the levels of such enzymes in cells diverts
monoclonal antibodies from the fucosylation pathway (leading to
decreased core fucosylation), and having increased amount of
bisecting N-acetylglucosamines. RNAi typically also targets FUT8
gene expression, leading to decreased mRNA transcript levels or
knocking out gene expression entirely. Any of these methods can be
used to generate a cell line that would be able to produce a
non-fucosylated antibody, e.g., a humanized, chimeric or veneered
SG16.17 antibody.
[0131] Many methods are available to determine the amount of
fucosylation on an antibody. Methods include, e.g., LC-MS via
PLRP-S chromatography and electrospray ionization quadrupole TOF
MS.
IV. Nucleic Acids
[0132] The invention further provides nucleic acids encoding any of
the humanized heavy and light chains described above. Typically,
the nucleic acids also encode a signal peptide fused to the mature
heavy and light chains. Coding sequences on nucleic acids can be in
operable linkage with regulatory sequences to ensure expression of
the coding sequences, such as a promoter, enhancer, ribosome
binding site, transcription termination signal and the like. The
nucleic acids encoding heavy and light chains can occur in isolated
form or can be cloned into one or more vectors. The nucleic acids
can be synthesized by for example, solid state synthesis or PCR of
overlapping oligonucleotides. Nucleic acids encoding heavy and
light chains can be joined as one contiguous nucleic acid, e.g.,
within an expression vector, or can be separate, e.g., each cloned
into its own expression vector.
V. Antibody Drug Conjugates
[0133] Anti-MCMA antibodies can be conjugated to cytotoxic moieties
to form antibody-drug conjugates (ADCs). Particularly suitable
moieties for conjugation to antibodies are cytotoxic agents (e.g.,
chemotherapeutic agents), prodrug converting enzymes, radioactive
isotopes or compounds, or toxins (these moieties being collectively
referred to as therapeutic agents or drugs). For example, an
anti-BCMA antibody can be conjugated to a cytotoxic agent such as a
chemotherapeutic agent, or a toxin (e.g., a cytostatic or cytocidal
agent such as, e.g., abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin). Examples of useful classes of cytotoxic agents
include, for example, DNA minor groove binders, DNA alkylating
agents, and tubulin inhibitors. Exemplary cytotoxic agents include,
for example, auristatins, camptothecins, duocarmycins, etoposides,
maytansines and maytansinoids (e.g., DM1 and DM4), taxanes,
benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines (PBDs),
indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vinca
alkaloids. Techniques for conjugating therapeutic agents to
proteins, and in particular to antibodies, are well-known. (See,
e.g., Alley et al., Current Opinion in Chemical Biology 2010
14:1-9; Senter, Cancer 1., 2008, 14(3):154-169.)
[0134] The therapeutic agent (e.g., cytotoxic agent) can be
conjugated to the antibody in a manner that reduces its activity
unless it is detached from the antibody (e.g., by hydrolysis, by
antibody degradation, or by a cleaving agent). Such therapeutic
agent can be attached to the antibody via a linker. A therapeutic
agent conjugated to a linker is also referred to herein as a drug
linker. The nature of the linker can vary widely. The components
that make up the linker are chosen on the basis of their
characteristics, which may be dictated in part, by the conditions
at the site to which the conjugate is delivered.
[0135] The therapeutic agent can be attached to the antibody with a
cleavable linker that is sensitive to cleavage in the intracellular
environment of the anti-BCMA-expressing cancer cell but is not
substantially sensitive to the extracellular environment, such that
the conjugate is cleaved from the antibody when it is internalized
by the anti-BCMA-expressing cancer cell (e.g., in the endosomal or,
for example by virtue of pH sensitivity or protease sensitivity, in
the lysosomal environment or in the caveolear environment). The
therapeutic agent can also be attached to the antibody with a
non-cleavable linker.
[0136] As indicated, the linker may comprise a cleavable unit. In
some such embodiments, the structure and/or sequence of the
cleavable unit is selected such that it is cleaved by the action of
enzymes present at the target site (e.g., the target cell). In
other embodiments, cleavable units that are cleavable by changes in
pH (e.g. acid or base labile), temperature or upon irradiation
(e.g. photolabile) may also be used.
[0137] In some embodiments, the cleavable unit may comprise one
amino acid or a contiguous sequence of amino acids. The amino acid
sequence may be the target substrate for an enzyme.
[0138] In some aspects, the cleavable unit is a peptidyl unit and
is at least two amino acids long. Cleaving agents can include
cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker,
1999, Pharm. Therapeutics 83:67-123). Most typical are cleavable
unit that are cleavable by enzymes that are present in anti-BCMA
expressing cells, i.e., an enzyme cleavable linker. Accordingly,
the linker can be cleaved by an intracellular peptidase or protease
enzyme, including a lysosomal or endosomal protease. For example, a
linker that is cleavable by the thiol-dependent protease
cathepsin-B, which is highly expressed in cancerous tissue, can be
used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide or a
Val-Ala peptide).
[0139] In some embodiments, the linker will comprise a cleavable
unit (e.g., a peptidyl unit) and the cleavable unit will be
directly conjugated to the therapeutic agent. In other embodiments,
the cleavable unit will be conjugated to the therapeutic agent via
an additional functional unit, e.g., a self-immolative spacer unit
or a non-self-immolative spacer unit. A non self-immolative spacer
unit is one in which part or all of the spacer unit remains bound
to the drug unit after cleavage of a cleavable unit (e.g., amino
acid) from the antibody drug conjugate. To liberate the drug, an
independent hydrolysis reaction takes place within the target cell
to cleave the spacer unit from the drug.
[0140] With a self-immolative spacer unit, the drug is released
without the need for drug for a separate hydrolysis step. In one
embodiment, wherein the linker comprises a cleavable unit and a
self immolative group, the cleavable unit is cleavable by the
action of an enzyme and after cleavage of the cleavable unit, the
self-immolative group(s) release the therapeutic agent. In some
embodiments, the cleavable unit of the linker will be directly or
indirectly conjugated to the therapeutic agent on one end and on
the other end will be directly or indirectly conjugated to the
antibody. In some such embodiments, the cleavable unit will be
directly or indirectly (e.g., via a self-immolative or
non-self-immolative spacer unit) conjugated to the therapeutic
agent on one end and on the other end will be conjugated to the
antibody via a stretcher unit. A stretcher unit links the antibody
to the rest of the drug and/or drug linker. In one embodiment, the
connection between the antibody and the rest of the drug or drug
linker is via a maleimide group, e.g., via a maleimidocaproyl
linker. In some embodiments, the antibody will be linked to the
drug via a disulfide, for example the disulfide linked maytansinoid
conjugates SPDB-DM4 and SPP-DM1.
[0141] The connection between the antibody and the linker can be
via a number of different routes, e.g., through a thioether bond,
through a disulfide bond, through an amide bond, or through an
ester bond. In one embodiment, the connection between the anti-BCMA
antibody and the linker is formed between a thiol group of a
cysteine residue of the antibody and a maleimide group of the
linker. In some embodiments, the interchain bonds of the antibody
are converted to free thiol groups prior to reaction with the
functional group of the linker. In some embodiments, a cysteine
residue is an introduced into the heavy or light chain of an
antibody and reacted with the linker. Positions for cysteine
insertion by substitution in antibody heavy or light chains include
those described in Published U.S. Application No. 2007-0092940 and
International Patent Publication WO2008070593, each of which are
incorporated by reference herein in its entirety and for all
purposes.
[0142] In some embodiments, the antibody-drug conjugates have the
following formula I:
L-(LU-D).sub.p (I)
[0143] wherein L is an anti-BCMA antibody, LU is a Linker unit and
D is a Drug unit (i.e., the therapeutic agent). The subscript p
ranges from 1 to 20. Such conjugates comprise an anti-BCMA antibody
covalently linked to at least one drug via a linker. The Linker
Unit is connected at one end to the antibody and at the other end
to the drug.
[0144] The drug loading is represented by p, the number of drug
molecules per antibody. Drug loading may range from 1 to 20 Drug
units (D) per antibody. In some aspects, the subscript p will range
from 1 to 20 (i.e., both integer and non-integer values from 1 to
20). In some aspects, the subscript p will be an integer from 1 to
20, and will represent the number of drug-linkers on a singular
antibody. In other aspects, p represents the average number of
drug-linker molecules per antibody, e.g., the average number of
drug-linkers per antibody in a reaction mixture or composition
(e.g., pharmaceutical composition), and can be an integer or
non-integer value. Accordingly, in some aspects, for compositions
(e.g., pharmaceutical compositions), p represents the average drug
loading of the antibody-drug conjugates in the composition, and p
ranges from 1 to 20.
[0145] In some embodiments, p is from about 1 to about 8 drugs per
antibody. In some embodiments, p is 1. In some embodiments, p is 2.
In some embodiments, p is from about 2 to about 8 drugs per
antibody. In some embodiments, p is from about 2 to about 6, 2 to
about 5, or 2 to about 4 drugs per antibody. In some embodiments, p
is about 2, about 4, about 6 or about 8 drugs per antibody.
[0146] The average number of drugs per antibody unit in a
preparation from a conjugation reaction may be characterized by
conventional means such as mass spectroscopy, ELISA assay, HIC, and
HPLC. The quantitative distribution of conjugates in terms of p may
also be determined.
[0147] Exemplary antibody-drug conjugates include auristatin based
antibody-drug conjugates, i.e., conjugates wherein the drug
component is an auristatin drug. Auristatins bind tubulin, have
been shown to interfere with microtubule dynamics and nuclear and
cellular division, and have anticancer activity. Typically the
auristatin based antibody-drug conjugate comprises a linker between
the auristatin drug and the anti-BCMA antibody. The auristatins can
be linked to the anti-BCMA antibody at any position suitable for
conjugation to a linker. The linker can be, for example, a
cleavable linker (e.g., a peptidyl linker) or a non-cleavable
linker (e.g., linker released by degradation of the antibody). The
auristatin can be auristatin E or a derivative thereof. The
auristatin can be, for example, an ester formed between auristatin
E and a keto acid. For example, auristatin E can be reacted with
paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and
AEVB, respectively. Other typical auristatins include MMAF
(monomethyl auristatin F), and MMAE (monomethyl auristatin E). The
synthesis and structure of exemplary auristatins are described in
U.S. Publication Nos. U.S. Pat. Nos. 7,659,241, 7,498,298,
2009-0111756, 2009-0018086, and U.S. Pat. No. 7,968,687 each of
which is incorporated herein by reference in its entirety and for
all purposes.
[0148] Exemplary auristatin based antibody-drug conjugates include
vcMMAE, vcMMAF and mcMMAF antibody-drug conjugates as shown below
wherein Ab is an antibody as described herein and val-cit
represents the valine-citrulline dipeptide:
##STR00003##
or a pharmaceutically acceptable salt thereof. The drug loading is
represented by p, the number of drug-linker molecules per antibody.
Depending on the context, p can represent the average number of
drug-linker molecules per antibody, also referred to the average
drug loading. The variable p ranges from 1 to 20 and is preferably
from 1 to 8. In some preferred embodiments, when p represents the
average drug loading, p ranges from about 2 to about 5. In some
embodiments, p is about 2, about 3, about 4, or about 5. In some
aspects, the antibody is conjugated to the linker via a sulfur atom
of a cysteine residue. In some aspects, the cysteine residue is one
that is engineered into the antibody. In other aspects, the
cysteine residue is an interchain disulfide cysteine residue.
[0149] Exemplary antibody-drug conjugates include PBD based
antibody-drug conjugates; i.e., antibody-drug conjugates wherein
the drug component is a PBD drug.
[0150] PBDs are of the general structure:
##STR00004##
[0151] They differ in the number, type and position of
substituents, in both their aromatic A rings and pyrrolo C rings,
and in the degree of saturation of the C ring. In the B-ring there
is either an imine (N.dbd.C), a carbinolamine (NH--CH(OH)), or a
carbinolamine methyl ether (NH--CH(OMe)) at the N10-C11 position,
which is the electrophilic center responsible for alkylating DNA.
All of the known natural products have an (S)-configuration at the
chiral C11a position which provides them with a right-handed twist
when viewed from the C ring towards the A ring. This gives them the
appropriate three-dimensional shape for isohelicity with the minor
groove of B-form DNA, leading to a snug fit at the binding site.
The ability of PBDs to form an adduct in the minor groove enables
them to interfere with DNA processing, hence their use as antitumor
agents.
[0152] The biological activity of these molecules can be
potentiated by joining two PBD units together through their
C8/C'-hydroxyl functionalities via a flexible alkylene linker. The
PBD dimers are thought to form sequence-selective DNA lesions such
as the palindromic 5'-Pu-GATC-P.gamma.-3' interstrand cross-link,
which is thought to be mainly responsible for their biological
activity.
[0153] In some embodiments, PBD based antibody-drug conjugates
comprise a PBD dimer linked to an anti-BCMA antibody. The monomers
that form the PBD dimer can be the same or different, i.e.,
symmetrical or unsymmetrical. The PBD dimer can be linked to the
anti-BCMA antibody at any position suitable for conjugation to a
linker. For example, in some embodiments, the PBD dimer will have a
substituent at the C2 position that provides an anchor for linking
the compound to the anti-BCMA antibody. In alternative embodiments,
the N10 position of the PBD dimer will provide the anchor for
linking the compound to the anti-BCMA antibody.
[0154] Typically the PBD based antibody-drug conjugate comprises a
linker between the PBD drug and the anti-BCMA antibody. The linker
may comprise a cleavable unit (e.g., an amino acid or a contiguous
sequence of amino acids that is a target substrate for an enzyme)
or a non-cleavable linker (e.g., linker released by degradation of
the antibody). The linker may further comprise a maleimide group
for linkage to the antibody, e.g., maleimidocaproyl. The linker
may, in some embodiments, further comprise a self-immolative group,
such as, for example, a p-aminobenzyl alcohol (PAB) unit.
[0155] An exemplary PBD for use as a conjugate is described in
International Application No. WO 2011/130613 and is as follows
wherein the wavy line indicates the site of attachment to the
linker:
##STR00005##
or a pharmaceutically acceptable salt thereof. An exemplary linker
is as follows wherein the wavy line indicates the site of
attachment to the drug and the antibody is linked via the maleimide
group.
##STR00006##
[0156] Exemplary PBDs based antibody-drug conjugates include
antibody-drug conjugates as shown below wherein Ab is an antibody
as described herein:
##STR00007##
or a pharmaceutically acceptable salt thereof. The drug loading is
represented by p, the number of drug-linker molecules per antibody.
Depending on the context, p can represent the average number of
drug-linker molecules per antibody, also referred to the average
drug loading. The variable p ranges from 1 to 20 and is preferably
from 1 to 8. In some preferred embodiments, when p represents the
average drug loading, p ranges from about 2 to about 5. In some
embodiments, p is about 2, about 3, about 4, or about 5. In some
aspects, the antibody is conjugated to the drug linker via a sulfur
atom of a cysteine residue that is engineered into the antibody. In
some aspects, the cysteine residue is engineered into the antibody
at position 239 (IgG1) as determined by the EU index (Kabat,
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987 and 1991).
VI. Animal Models of Immunological Disorders or BCMA-Expressing
Cancers
[0157] The anti-BCMA antibodies or derivatives can be tested or
validated in animal models of immunological disorders or
BCMA-expressing cancers. Examples for animal models of systemic and
organ-specific autoimmune diseases including diabetes, lupus,
systemic sclerosis, Sjogren's Syndrome, experimental autoimmune
encephalomyelitis (multiple sclerosis), thyroiditis, myasthenia
gravis, arthritis, uveitis, inflammatory bowel disease have been
described by Bigazzi, "Animal Models of Autoimmunity: Spontaneous
and Induced," in The Autoimmune Diseases (Rose and Mackay eds.,
Academic Press, 1998) and in "Animal Models for Autoimmune and
Inflammatory Disease," in Current Protocols in Immunology (Coligan
et al. eds., Wiley and Sons, 1997).
[0158] Allergic conditions, e.g., asthma and dermatitis, can also
be modeled in rodents. Airway hypersensitivity can be induced in
mice by ovalbumin (Tomkinson et al., 2001, J. Immunol.
166:5792-800) or Schistosoma mansoni egg antigen (Tesciuba et al.,
2001, J. Immunol. 167:1996-2003). The Nc/Nga strain of mice show
marked increase in serum IgE and spontaneously develop atopic
dermatitis-like leisons (Vestergaard et al., 2000, Mol. Med. Today
6:209-10; Watanabe et al., 1997, Int. Immunol. 9:461-66; Saskawa et
al., 2001, Int. Arch. Allergy Immunol. 126:239-47).
[0159] Injection of immuno-competent donor lymphocytes into a
lethally irradiated histo-incompatible host is a classical approach
to induce GVHD in mice. Alternatively, the parent B6D2F1 murine
model provides a system to induce both acute and chronic GVHD. In
this model the B6D2F1 mice are F1 progeny from a cross between the
parental strains of C57BL/6 and DBA/2 mice. Transfer of DBA/2
lymphoid cells into non-irradiated B6D2F1 mice causes chronic GVHD,
whereas transfer of C57BL/6, C57BL/10 or B10.D2 lymphoid cells
causes acute GVHD (Slayback et al., 2000, Bone Marrow Transpl.
26:931-938; Kataoka et al., 2001, Immunology 103:310-318).
[0160] Additionally, both human hematopoietic stem cells and mature
peripheral blood lymphoid cells can be engrafted into SCID mice,
and these human lympho-hematopoietic cells remain functional in the
SCID mice (McCune et al., 1988, Science 241:1632-1639; Kamel-Reid
and Dick, 1988, Science 242:1706-1709; Mosier et al., 1988, Nature
335:256-259). This has provided a small animal model system for the
direct testing of potential therapeutic agents on human lymphoid
cells. (See, e.g., Tournoy et al., 2001, J. Immunol.
166:6982-6991).
[0161] Moreover, small animal models to examine the in vivo
efficacies of the anti-BCMA antibodies or derivatives can be
created by implanting BCMA-expressing human tumor cell lines into
appropriate immunodeficient rodent strains, e.g., athymic nude mice
or SCID mice. Examples of BCMA-expressing human lymphoma cell lines
include, for example, Daudi (Ghetie et al., 1994, Blood 83:1329-36;
Ghetie et al., 1990, Int. J. Cancer 15:481-85; de Mont et al.,
2001, Cancer Res. 61:7654-59), Ramos (Ma et al., 2002, Leukemia
16:60-6; Press et al., 2001, Blood 98:2535-43), HS-Sultan (Cattan
and Maung, 1996, Cancer Chemother. Pharmacol. 38:548-52; Cattan and
Douglas, 1994, Leuk. Res. 18:513-22), Raji (Ochakovskaya et al.,
2001, Clin. Cancer Res. 7:1505-10; Breisto et al., 1999, Cancer
Res. 59:2944-49), and CA46 (Kreitman et al., 1999, Int. J. Cancer
81:148-55). Non-limiting example of a BCMA-expressing Hodgkin's
lymphoma line is L540cy (Barth et al., 2000, Blood 95:3909-14; Wahl
et al., 2002, Cancer Res. 62:3736-42). Non-limiting examples of
BCMA expressing human renal cell carcinoma cell lines include 786-O
(Ananth et al., 1999, Cancer Res. 59:2210-16; Datta et al., 2001,
Cancer Res. 61:1768-75), ACHN (Hara et al., 2001, J. Urol.
166:2491-94; Miyake et al., 2002, J. Urol. 167:2203-08), Caki-1
(Prewett et al., 1998, Clin. Cancer Res. 4:2957-66; Shi and
Siemann, 2002, Br. J. Cancer 87:119-26), and Caki-2 (Zellweger et
al., 2001, Neoplasia 3:360-67). Non-limiting examples of
BCMA-expressing nasopharyngeal carcinoma cell lines include C15 and
C17 (Burson et al., 1988, Int. J. Cancer 42:599-606; Bernheim et
al., 1993, Cancer Genet. Cytogenet. 66:11-5). Non-limiting examples
of BCMA-expressing human glioma cell lines include U373 (Palma et
al., 2000, Br. J. Cancer 82:480-7) and U87MG (Johns et al., 2002,
Int. J. Cancer 98:398-408). These tumor cell lines can be
established in immunodeficient rodent hosts either as solid tumor
by subcutaneous injections or as disseminated tumors by intravenous
injections. Once established within a host, these tumor models can
be applied to evaluate the therapeutic efficacies of the anti-BCMA
antibody or derivatives as described herein on modulating in vivo
tumor growth.
VII. Therapeutic Applications
[0162] The anti-BCMA antibodies of the invention can be used to
treat cancer. Some such cancers show detectable levels of BCMA
measured at either the protein (e.g., by immunoassay using one of
the exemplified antibodies) or mRNA level. Some such cancers show
elevated levels of BCMA relative to noncancerous tissue of the same
type, preferably from the same patient. An exemplary level of BCMA
on cancer cells amenable to treatment is 5000-150000 BCMA molecules
per cell, although higher or lower levels can be treated.
Optionally, a level of BCMA in a cancer is measured before
performing treatment.
[0163] Cancers treatable with antibodies of the invention include
solid tumors and hematological cancers, such as leukemias and
lymphomas. The antibodies are particularly suitable for cancers of
B-cells. Examples of cancers treatable with the antibodies include:
adult and pediatric acute myeloid leukemia (AML), chronic myeloid
leukemia (CML), acute lymphocytic leukemia (ALL), chronic
lymphocytic leukemia (CLL) and secondary leukemia; non-Hodgkin's
lymphoma (NHL) and Hodgkin's disease; myelodysplastic syndromes
(MDS), myeloproliferative syndromes (MPS) multiple myeloma,
Waldenstrom's macroglobulinemia or Burkett's lymphoma., malignant
plasma cell neoplasms, BCMA+high-grade lymphoma, Kahler's disease
and myelomatosis; plasma cell leukemia; plasmacytoma; B-cell
prolymphocytic leukemia; hairy cell leukemia; follicular lymphoma
(including follicular non-Hodgkin's lymphoma types); Burkitt's
lymphoma (Endemic Burkitt's lymphoma; sporadic Burkitt's lymphoma):
marginal zone lymphoma (Mucosa-Associated Lymphoid Tissue: MALT 1
MALToma; Monocytoid B cell lymphoma; splenic lymphoma with villous
lymphocytes); mantle cell lymphoma; large cell lymphoma (diffuse
large cell; diffuse mixed cell; immunoblastic lymphoma; primary
mediastinal B cell cymphoma; angiocentric lymphoma pulmonary B
cell): small lymphocytic lymphoma (SLL); recursor B-lymphoblastic
lymphoma; myeloid leukemia (granulocytic; myelogenous; acute
myeloid leukemia; chronic myeloid leukemia; subacute myeloid
leukemia; myeloid sarcoma; chloroma; granulocytic sarcoma; acute
promyelocytic leukemia; acute myelomonocytic leukemia);
Waldenstrom's macroglobulinemia, or other B-cell leukemia or
lymphoma.
[0164] The antibodies of the invention are also useful for immune
disorders mediated by immune cells expressing BCMA, particularly
B-cell mediated disorders. Examples of such diseases include
rheumatoid arthritis, systemic lupus E (SLE), Type I diabetes,
asthma, atopic dermitus, allergic rhinitis, thrombocytopenic
purpura, multiple sclerosis, psoriasis, Sjorgren's syndrome,
Hashimoto's thyroiditis, Grave's disease, primary biliary
cirrhosis, Wegener's granulomatosis, tuberculosis, and graft versus
host disease immune-mediated thrombocytopenia, haemolytic anaemia,
bullous pemphigoid, myasthenia gravis, Graves' disease, Addison's
disease, pemphigus foliaceus, psoriasis, psoriatic arthritis, and
ankylosing spondylitis.
[0165] Anti-BCMA antibodies alone or as drug-conjugates thereof,
are administered in an effective regime meaning a dosage, route of
administration and frequency of administration that delays the
onset, reduces the severity, inhibits further deterioration, and/or
ameliorates at least one sign or symptom of cancer. If a patient is
already suffering from cancer, the regime can be referred to as a
therapeutically effective regime. If the patient is at elevated
risk of the cancer relative to the general population but is not
yet experiencing symptoms, the regime can be referred to as a
prophylactically effective regime. In some instances, therapeutic
or prophylactic efficacy can be observed in an individual patient
relative to historical controls or past experience in the same
patient. In other instances, therapeutic or prophylactic efficacy
can be demonstrated in a preclinical or clinical trial in a
population of treated patients relative to a control population of
untreated patients.
[0166] Exemplary dosages for a monoclonal antibody are 0.1 mg/kg to
50 mg/kg of the patient's body weight, more typically 1 mg/kg to 30
mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12
mg/kg, or 1 mg/kg to 10 mg/kg1, or 2 mg/kg to 30 mg/kg, 2 mg/kg to
20 mg/kg, 2 mg/kg to 15 mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to
10 mg/kg, or 3 mg/kg to 30 mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to
15 mg/kg, 3 mg/kg to 12 mg/kg, or 3 mg/kg to 10 mg/kg. Exemplary
dosages for active monoclonal antibody drug conjugates thereof,
e.g., auristatins, are 1 mg/kg to 7.5 mg/kg, or 2 mg/kg to 7.5
mg/kg or 3 mg/kg to 7.5 mg/kg of the subject's body weight, or
0.1-20, or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7,
8, 9 or 10 mg/kg) or 10-1500 or 200-1500 mg as a fixed dosage.
Exemplary dosages for highly active monoclonal antibody drug
conjugates thereof, e.g., PBDs, are 1.0 .mu.g/kg to 1.0 mg/kg, or
1.0 .mu.g/kg to 500.0 .mu.g/kg of the subject's body weight. In
some methods, the patient is administered then antibody or ADC
every two, three or four weeks. The dosage depends on the frequency
of administration, condition of the patient and response to prior
treatment, if any, whether the treatment is prophylactic or
therapeutic and whether the disorder is acute or chronic, among
other factors.
[0167] Administration can be parenteral, intravenous, oral,
subcutaneous, intra-arterial, intracranial, intrathecal,
intraperitoneal, topical, intranasal or intramuscular.
Administration can also be localized directly into a tumor.
Administration into the systemic circulation by intravenous or
subcutaneous administration is preferred. Intravenous
administration can be, for example, by infusion over a period such
as 30-90 min or by a single bolus injection.
[0168] The frequency of administration depends on the half-life of
the antibody or antibody-drug conjugate in the circulation, the
condition of the patient and the route of administration among
other factors. The frequency can be daily, weekly, monthly,
quarterly, or at irregular intervals in response to changes in the
patient's condition or progression of the cancer being treated. An
exemplary frequency for intravenous administration is between twice
a week and quarterly over a continuous course of treatment,
although more or less frequent dosing is also possible. Other
exemplary frequencies for intravenous administration are between
once weekly or once monthly over a continuous course of treatment,
although more or less frequent dosing is also possible. For
subcutaneous administration, an exemplary dosing frequency is daily
to monthly, although more or less frequent dosing is also
possible.
[0169] The number of dosages administered depends on the nature of
the cancer or autoimmune disease (e.g., whether presenting acute or
chronic symptoms) and the response of the disorder to the
treatment. For acute disorders or acute exacerbations of a chronic
disorder between 1 and 10 doses are often sufficient. Sometimes a
single bolus dose, optionally in divided form, is sufficient for an
acute disorder or acute exacerbation of a chronic disorder.
Treatment can be repeated for recurrence of an acute disorder or
acute exacerbation. For chronic disorders, an antibody can be
administered at regular intervals, e.g., weekly, fortnightly,
monthly, quarterly, every six months for at least 1, 5 or 10 years,
or the life of the patient.
[0170] Pharmaceutical compositions for parenteral administration
are preferably sterile and substantially isotonic and manufactured
under GMP conditions. Pharmaceutical compositions can be provided
in unit dosage form (i.e., the dosage for a single administration).
Pharmaceutical compositions can be formulated using one or more
physiologically acceptable carriers, diluents, excipients or
auxiliaries. The formulation depends on the route of administration
chosen. For injection, antibodies can be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as
Hank's solution, Ringer's solution, or physiological saline or
acetate buffer (to reduce discomfort at the site of injection). The
solution can contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Alternatively antibodies can
be in lyophilized form for constitution with a suitable vehicle,
e.g., sterile pyrogen-free water, before use. The concentration of
antibody in a liquid formulation can be e.g., 0.01-10 mg/ml, such
as 1.0 mg/ml.
[0171] Treatment with antibodies of the invention can be combined
with chemotherapy, radiation, stem cell treatment, surgery other
treatments effective against the disorder being treated. Useful
classes of other agents that can be administered with antibodies to
BCMA include, for example, antibodies to other receptors expressed
on cancerous cells, antitubulin agents (e.g., auristatins), DNA
minor groove binders (e.g., PBDs), DNA replication inhibitors,
alkylating agents (e.g., platinum complexes such as cis-platin,
mono(platinum), bis(platinum) and tri-nuclear platinum complexes
and carboplatin), anthracyclines, antibiotics, antifolates,
antimetabolites, chemotherapy sensitizers, duocarmycins,
etoposides, fluorinated pyrimidines, ionophores, lexitropsins,
nitrosoureas, platinols, pre-forming compounds, purine
antimetabolites, puromycins, radiation sensitizers, steroids,
taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.
The same additional treatments just mentioned for cancer can also
be used for immune mediated disorders. Additional agents for immune
mediate disorders include immune suppressors such as mast cell
degranulation inhibitors, anti-histamines, corticosteroids, NSAIDs,
azathioprine, cyclophosphamide, leukeran, and cyclosporine and
biologic anti-inflammatory agents, such as Tysabri.RTM. or
Humira.RTM..
[0172] Treatment with anti-BCMA antibodies, optionally in
combination with any of the other agents or regimes described above
alone or as an antibody drug conjugate, can increase the median
progression-free survival or overall survival time of patients with
cancer, especially when relapsed or refractory, by at least 30% or
40% but preferably 50%, 60% to 70% or even 100% or longer, compared
to the same treatment (e.g., chemotherapy) but without the
anti-BCMA antibody. In addition or alternatively, treatment (e.g.,
standard chemotherapy) including the anti-BCMA antibody, alone or
as an antibody-drug conjugate, can increase the complete response
rate, partial response rate, or objective response rate
(complete+partial) of patients with tumors by at least 30% or 40%
but preferably 50%, 60% to 70% or even 100% compared to the same
treatment (e.g., chemotherapy) but without the anti-BCMA
antibody.
[0173] Typically, in a clinical trial (e.g., a phase II, phase
II/III or phase III trial), the aforementioned increases in median
progression-free survival and/or response rate of the patients
treated with standard therapy plus the anti-BCMA antibody, relative
to the control group of patients receiving standard therapy alone
(or plus placebo), are statistically significant, for example at
the p=0.05 or 0.01 or even 0.001 level. The complete and partial
response rates are determined by objective criteria commonly used
in clinical trials for cancer, e.g., as listed or accepted by the
National Cancer Institute and/or Food and Drug Administration.
VIII. Other Applications
[0174] The anti-BCMA antibodies disclosed herein can be used for
detecting BCMA in the context of clinical diagnosis or treatment or
in research. Expression of BCMA on a cancer provides an indication
that the cancer is amenable to treatment with the antibodies of the
present invention. The antibodies can also be sold as research
reagents for laboratory research in detecting cells bearing BCMA
and their response to various stimuli. In such uses, monoclonal
antibodies can be labeled with fluorescent molecules, spin-labeled
molecules, enzymes or radioisotypes, and can be provided in the
form of kit with all the necessary reagents to perform the assay
for BCMA. The antibodies can also be used to purify BCMA protein,
e.g., by affinity chromatography.
[0175] Any feature, step, element, embodiment, or aspect of the
invention can be used in combination with any other unless
specifically indicated otherwise. Although the present invention
has been described in some detail by way of illustration and
example for purposes of clarity and understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the appended claims.
EXAMPLES
Example 1: Antibody Development
Preparation of Recombinant BCMA Extracellular Domain (BCMA ECD)
[0176] The extracellular domain (ECD) of human (amino acids 1-51)
and mouse BCMA (amino acids 1-46) were cloned and expressed as a
GST fusion protein (pGEX4T1; Amersham Biosciences). Purified BCMA
ECD was obtained by capturing the BCMA fusion protein with
glutathione-Sepharose and releasing the BCMA ECD by protease
digestion with thrombin. Thrombin was subsequently removed by
benzamidine sepharose.
Identification of BCMA Expression on Malignant B-Cell Lines
[0177] Quantitative flow cytometry was performed on multiple
myeloma cell lines using Vicky-1, a commercial antibody for BCMA
(Alexis Biotechnology). Results showed that BCMA is prevalent among
myeloma lines tested. NCI H929 showed positive cell surface
staining for BCMA but lacked expression of either BR3 or TACI.
Because NCI H929 expressed BCMA but not BR3 or TACI, it was used
for cell-based screening of the BCMA hybridomas.
Development of a Transfected BCMA Cell Line.
[0178] Stable cell lines were developed by transfecting HEK 293
cells with either a full-length BCMA clone or an empty vector. Flow
cytometry confirmed positive expression of BCMA on the surface of
the BCMA transfected (293: BCMA) but not the vector empty control
plasmid (293: vector). These cell lines were subsequently used as a
tool to confirm the specificity of cloned BCMA antibodies.
Example 2: Immunization and Screening of Uncloned Hybridoma
Wells
Immunization and Screening of Antiserum
[0179] Our immunization strategy used amino acids 1-50 of the BCMA
ECD so that epitopes internal and external to ligand binding domain
could be targeted by antibodies (FIGS. 1A and 1B) KLH-conjugated
BCMA ECD was generated from a commercial source (Alexis
Biochemicals). Rats were immunized KLH-conjugated BCMA using
Titermax adjuvant until a maximum immune response was detected by
ELISA. Immunized rats serum was also screened for ability to block
APRIL binding in a plate-based assay. Rat 2-3 was selected for
fusion because the antiserum had a significant titer of human BCMA
antibodies and it displayed robust blocking activity.
[0180] Spleen cells from rat 2-3 were harvested, fused to
X-63.Ag8.653.3.12.11 mouse myeloma cells and selected as described
(Goding, 1989). Culture supernatants from the resulting hybridomas
were screened by ELISA using purified hBCMA-GST (see flow chart in
FIG. 2). Eighty positive wells were identified and selected for
expansion. Sixty of the eighty positives wells continued to showed
an OD>0.5 by ELISA following expansion. These sixty uncloned
hybridoma wells were then screened in secondary assays for
cell-based binding, ligand blockade activity, and cross-reactivity
to mouse BCMA. This led to the identification of twelve lead BCMA
hybridoma wells. Cell binding data and ligand blockade activity
from these twelve lead wells is summarized in FIG. 3. Hybridoma
well 17 showed cell binding and ligand blockade activity that
superseded the commercial monoclonal Vicky-1 (Alexis Biochemicals).
Eight wells (indicated with a red asterisk in FIG. 3) were taken
forward for cloning based on their ability to bind BCMA-positive
cells or block ligand binding.
Example 3: Characterization of Clonal Hybridomas
Cell Binding and Ligand Blockade Activity.
[0181] Hybridoma wells 11, 17, 20, 29, 40, 45 and 70 were taken
through 2 rounds of limited dilution cloning. From this point
forward, the antibodies will be designated with the formal clone ID
shown in Table 1. The specific binding of the antibodies to 293:
BCMA cells but not to the 293: vector control cells confirms that
the antibodies are binding to BCMA.
TABLE-US-00001 TABLE 1 Formal Clone IDs. Uncloned Designation
Cloned ID 11 SG16.11 17 SG16.17 20 SG16.20 29 SG16.29 40 SG16.40 45
SG16.45 70 SG16.70
[0182] Ligand blockade activity of the new BCMA antibodies was
compared using supernatant from the uncloned master well,
supernatant from the cloned well and purified antibody from a
cloned well (FIG. 4). A commercial antibody was used as a positive
control. SG-16.17 gave significant blocking of APRIL binding using
culture supernatant from the cloned hybridoma well. A titration of
the SG16.17 blockade of APRIL binding was performed in a separate
experiment using purified SG16.17 and the commercial antibody (FIG.
5). Purified SG16.17 displayed improved blocking activity across
similar concentrations when compared to the commercial antibody.
SG-16.45 showed dose-dependent inhibition of April binding although
not as strongly as SG-16.17. Ligand blockade activity for the
remaining BCMA antibodies (SG-16.11, SG16.20, SG16.29, SG16.40, and
SG16.70) was more modest. Certain blocking BCMA antibodies show
>75% inhibition of APRIL binding as was observed with SG-16.17.
More "modest" blocking antibodies including SG-16.11, SG-16.20,
SG-16.29, SG-16.40, and SG-16.70 showed about 30% inhibition for
APRIL binding (FIG. 4).
[0183] The ability of BAFF to bind immobilized BCMA was also
analyzed in the presence and absence of purified BCMA antibodies.
Pretreatment with BCMA antibodies SG16.17, SG16.40, SG16.20 and
SG17.70 all resulted in a titratable inhibition of BAFF binding to
BCMA (FIG. 6). The relative inhibition was determined by binding
BAFF to immobilized BCMA in the absence of antibody treatment (FIG.
6, asterisk). Taken together, the data in FIGS. 5 and 6 shows that
BCMA antibodies can block ligand binding of APRIL and BAFF to BCMA
and thereby interfere with B cell survival signals.
Example 4: Testing SG16.17 and SG16.45 Antibodies for ADCC and
Cytotoxicity as an ADC
[0184] The SG16.17 antibody was converted into a rat-human chimeric
IgG by fusing the rat V.sub.H and V.sub.L domains to wild-type
human IgG1 heavy chain and .kappa. light chain constant domains,
respectively. The chimerized antibody, designated cSG16.17
wild-type, showed similar antigen binding properties when compared
with the parental antibody SG16.17. Next, we installed Fc
mutations, S239D:A330L:I332E, known to enhance ADCC, to generate
cSG16.17 mutant. Similar to cSG16.17 wild-type, generation of the
Fc triple mutant did not alter the antigen-binding properties of
cSG16.17 mutant. Evaluation of cSG16.17 wild-type and cSG16.17
mutant in an ADCC assay with purified natural killer cells resulted
in dose-dependent lysis of JJN3 and U266 cells whereas no
significant lysis was observed with a nonbinding human IgG control.
The cSG16.17 wild-type antibody displayed limited ADCC activity on
JJN3 cells, which was increased .about.100-fold in potency and
>2-fold in efficacy (maximal lysis) by cSG16.17 mutant.
Similarly, for U266 cells, the ADCC activity of cSG16.17 mutant was
enhanced .about.100-fold in potency and 2-fold in efficacy compared
with the parent chimeric antibody. The concentration of cSG16.17
mutant required for maximal lysis of both JJN3 and U266 cells was
.about.100 pmol/L. In contrast, the dissociation constant (K.sub.D)
of cSG16.17 on JJN3 and U266 cells was estimated as 15 and 10
nmol/L, respectively. Thus, maximal lysis by cSG16.17 mutant was
achieved at concentrations well below those required to reach
saturation binding.
[0185] We assessed the ability of SG16.17 and SG16.45 to induce
cytotoxicity as ADCs using vcMMAF with a stoichiometry of eight
drugs per antibody. SG16.17 or SG16.45-vcMMAF8 was potently
cytotoxic against H929 cells. No decline in cell viability was
observed using a nonbinding control ADC or unconjugated antibodies.
We also examined the potency of SG16.17 ADC across other MM cell
lines, including JJN3 and U266 cell lines. SG16.17-vcMMAF8 showed
consistent and high potency (IC.sub.50 values .ltoreq.130 pmol/L)
across all three MM cell lines whereas SG16.45-vcMMAF8 showed more
variability and less overall potency.
Example 5: Testing SG16.17 Antibody for Binding to Fc.gamma.RIIIa,
and Signaling Through Fc.gamma.RIIIa
[0186] For the binding assay, CHO cells were transfected with
Fc.gamma.RIIIa (hCD16) and binding of labelled h00 antibody
measured in competition with chimeric SG16.17 with wild type IgG1
and IgG1 S239D, A330L, I332E genotype, and various IgG1 control
antibodies. FIG. 12 shows that chimeric SG16.17 competed more
strongly than two control antibodies, rituximab and cOKT9. The
mutant form of SG16.17 competed more strongly than the wild type
IgG1 form. The signaling assay uses U266 target cells expressing
BCMA, Jurkat effector cells expressing Fc.gamma.RIIIa and
engineered to express a luciferase reporter from a NFAT response
element and Bio-Glo indicator. cSG16.17 G1 WT & S239D, A330L,
I332E both elicited Fc.gamma.RIIIa signaling with that from the
S239D, A330L, I332E form being stronger (FIG. 13).
Example 6: Humanization of SG16.17
TABLE-US-00002 [0187] TABLE 2 Humanizing Mutations in hSG16.17
Heavy Chain Variants HV Exon vH Acceptor Donor Framework Acceptor
CDR Variant Sequence Residues Residues hvH1 HV1-2/HJ3 H8, H20, H48,
H67, none H69, H71, H73, H76, H80, H88, H91, H93 hvH2 HV1-2/HJ3
H20, H48, H69, H71, H34, H50, H58, H73, H76, H80, H88, H60, H61,
H62, H91, H93 H64, H65 hvH3 HV1-2/HJ3 H20, H48, H67, H69, H58, H60,
H71, H73, H76, H80, H61, H62, H88, H91, H93 H64, H65 hvH4 HV1-2/HJ3
H48, H67, H69, H71, H34, H50, H58, H73, H76, H80, H88, H60, H61,
H62, H91, H93 H64, H65 hvH5 HV1-46/HJ3 H48, H67, H71, H73, none
H76, H78, H80, H91, H93 hvH6 HV1-46/HJ3 H8, H20, H48, H71, none
H73, H76, H78, H80, H91, H93
TABLE-US-00003 TABLE 3 Humanizing Mutations in hSG16.17 Kappa Light
Chain Variants KV Exon vK Acceptor Donor Framework Acceptor CDR
Variant Sequence Residues Residues hVK2 KV1-12/KJ5 L46, L48, L87
L53 hVK3 KV1-12/KJ5 L46, L48, L87 L24, L53 hVK4 KV1-12/KJ5 L46,
L48, L78, L85, L87 none hVK5 KV1-12/KJ5 L40, L46, L48, L87 L24,
L53
TABLE-US-00004 TABLE 4 Specific Framework Mutations in hSG16.17
Heavy Chain Variants % Variant H8 H20 H48 H67 H69 H71 H73 H76 H78
H80 H88 H91 H93 Human hvH1 R* L* I* A* M* A* K* N* A V* A* F* T*
79.6 hvH2 G L* I* V M* A* K* N* A V* A* F* T* 88.8 hvH3 G L* I* A*
M* A* K* N* A V* A* F* T* 86.7 hvH4 G V I* A* M* A* K* N* A V* A*
F* T* 88.8 hvH5 G V I* A* M A* K* N* A* V* A F* T* 78.6 hvH6 R* L*
I* V M A* K* N* A* V* A F* T* 85.7 *Rat residues
TABLE-US-00005 TABLE 5 Specific Framework Mutations in hSG16.17
Kappa Light Chain Variants Variant L40 L46 L48 L78 L85 L87 % Human
hvK2 P V* V* L T F* 86.3 hvK3 P V* V* L T F* 87.4 hvK4 P V* V* M*
D* F* 83.2 hvK5 S* V* V* L T F* 86.3 *Rat residues
[0188] The rat heavy and light chain variable regions of the rat
hybridoma expressing SG16.17 were sequenced. HV1-2/HJ3 (SEQ ID NO:
9) or HV1-46/HJ3 (SEQ ID NO: 10) was used as the human acceptor
sequence for the heavy chain and KV1-12/KJ5 (SEQ ID NO: 18) was
used as the human acceptor sequence for the light chain.
[0189] Positions differing between rat donor and human acceptor
sequences included H8, H20, H48, H67, H69, H71, H76, H78, H80, H88,
H91, H93, L40, L46, L48, L78, L85 and L87. Different permutations
of these residues were included as back mutations in different
humanized heavy chain and light chain sequences. Several rat
residues in the Kabat CDRs were also tested for replacement with
corresponding residues of the human acceptor sequences. The
positions of these residues were H34, H50, H58, H60, H61, H62, H64
and H65, and L24 and L53. Six humanized heavy chain variants and
four humanized light chain variants were designed and expressed.
Tables 2 and 3 indicate the human acceptor sequence, back mutations
(donor framework residues), and CDR substitutions (Acceptor CDR
residues) in each humanized variant chain. Tables 4 and 5 indicate
the amino acids occupying each of the positions considered for back
mutation in each of the humanized variant chain. These tables also
indicate the percent of residue identical to the closest human
germline sequence. According to recent INN Guidelines only
antibodies with at least 85% identity to a human germline sequence
in both heavy and light chains can be referred to as humanized.
FIGS. 7-9 show alignments of humanized heavy chain variable regions
with the rat variable region and human acceptor sequences. FIGS. 10
and 11 show alignment of the humanized light chain variable regions
with the rat variable region and human acceptor sequences. The
C-terminal arginine (R) of the variable light chains can
alternatively be regarded as the N-terminal arginine of the light
chain constant region.
[0190] The six humanized heavy chains and four humanized light
chains were tested in all 24 possible permutations for binding to
BCMA expressed on NCI-H929 cells, which express about 50,000
molecules of BCMA per cell. The results are shown in Table 6 below.
In brief, all of the humanized light chains showed good binding. Of
the humanized heavy chains, variants VH1, VH3 and VH5 all showed
improved binding compared with either chimeric or rat SG16.17
antibody.
TABLE-US-00006 TABLE 6 Humanized Antibodies hSG16.17 Binding to
BCMA Expressed on NCI-H929 Cells hSG16.17 vH vK NCI-H929 3-pt Assay
1 vH1 vK2 ++++ 2 vH1 vK3 ++++ 3 vH1 vK4 ++++ 4 vH1 vK5 ++++ 5 vH2
vK2 - 6 vH2 vK3 - 7 vH2 vK4 - 8 vH2 vK5 - 9 vH3 vK2 ++++ 10 vH3 vK3
++++ 11 vH3 vK4 ++++ 12 vH3 vK5 ++++ 13 vH4 vK2 - 14 vH4 vK3 - 15
vH4 vK4 - 16 vH4 vK5 - 17 vH5 vK2 ++++ 18 vH5 vK3 ++++ 19 vH5 vK4
++++ 20 vH5 vK5 ++++ 21 vH6 vK2 ++ 22 vH6 vK3 ++ 23 vH6 vK4 ++ 24
vH6 vK5 ++ cSG16.17 +++ rSG16.17 +++
[0191] The humanized antibodies performing best on the NCI-H929
assay (i.e., those containing VH1, VH3 or VH5 heavy chains, were
further tested for binding to U266 cells at a full range of
concentration points. In this assay, humanized antibodies
containing VH1 heavy chains (regardless of which humanized light
chain variant was included) showed enhanced binding relative to rat
or chimeric SG16.17. Humanized antibodies containing VH3 or VH5
heavy chains (regardless of which humanized light chain variant was
included) showed the same binding within experimental error as rat
or chimeric SG16.17 binding. Humanized antibodies containing VH2 or
VH6 variable regions showed reduced binding relative to rat or
chimeric SG16.17 regardless of which humanized light chain variant
was included.
[0192] The humanized antibodies performing best on the NCI-H929
assay were also compared for protein expression level, monomer
level and percentage sequence identity to human germ line as shown
in Table 7 below.
TABLE-US-00007 TABLE 7 hBCMA Transient aSEC .gtoreq.85% human (vH,
vK) Lead hSG16.17 vH vK Binding Titer (mg/L) (% Monomer) & INN
Designation Selection 1 vH1 vK2 ++++ 139 90.4 79.6 86.3 Mix Y 2 vH1
vK3 ++++ 126 89.6 79.6 87.4 Mix Y 3 vH1 vK4 ++++ 80 94.6 79.6 83.2
Chimeric N 4 vH1 vK5 ++++ 119 89.5 79.6 86.3 Mix N 9 vH3 vK2 ++++
129 94.1 86.7 86.3 Humanized Y 10 vH3 vK3 ++++ 116 94.1 86.7 87.4
Humanized Y 11 vH3 vK4 ++++ 82 95.2 86.7 83.2 Mix Y 12 vH3 vK5 ++++
117 93.5 86.7 86.3 Humanized Y 17 vH5 vK2 ++++ 97 96.2 78.6 86.3
Mix Y 18 vH5 vK3 ++++ 86 96.1 78.6 87.4 Mix Y 19 vH5 vK4 ++++ 65
96.5 78.6 83.2 Chimeric N 20 vH5 vK5 ++++ 73 95.0 78.6 86.3 Mix
Y
[0193] The VH3 VK2 humanized antibody was selected as the lead
humanized antibody based on it having the same binding affinity for
human BCMA as rat and mouse SG16.17 antibodies (within experimental
error); greater than 85% identity to human germline sequence in
both heavy and light chain variable regions, good expression and
high percentage of monomers.
Example 7: Humanization of SG16.45
TABLE-US-00008 [0194] TABLE 8 Humanizing Mutations in hSG16.45
Heavy Chain Variants HV Exon vH Acceptor Donor Framework Acceptor
CDR Variant Sequence Residues Residues hvH1 HV3-23/HJ3 H30, H37,
H48, H93, none H94, H107 hvH2 HV3-23/HJ3 H30, H37, H48, H93, H50,
H60 H94, H107 hvH3 HV3-23/HJ3 H30, H37, H48, H76, H50, H60 H93,
H94, H107 hvH4 HV3-23/HJ3 H30, H48, H76, H93, H50 H94 hvH5
HV3-74/HJ3 H30, H93, H94 H50 hvH6 HV3-9/HJ3 H30, H93, H94 H50,
H60
TABLE-US-00009 TABLE 9 Humanizing Mutations in hSG16.45 Kappa Light
Chain Variants KV Exon vK Acceptor Donor Framework Acceptor CDR
Variant Sequence Residues Residues hvK1 KV3-20/KJ2 L14, L19, L21.
L38, L24, L26 L58, 171, 178 hvK2 KV3-20/KJ2 none L24, L26 hvK3
KV3-20/KJ2 L21, L38, L58, L71 L24, L26 hvK5 KV3-20/KJ2 L38, L71
none
TABLE-US-00010 TABLE 10 Specific Framework Mutations in hSG16.45
Heavy Chain Variants Variant H30 H37 H48 H76 H93 H94 H107 % Human
hvH1 N* I* I* N T* S* V* 86.5 hvH2 N* I* I* N T* S* V* 88.5 hvH3 N*
I* I* S* T* S* V* 87.5 hvH4 N* V I* S* T* S* T 87.5 hvH5 N* V V N
T* S* T 88.5 hvH6 N* V V N T* S* T 88.5 *Rat residues
TABLE-US-00011 TABLE 11 Specific Framework Mutations in hSG16.45
Kappa Light Chain Variants Variant L14 L19 L21 L38 L58 L71 L78 %
Human hvK1 A* V* I* H* V* Y* M* 79.2 hvK2 L A L Q I F L 86.5 hvK3 L
A I* H* V* Y* L 82.3 hvK5 L A L H* I Y* L 82.3 *Rat residues
[0195] The rat heavy and light chain variable regions of the rat
hybridoma expressing SG16.45 were sequenced. HV3-23/HJ3 (SEQ ID NO:
24) was used as the human acceptor sequence for the heavy chain and
KV3-20/KJ2 (SEQ ID NO: 34) was used as the human acceptor sequence
for the light chain.
[0196] Variable region framework positions differing between rat
donor and human acceptor sequences included H30, H37, H48, H67,
H93, H94 and H107 and positions L14, L19, L21, L38, L58, L71 and
L78. Different permutations of these residues were included as back
mutations in different humanized heavy chain and light chain
sequences. Several rat residues in the Kabat CDRs were also tested
for replacement with corresponding residues of the human acceptor
sequences. The positions of these residues were H50, H60, L24 and
L26. Six humanized heavy chain variants and four humanized light
chain variants were designed and expressed. Tables 8 and 9 indicate
the human acceptor sequence, back mutations (donor framework
residues), and CDR substitutions (Acceptor CDR residues) in each
humanized variant chain. Tables 10 and 11 indicate the amino acids
occupying each of the positions considered for back mutation in
each of the humanized variant chain. These tables also indicate the
percent of residue identical to the closest human germline
sequence. According to recent INN Guidelines only antibodies with
at least 85% identity to a human germline sequence in both heavy
and light chains can be referred to as humanized. FIGS. 14-17 show
an alignment of humanized heavy chain variable regions with the rat
variable region and human acceptor sequences. FIGS. 18 and 19 show
alignments of the light chain variable regions. The C-terminal
arginine (R) of the variable light chains can alternatively be
regarded as the N-terminal arginine of the light chain constant
region.
[0197] The six humanized heavy chains and four humanized light
chains were tested in all 24 possible permutations for binding to
BCMA expressed on NCI-H929 cells, which express about 50,000
molecules of BCMA per cell. The results are shown in Table 12
below.
TABLE-US-00012 TABLE 12 Humanized Antibodies hSG16.45 Binding to
BCMA Expressed on NCI-H929 Cells hSG16.45 vH vK NCI-H929 3-pt Assay
1 vH1 vK1 +++ 2 vH1 vK2 +++ 3 vH1 vK3 +++ 4 vH1 vK5 +++ 5 vH2 vK1 -
6 vH2 vK2 - 7 vH2 vK3 - 8 vH2 vK5 - 9 vH3 vK1 - 10 vH3 vK2 - 11 vH3
vK3 - 12 vH3 vK5 ++ 13 vH4 vK1 + 14 vH4 vK2 + 15 vH4 vK3 + 16 vH4
vK5 ++ 17 vH5 vK1 ++ 18 vH5 vK2 ++ 19 vH5 vK3 ++ 20 vH5 vK5 ++ 21
vH6 vK1 + 22 vH6 vK2 + 23 vH6 vK3 + 24 vH6 vK5 ++ cSG16.45 +++
rSG16.45 +++
[0198] The humanized antibodies performing best on the NCI-H929
assay, were further tested for binding to U266 cells at a full
range of concentration points, as well as for expression and
monomer content, as well as sequence identity to human germline
(Table 13).
TABLE-US-00013 TABLE 13 IgG aSEC VH VK hSG16.45 VH VK hBCMA mg % %
% INN 1 VH1 VK1 +++ 0.67 94.5 86.5 79.2 Mix 3 VH1 VK3 +++ 0.54 94.6
86.5 82.3 Mix 4 VH1 VK5 +++ 0.16 76.0 86.5 82.3 Mix 17 VH5 VK1 ++
0.64 94.4 88.5 79.2 Mix 18 VH5 VK2 ++ 0.65 93.7 88.5 86.5 Hu 19 VH5
VK3 ++ 0.64 94.1 88.5 82.3 Mix
[0199] The VH5 VK2, VH1 VK1 and VH1 VK3 were the best antibodies
overall based on binding affinity for human, sequence identity to
human germline sequence in both heavy and light chain variable
regions, good expression and high percentage of monomers VH1 VK1
and VH1 VK3 have somewhat higher binding (the same as rat or
chimeric within experimental error) but lower sequence identity to
human germline.
Example 8: Synthesis of a Reduced-Fucosylated hSG16.17 or hSG16.45
Antibody
[0200] The hSG16.17 VH3 VK2 or hSG16.45 VH5 VK2 antibody was
expressed in CHO cells. A fucosylation inhibitor, 2-fluorofucose,
was included in the cell culture media during the production of
antibodies resulted in non-fucosylated antibody. See, e.g., Okeley
et al., Proc. Nat'l Acad. Sci. 110:5404-55409 (2013). The base
media for cell growth was fucose free and 2-flurofucose was added
to the media to inhibit protein fucosylation. Ibid. Incorporation
of fucose into antibodies was measured by LC-MS via PLRP-S
chromatography and electrospray ionization quadrople TOF MS.
Ibid.
Example 9: In Vivo Activity of hSG16.17-SEA in SCID or NSG Mice
[0201] FIGS. 20A-C showed in vivo activity of multi dosed
hSG16.17-SEA in MM1S disseminated tumor model in SCID mice. Animals
were implanted with MM1S cells IV, and antibody dosing was
initiated 9 days post implant. Animal survival was followed over
time. N=8 animals per group. BCMA copy#=7,000, CD38 copy#=14,000.
A) 1 mg/kg weekly ip for 5 weeks B) 3 mg/kg weekly ip for 5 weeks
and C) 10 mg/kg weekly ip for 5 weeks. SCID animals contain
effector cells to mediate ADCC and ADCP. Data in this figure show
that hSG16.17 SEA improves survival comparable to daratumumab (CD38
targeted Ab. Non-binding h00 control showed no activity.
[0202] FIGS. 21A-C showed In vivo activity of single dosed
hSG16.17-SEA in EJM disseminated tumor model in NSG mice. NSG
animals contain no NK cells and minimally active macrophages
Animals were implanted with EJM cells IV, and a single dose of
antibody was given ip 5 days post implant. Animal survival was
followed over time. N=8 animals per group. BCMA copy#=45,000. CD38
copy#=47,000. CS1 copy#=14,000. A) 1 mg/kg dose B) 3 mg/kg dose C)
10 mg/kg dose. Data in this figure show that hSG16.17 SEA increases
survival to an equal or greater extent than daratumumab (CD38
targeted Ab) and elotuzumab (CS1 targeted Ab). WT SG16.17 can also
induce increased survival. Non-binding h00 control showed no
activity at the highest dose. Since there are minimal effector
cells in these animals, activity of WT and SEA hSG16.17 antibodies
is likely due to blocking of the APRIL and BAFF proliferation
signals.
[0203] FIG. 22 showed in vivo activity of multi dosed hSG16.17-SEA
in NCI-H929-luciferase disseminated tumor model in NSG mice. NSG
animals were implanted with NCI-H929 luciferase cells. Antibody
dosing was initiated 21 days post implant when bioluminescence was
observed in the bone marrow. Dosed ip weekly for 5 doses total. N=5
animals per group. BCMA copy#=25,000. CD38 copy#=45,000. CS1
copy#=3,000. Average luminescence is plotted over time in
comparison to untreated and naive animals. hSG16.17 SEA displayed
much better activity compared to daratumumab (CD38 targeted Ab) and
elotuzumab (CS1 targeted Ab). The increased luminescence observed
in the hSG16.17-SEA 10 mg/kg group is driven by a single
animal.
[0204] FIGS. 23A and 23B showed in vivo activity of single dosed
hSG16.17-SEA in NCI-H929-luciferase disseminated tumor model in NSG
mice. NSG animals were implanted with NCI-H929 luciferase cells.
Antibody dosing was initiated 21 days post injection when
bioluminescence was observed in the bone marrow. Dosed once IP. N=5
animals per group. A) 3 mg/kg WT vs SEA antibodies. B) Dose range
of hSG16.17 SEA. Data in this figure show that hSG16.17 SEA can be
active at 0.3 mg/kg single dose and hSG16.17SEA can be more active
than its WT (fucosylated) counterpart.
[0205] FIGS. 23A and 23B showed in vivo activity of single dosed
hSG16.17-SEA in NCI-H929-luciferase disseminated tumor model in NSG
mice. NSG animals were implanted with NCI-H929 luciferase cells.
Antibody dosing was initiated 21 days post injection when
bioluminescence was observed in the bone marrow. Dosed once IP. N=5
animals per group. A) 3 mg/kg WT vs SEA antibodies. B) Dose range
of hSG16.17 SEA. Data in this figure show that hSG16.17 SEA can be
active at 0.3 mg/kg single dose and hSG16.17SEA can be more active
than its WT (fucosylated) counterpart. Effects on luminescence
translates to prolonged animal survival (data not shown).
[0206] FIG. 24 In vivo activity of single dosed hSG16.17-SEA in
MOLP-8-luciferase disseminated tumor model in SCID mice. SCID
animals were implanted with MOLP-8 luciferase cells by IV. Antibody
dosing was initiated 13 days post injection when bioluminescence
was observed in the bone marrow. Dosed once IP. N=5 animals per
group. BCMA copy#=2,000. Luminescence is plotted over time. These
data show that even with only 2000 BCMA copies the hSG16.17-SEA
displays significant antitumor activity. Deglycosylated SEA BCMA
antibody, which does not bind Fc.gamma.RII or Fc.gamma.RIII, showed
no activity similar to h00 SEA non-binding control. This reveals
the importance of Fc mediated activity in this model.
[0207] FIG. 25 The SG16.17 SEA antibody displays improved ADCC
activity on MM1R target cells in comparison to WT antibody in
vitro. NK cells were isolated from PBMCs via negative selection
using an EasySep Human NK cell enrichment kit, and resulting CD16+
cells were quantitated. Multiple myeloma MM1R ADCC target cells
were labeled with chromium-51 for 1 hr. A dilution series of
antibodies was added to the assay plate, followed by target cells
(T) and NK effector cells (E) at a 13:1 E:T ratio. Lysis was
calculated based on total and spontaneous release controls after 4
hrs at 37.degree. C. These data show a significant improvement in
ADCC activity of the afucosylated SEA SG16.17 antibody over WT
antibody as well as clinical antibodies, daratumumba and
elotuzumab.
[0208] Although the invention has been described in detail for
purposes of clarity of understanding, certain modifications may be
practiced within the scope of the appended claims. All publications
including accession numbers, websites and the like, and patent
documents cited in this application are hereby incorporated by
reference in their entirety for all purposes to the same extent as
if each were so individually denoted. To the extent difference
version of a sequence, website or other reference may be present at
different times, the version associated with the reference at the
effective filing date is meant. The effective filing date means the
earliest priority date at which the accession number at issue is
disclosed. Unless otherwise apparent from the context any element,
embodiment, step, feature or aspect of the invention can be
performed in combination with any other.
Sequence CWU 1
1
1931994DNAHomo sapiens 1aagactcaaa cttagaaact tgaattagat gtggtattca
aatccttagc tgccgcgaag 60acacagacag cccccgtaag aacccacgaa gcaggcgaag
ttcattgttc tcaacattct 120agctgctctt gctgcatttg ctctggaatt
cttgtagaga tattacttgt ccttccaggc 180tgttctttct gtagctccct
tgttttcttt ttgtgatcat gttgcagatg gctgggcagt 240gctcccaaaa
tgaatatttt gacagtttgt tgcatgcttg cataccttgt caacttcgat
300gttcttctaa tactcctcct ctaacatgtc agcgttattg taatgcaagt
gtgaccaatt 360cagtgaaagg aacgaatgcg attctctgga cctgtttggg
actgagctta ataatttctt 420tggcagtttt cgtgctaatg tttttgctaa
ggaagataaa ctctgaacca ttaaaggacg 480agtttaaaaa cacaggatca
ggtctcctgg gcatggctaa cattgacctg gaaaagagca 540ggactggtga
tgaaattatt cttccgagag gcctcgagta cacggtggaa gaatgcacct
600gtgaagactg catcaagagc aaaccgaagg tcgactctga ccattgcttt
ccactcccag 660ctatggagga aggcgcaacc attcttgtca ccacgaaaac
gaatgactat tgcaagagcc 720tgccagctgc tttgagtgct acggagatag
agaaatcaat ttctgctagg taattaacca 780tttcgactcg agcagtgcca
ctttaaaaat cttttgtcag aatagatgat gtgtcagatc 840tctttaggat
gactgtattt ttcagttgcc gatacagctt tttgtcctct aactgtggaa
900actctttatg ttagatatat ttctctaggt tactgttggg agcttaatgg
tagaaacttc 960cttggtttca tgattaaact cttttttttc ctga 9942184PRTHomo
sapiens 2Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe
Asp Ser1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys
Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn
Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Ile Leu
Trp Thr Cys Leu Gly Leu Ser Leu 50 55 60 Ile Ile Ser Leu Ala Val
Phe Val Leu Met Phe Leu Leu Arg Lys Ile65 70 75 80 Asn Ser Glu Pro
Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu 85 90 95 Leu Gly
Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu 100 105 110
Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys 115
120 125 Glu Asp Cys Ile Lys Ser Lys Pro Lys Val Asp Ser Asp His Cys
Phe 130 135 140 Pro Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu Val
Thr Thr Lys145 150 155 160 Thr Asn Asp Tyr Cys Lys Ser Leu Pro Ala
Ala Leu Ser Ala Thr Glu 165 170 175 Ile Glu Lys Ser Ile Ser Ala Arg
180 3106PRThomo sapiens 3Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85
90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 4330PRThomo
sapiens 4Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115
120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320 Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 330 5329PRThomo sapiens 5Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165
170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu225 230 235 240 Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325
6330PRThomo sapiens 6Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Cys Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 7329PRThomo sapiens
7Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5
10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Cys Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240 Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320 Gln Lys Ser Leu Ser Leu Ser
Pro Gly 325 8121PRTRattus norvegicus 8Gln Val Asn Leu Leu Gln Ser
Arg Ala Ala Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys Leu Ser
Cys Glu Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile His
Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly
Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Lys Tyr Asn Glu Asn Phe 50 55
60 Lys Thr Lys Ala Thr Met Thr Ala Asp Lys Ser Thr Asn Thr Ala
Tyr65 70 75 80 Val Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Thr
Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe
Phe Asp Phe Trp Gly 100 105 110 Pro Gly Thr Lys Val Thr Val Ser Ser
115 120 9109PRThomo sapiens 9Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile
Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ser Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Val Val Tyr Tyr Cys
85 90 95 Ala Arg Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 100
105 10109PRThomo sapiens 10Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 100 105
11121PRTArtificial SequencehSG16.17 vH1 11Gln Val Gln Leu Val Gln
Ser Arg Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Lys Tyr Asn Glu Asn Phe 50
55 60 Lys Thr Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Asn Thr Ala
Tyr65 70 75 80 Val Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe
Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser
115 120 12121PRTArtificial SequencehSG16.17 vH2 12Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asn
Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Ala Asp Lys Ser Ile Asn Thr Ala Tyr65 70 75 80
Val Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85
90 95 Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe Trp
Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120
13121PRTArtificial SequencehSG16.17 vH3 13Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Asn Thr Ala
Tyr65 70 75 80 Val Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe
Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser
115 120 14121PRTArtificial SequencehSG16.17 vH4 14Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Arg Ile Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Asn
Thr Ala Tyr65 70 75 80 Val Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr
Ala Val Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp Glu Arg Val Thr
Gly Phe Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val
Ser Ser 115 120 15121PRTArtificial SequencehSG16.17 vH5 15Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Lys Tyr Asn
Glu Asn Phe 50 55 60 Lys Thr Arg Ala Thr Met Thr Ala Asp Lys Ser
Thr Asn Thr Ala Tyr65 70 75 80 Val Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp Glu Arg
Val Thr Gly Phe Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 16121PRTArtificial SequencehSG16.17 vH6
16Gln Val Gln Leu Val Gln Ser Arg Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Ile Ile Asn Pro Asn Ser Gly Tyr Thr Ser
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ala Asp
Lys Ser Thr Asn Thr Ala Tyr65 70 75 80 Val Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp
Glu Arg Val Thr Gly Phe Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 17108PRTRattus norvegicus 17Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly1 5 10 15
Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Asp Ile Ser Asp Asp 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Val Leu
Val 35 40 45 Tyr Thr Thr Ser Arg Leu Gln Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Arg Phe Ser Leu Lys Ile
Ile Val Met Gln Pro65 70 75 80 Glu Asp Glu Ala Asp Tyr Phe Cys Gln
Gln Thr Tyr Lys Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Arg
Leu Asp Leu Lys Arg 100 105 18106PRThomo sapiens 18Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala
Asn Ser Phe Pro Phe 85 90 95 Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 19108PRTArtificial SequencehSG16.17 vK2 19Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Asp Ile Ser Asp Asp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Val
35 40 45 Tyr Thr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln
Thr Tyr Lys Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 20108PRTArtificial SequencehSG16.17 vK3
20Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Ser Asp
Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Val Leu Val 35 40 45 Tyr Thr Thr Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe
Cys Gln Gln Thr Tyr Lys Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg 100 105 21108PRTArtificial
SequencehSG16.17 vK4 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala
Ser Glu Asp Ile Ser Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Val Leu Val 35 40 45 Tyr Thr Thr Ser Arg
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Met Gln Pro65 70 75 80 Glu
Asp Phe Ala Asp Tyr Phe Cys Gln Gln Thr Tyr Lys Phe Pro Pro 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105
22108PRTArtificial SequencehSG16.17 vK5 22Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Glu Asp Ile Ser Asp Asp 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Val Leu Val 35 40 45
Tyr Thr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr Lys
Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 23116PRTRattus norvegicus 23Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15 Ser Leu Lys Leu Ser
Cys Val Ala Ser Gly Phe Thr Phe Asn Asp His 20 25 30 Trp Met Thr
Trp Ile Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Ser
Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr Phe Asp Tyr Trp Gly
Gln Gly Val Met Val 100 105 110 Thr Val Ser Ser 115 24109PRThomo
sapiens 24Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp His 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Asn Thr Gly Gly
Ala Thr Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 100 105 25109PRThomo
sapiens 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp His 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Val Trp Val 35 40 45 Ser Ser Ile Thr Asn Thr Gly Gly
Ala Thr Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 100 105 26109PRThomo
sapiens 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp His 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Asn Thr Gly Gly
Ala Thr Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 100 105
27116PRTArtificial SequencehSG16.45 vH1 27Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp His 20 25 30 Trp Met
Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Ser Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr Phe Asp Tyr Trp Gly
Gln Gly Val Met Val 100 105 110 Thr Val Ser Ser 115
28116PRTArtificial SequencehSG16.45 vH2 28Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp His 20 25 30 Trp Met
Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Ser Ala Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr Phe Asp Tyr Trp Gly
Gln Gly Val Met Val 100 105 110 Thr Val Ser Ser 115
29116PRTArtificial SequencehSG16.45 vH3 29Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp His 20 25 30 Trp Met
Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Ser Ala Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr Phe Asp Tyr Trp Gly
Gln Gly Val Met Val 100 105 110 Thr Val Ser Ser 115
30116PRTArtificial SequencehSG16.45 vH4 30Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp His 20 25 30 Trp Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Ser Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115
31116PRTArtificial SequencehSG16.45 vH5 31Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp His 20 25 30 Trp Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45
Ser Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Met Val 100 105 110
Thr Val Ser Ser 115 32116PRTArtificial SequencehSG16.45 vH6 32Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp His
20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Gly Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ser Pro Gly Leu Tyr
Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser
115 33107PRTRattus norvegicus 33Glu Ile Val Leu Thr Gln Ser Pro Thr
Thr Thr Ala Ala Ser Pro Gly1 5 10 15 Glu Lys Val Thr Ile Thr Cys
Leu Ala Thr Ser Ser Val Ser Val Met 20 25 30 Tyr Trp Tyr Gln His
Lys Ser Gly Ala Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser
Ser Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Thr Met Glu Ala Glu65 70 75
80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Asp Pro Pro Thr
85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105
34107PRThomo sapiens 34Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Leu Ala
Thr Ser Ser Val Ser Val Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Ser Leu
Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu65 70 75 80 Asp
Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Asp Pro Pro Thr 85 90
95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
35107PRTArtificial SequencehSG16.45 vK1 35Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Ala Ser Pro Gly1 5 10 15 Glu Arg Val Thr
Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Val Met 20 25 30 Tyr Trp
Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45
Ser Thr Ser Ser Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50
55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg Met Glu Pro
Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Asp
Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 36107PRTArtificial SequencehSG16.45 vK2 36Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Val Met 20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35
40 45 Ser Thr Ser Ser Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu Pro Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser
Ser Asp Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg 100 105 37107PRTArtificial SequencehSG16.45 vK3 37Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Val Met 20
25 30 Tyr Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr 35 40 45 Ser Thr Ser Ser Leu Ala Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Arg Leu Glu Pro Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln
Trp Ser Ser Asp Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 38107PRTArtificial SequencehSG16.45 vK5
38Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Leu Ala Thr Ser Ser Val Ser Val
Met 20 25 30 Tyr Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Ser Thr Ser Ser Leu Ala Ser Gly Ile Pro Asp
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr
Ile Ser Arg Leu Glu Pro Glu65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
His Gln Trp Ser Ser Asp Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 395PRTRattus norvegicus 39Asp Tyr
Tyr Ile His1 5 4017PRTRattus norvegicus 40Tyr Ile Asn Pro Asn Ser
Gly Tyr Thr Lys Tyr Asn Glu Asn Phe Lys1 5 10 15 Thr4112PRTRattus
norvegicus 41Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10
428PRTRattus norvegicus 42Ile Asn Pro Asn Ser Gly Tyr Thr1 5
4314PRTRattus norvegicus 43Thr Arg Tyr Met Trp Glu Arg Val Thr Gly
Phe Phe Asp Phe1 5 10 445PRThomo sapiens 44Gly Tyr Tyr Met His1 5
4517PRThomo sapiens 45Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr
Ala Gln Lys Phe Gln1 5 10 15 Gly468PRThomo sapiens 46Ile Asn Pro
Asn Ser Gly Gly Thr1 5 475PRThomo sapiens 47Ser Tyr Tyr Met His1 5
4817PRThomo sapiens 48Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr
Ala Gln Lys Phe Gln1 5 10 15 Gly498PRThomo sapiens 49Ile Asn Pro
Ser Gly Gly Ser Thr1 5 505PRTArtificial SequencehSG16.17 vH1 Kabat
CDR1 50Asp Tyr Tyr Ile His1 5 5117PRTArtificial SequencehSG16.17
vH1 Kabat CDR2 51Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Lys Tyr Asn
Glu Asn Phe Lys1 5 10 15 Thr5212PRTArtificial SequencehSG16.17 vH1
Kabat CDR3 52Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10
538PRTArtificial SequencehSG16.17 vH1 IMGT CDR1 53Ile Asn Pro Asn
Ser Gly Tyr Thr1 5 5414PRTArtificial SequencehSG16.17 vH1 IMGT CDR2
54Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10
555PRTArtificial SequencehSG16.17 vH2 Kabat CDR1 55Asp Tyr Tyr Met
His1 5 5617PRTArtificial SequencehSG16.17 vH2 Kabat CDR2 56Arg Ile
Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15
Gly5712PRTArtificial SequencehSG16.17 vH2 Kabat CDR3 57Tyr Met Trp
Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 588PRTArtificial
SequencehSG16.17 vH2 IMGT CDR1 58Ile Asn Pro Asn Ser Gly Tyr Thr1 5
5914PRTArtificial SequencehSG16.17 vH2 IMGT CDR2 59Thr Arg Tyr Met
Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 605PRTArtificial
SequencehSG16.17 vH3 Kabat CDR1 60Asp Tyr Tyr Ile His1 5
6117PRTArtificial SequencehSG16.17 vH3 Kabat CDR2 61Tyr Ile Asn Pro
Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15
Gly6212PRTArtificial SequencehSG16.17 vH3 Kabat CDR3 62Tyr Met Trp
Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 638PRTArtificial
SequencehSG16.17 vH3 IMGT CDR1 63Ile Asn Pro Asn Ser Gly Tyr Thr1 5
6414PRTArtificial SequencehSG16.17 vH3 IMGT CDR2 64Thr Arg Tyr Met
Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 655PRTArtificial
SequencehSG16.17 vH4 Kabat CDR1 65Asp Tyr Tyr Met His1 5
6617PRTArtificial SequencehSG16.17 vH4 Kabat CDR2 66Arg Ile Asn Pro
Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15
Gly6712PRTArtificial SequencehSG16.17 vH4 Kabat CDR3 67Tyr Met Trp
Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 688PRTArtificial
SequencehSG16.17 vH4 IMGT CDR1 68Ile Asn Pro Asn Ser Gly Tyr Thr1 5
6914PRTArtificial SequencehSG16.17 vH4 IMGT CDR2 69Thr Arg Tyr Met
Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 705PRTArtificial
SequencehSG16.17 vH5 Kabat CDR1 70Asp Tyr Tyr Ile His1 5
7117PRTArtificial SequencehSG16.17 vH5 Kabat CDR2 71Tyr Ile Asn Pro
Asn Ser Gly Tyr Thr Lys Tyr Asn Glu Asn Phe Lys1 5 10 15
Thr7212PRTArtificial SequencehSG16.17 vH5 Kabat CDR3 72Tyr Met Trp
Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 738PRTArtificial
SequencehSG16.17 vH5 IMGT CDR1 73Ile Asn Pro Asn Ser Gly Tyr Thr1 5
7414PRTArtificial SequencehSG16.17 vH5 IMGT CDR2 74Thr Arg Tyr Met
Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 755PRTArtificial
SequencehSG16.17 vH6 Kabat CDR1 75Asp Tyr Tyr Met His1 5
7617PRTArtificial SequencehSG16.17 vH6 Kabat CDR2 76Ile Ile Asn Pro
Asn Ser Gly Tyr Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10 15
Gly7712PRTArtificial SequencehSG16.17 vH6 Kabat CDR3 77Tyr Met Trp
Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 788PRTArtificial
SequencehSG16.17 vH6 IMGT CDR1 78Ile Asn Pro Asn Ser Gly Tyr Thr1 5
7914PRTArtificial SequencehSG16.17 vH6 IMGT CDR2 79Thr Arg Tyr Met
Trp Glu Arg Val Thr Gly Phe Phe Asp Phe1 5 10 8011PRTRattus
norvegicus 80Leu Ala Ser Glu Asp Ile Ser Asp Asp Leu Ala1 5 10
817PRTRattus norvegicus 81Thr Thr Ser Arg Leu Gln Asp1 5
829PRTRattus norvegicus 82Gln Gln Thr Tyr Lys Phe Pro Pro Thr1 5
836PRTRattus norvegicus 83Glu Asp Ile Ser Asp Asp1 5 849PRTRattus
norvegicus 84Gln Gln Thr Tyr Lys Phe Pro Pro Thr1 5 8511PRThomo
sapiens 85Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 10
867PRThomo sapiens 86Ala Ala Ser Ser Leu Gln Ser1 5 877PRThomo
sapiens 87Gln Gln Ala Asn Ser Phe Pro1 5 886PRThomo sapiens 88Gln
Gly Ile Ser Ser Trp1 5 897PRThomo sapiens 89Gln Gln Ala Asn Ser Phe
Pro1 5 9011PRTArtificial SequencehSG16.17 vK2 Kabat CDR1 90Leu Ala
Ser Glu Asp Ile Ser Asp Asp Leu Ala1 5 10 917PRTArtificial
SequencehSG16.17 vK2 Kabat CDR2 91Thr Thr Ser Ser Leu Gln Ser1 5
929PRTArtificial SequencehSG16.17 vK2 Kabat CDR3 92Gln Gln Thr Tyr
Lys Phe Pro Pro Thr1 5 936PRTArtificial SequencehSG16.17 vK2 IMGT
CDR1 93Glu Asp Ile Ser Asp Asp1 5 949PRTArtificial SequencehSG16.17
vK2 IMGT CDR3 94Gln Gln Thr Tyr Lys Phe Pro Pro Thr1 5
9511PRTArtificial SequencehSG16.17 vK3 Kabat CDR1 95Arg Ala Ser Glu
Asp Ile Ser Asp Asp Leu Ala1 5 10 967PRTArtificial SequencehSG16.17
vK3 Kabat CDR2 96Thr Thr Ser Ser Leu Gln Ser1 5 979PRTArtificial
SequencehSG16.17 vK3 Kabat CDR3 97Gln Gln Thr Tyr Lys Phe Pro Pro
Thr1 5 986PRTArtificial SequencehSG16.17 vK3 IMGT CDR1 98Glu Asp
Ile Ser Asp Asp1 5 999PRTArtificial SequencehSG16.17 vK3 IMGT CDR3
99Gln Gln Thr Tyr Lys Phe Pro Pro Thr1 5 10011PRTArtificial
SequencehSG16.17 vK4 Kabat CDR1 100Leu Ala Ser Glu Asp Ile Ser Asp
Asp Leu Ala1 5 10 1017PRTArtificial SequencehSG16.17 vK4 Kabat CDR2
101Thr Thr Ser Arg Leu Gln Ser1 5 1029PRTArtificial
SequencehSG16.17 vK4 Kabat CDR3 102Gln Gln Thr Tyr Lys Phe Pro Pro
Thr1 5 1036PRTArtificial SequencehSG16.17 vK4 IMGT CDR1 103Glu Asp
Ile Ser Asp Asp1 5 1049PRTArtificial SequencehSG16.17 vK4 IMGT CDR3
104Gln Gln Thr Tyr Lys Phe Pro Pro Thr1 5 10511PRTArtificial
SequencehSG16.17 vK5 Kabat CDR1 105Arg Ala Ser Glu Asp Ile Ser Asp
Asp Leu Ala1 5 10 1067PRTArtificial SequencehSG16.17 vK5 Kabat CDR2
106Thr Thr Ser Ser Leu Gln Ser1 5 1079PRTArtificial
SequencehSG16.17 vK5 Kabat CDR3 107Gln Gln Thr Tyr Lys Phe Pro Pro
Thr1 5 1086PRTArtificial SequencehSG16.17 vK5 IMGT CDR1 108Glu Asp
Ile Ser Asp Asp1 5 1099PRTArtificial SequencehSG16.17 vK5 IMGT CDR3
109Gln Gln Thr Tyr Lys Phe Pro Pro Thr1 5 1105PRTRattus norvegicus
110Asp His Trp Met Thr1 5 11117PRTRattus norvegicus 111Ser Ile Thr
Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val Lys1 5 10 15
Gly1127PRTRattus norvegicus 112Pro Gly Leu Tyr Phe Asp Tyr1 5
1137PRTRattus norvegicus 113Gly Phe Thr Phe Asn Asp His1 5
1148PRTRattus norvegicus 114Ile Thr Asn Thr Gly Gly Ala Thr1 5
1159PRTRattus norvegicus 115Thr Ser Pro Gly Leu Tyr Phe Asp Tyr1 5
1165PRThomo sapiens 116Asp His Trp Met Thr1 5 11717PRThomo sapiens
117Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val Lys1
5 10 15 Gly1188PRThomo sapiens 118Gly Phe Thr Phe Ser Asp His Trp1
5 1198PRThomo sapiens 119Ile Thr Asn Thr Gly Gly Ala Thr1 5
1205PRThomo sapiens 120Asp His Trp Met Thr1 5 12117PRThomo sapiens
121Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val Lys1
5 10 15 Gly1228PRThomo sapiens 122Gly Phe Thr Phe Ser Asp His Trp1
5 1238PRThomo sapiens 123Ile Thr Asn Thr Gly Gly Ala Thr1 5
1245PRThomo sapiens 124Asp His Trp Met Thr1 5 12517PRThomo sapiens
125Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val Lys1
5 10 15 Gly1268PRThomo sapiens 126Gly Phe Thr Phe Asp Asp His Trp1
5 1278PRThomo sapiens 127Ile Thr Asn Thr Gly Gly Ala Thr1 5
1285PRTArtificial SequencehSG16.45 vH1 Kabat CDR1 128Asp His Trp
Met Thr1 5 12917PRTArtificial SequencehSG16.45 vH1 Kabat CDR2
129Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Leu Asp Ser Val Lys1
5 10 15 Gly1307PRTArtificial SequencehSG16.45 vH1 Kabat CDR3 130Pro
Gly Leu Tyr Phe Asp Tyr1 5 1318PRTArtificial SequencehSG16.45 vH1
IMGT CDR1 131Gly Phe Thr Phe Asn Asp His Trp1 5 1328PRTArtificial
SequencehSG16.45 vH1 IMGT CDR1 132Ile Thr Asn Thr Gly Gly Ala Thr1
5 1339PRTArtificial SequencehSG16.45 vH1 IMGT CDR1 133Thr Ser Pro
Gly Leu Tyr Phe Asp Tyr1 5 1345PRTArtificial SequencehSG16.45 vH2
Kabat CDR1 134Asp His Trp Met Thr1 5 13517PRTArtificial
SequencehSG16.45 vH2 Kabat CDR2 135Ala Ile Thr Asn Thr Gly Gly Ala
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly1367PRTArtificial
SequencehSG16.45 vH2 Kabat CDR3 136Pro Gly Leu Tyr Phe Asp Tyr1 5
1378PRTArtificial SequencehSG16.45 vH2 IMGT CDR1 137Gly Phe Thr Phe
Asn Asp His Trp1 5 1388PRTArtificial SequencehSG16.45 vH2 IMGT CDR2
138Ile Thr Asn Thr Gly Gly Ala Thr1 5 1399PRTArtificial
SequencehSG16.45 vH2 IMGT CDR3 139Thr Ser Pro Gly Leu Tyr Phe Asp
Tyr1 5 1405PRTArtificial SequencehSG16.45 vH3 Kabat CDR1 140Asp His
Trp Met Thr1 5 14117PRTArtificial SequencehSG16.45 vH3 Kabat
CDR2
141Ala Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys1
5 10 15 Gly1427PRTArtificial SequencehSG16.45 vH3 Kabat CDR3 142Pro
Gly Leu Tyr Phe Asp Tyr1 5 1438PRTArtificial SequencehSG16.45 vH3
IMGT CDR1 143Gly Phe Thr Phe Asn Asp His Trp1 5 1448PRTArtificial
SequencehSG16.45 vH3 IMGT CDR2 144Ile Thr Asn Thr Gly Gly Ala Thr1
5 1459PRTArtificial SequencehSG16.45 vH3 IMGT CDR3 145Thr Ser Pro
Gly Leu Tyr Phe Asp Tyr1 5 1465PRTArtificial SequencehSG16.45 vH4
Kabat CDR1 146Asp His Trp Met Thr1 5 14717PRTArtificial
SequencehSG16.45 vH4 Kabat CDR2 147Ser Ile Thr Asn Thr Gly Gly Ala
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly1487PRTArtificial
SequencehSG16.45 vH4 Kabat CDR3 148Pro Gly Leu Tyr Phe Asp Tyr1 5
1498PRTArtificial SequencehSG16.45 vH4 IMGT CDR1 149Gly Phe Thr Phe
Asn Asp His Trp1 5 1508PRTArtificial SequencehSG16.45 vH4 IMGT CDR2
150Ile Thr Asn Thr Gly Gly Ala Thr1 5 1519PRTArtificial
SequencehSG16.45 vH4 IMGT CDR3 151Thr Ser Pro Gly Leu Tyr Phe Asp
Tyr1 5 1525PRTArtificial SequencehSG16.45 vH5 Kabat CDR1 152Asp His
Trp Met Thr1 5 15317PRTArtificial SequencehSG16.45 vH5 Kabat CDR2
153Ser Ile Thr Asn Thr Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys1
5 10 15 Gly1547PRTArtificial SequencehSG16.45 vH5 Kabat CDR3 154Pro
Gly Leu Tyr Phe Asp Tyr1 5 1558PRTArtificial SequencehSG16.45 vH5
IMGT CDR1 155Gly Phe Thr Phe Asn Asp His Trp1 5 1568PRTArtificial
SequencehSG16.45 vH5 IMGT CDR2 156Ile Thr Asn Thr Gly Gly Ala Thr1
5 1579PRTArtificial SequencehSG16.45 vH5 IMGT CDR3 157Thr Ser Pro
Gly Leu Tyr Phe Asp Tyr1 5 1585PRTArtificial SequencehSG16.45 vH6
Kabat CDR1 158Asp His Trp Met Thr1 5 15917PRTArtificial
SequencehSG16.45 vH6 Kabat CDR2 159Gly Ile Thr Asn Thr Gly Gly Ala
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly1607PRTArtificial
SequencehSG16.45 vH6 Kabat CDR3 160Pro Gly Leu Tyr Phe Asp Tyr1 5
1618PRTArtificial SequencehSG16.45 vH6 IMGT CDR1 161Gly Phe Thr Phe
Asn Asp His Trp1 5 1628PRTArtificial SequencehSG16.45 vH6 IMGT CDR2
162Ile Thr Asn Thr Gly Gly Ala Thr1 5 1639PRTArtificial
SequencehSG16.45 vH6 IMGT CDR3 163Thr Ser Pro Gly Leu Tyr Phe Asp
Tyr1 5 16410PRTRattus norvegicus 164Leu Ala Thr Ser Ser Val Ser Val
Met Tyr1 5 10 1657PRTRattus norvegicus 165Ser Thr Ser Ser Leu Ala
Ser1 5 1669PRTRattus norvegicus 166His Gln Trp Ser Ser Asp Pro Pro
Thr1 5 1677PRTRattus norvegicus 167Ser Ser Val Ser Val Met Tyr1 5
1689PRTRattus norvegicus 168His Gln Trp Ser Ser Asp Pro Pro Thr1 5
16910PRThomo sapiens 169Leu Ala Thr Ser Ser Val Ser Val Met Tyr1 5
10 1707PRThomo sapiens 170Ser Thr Ser Ser Leu Ala Ser1 5
1719PRThomo sapiens 171His Gln Trp Ser Ser Asp Pro Pro Thr1 5
1727PRThomo sapiens 172Ser Ser Val Ser Val Met Tyr1 5 1739PRThomo
sapiens 173His Gln Trp Ser Ser Asp Pro Pro Thr1 5
17410PRTArtificial SequencehSG16.45 vK1 Kabat CDR1 174Arg Ala Ser
Ser Ser Val Ser Val Met Tyr1 5 10 1757PRTArtificial
SequencehSG16.45 vK1 Kabat CDR2 175Ser Thr Ser Ser Leu Ala Ser1 5
1768PRTArtificial SequencehSG16.45 vK1 Kabat CDR3 176His Gln Trp
Ser Ser Asp Pro Pro1 5 1777PRTArtificial SequencehSG16.45 vK1 IMGT
CDR1 177Ser Ser Val Ser Val Met Tyr1 5 1788PRTArtificial
SequencehSG16.45 vK1 IMGT CDR3 178His Gln Trp Ser Ser Asp Pro Pro1
5 17910PRTArtificial SequencehSG16.45 vK2 Kabat CDR1 179Arg Ala Ser
Ser Ser Val Ser Val Met Tyr1 5 10 1807PRTArtificial
SequencehSG16.45 vK2 Kabat CDR2 180Ser Thr Ser Ser Leu Ala Ser1 5
1819PRTArtificial SequencehSG16.45 vK2 Kabat CDR3 181His Gln Trp
Ser Ser Asp Pro Pro Thr1 5 1827PRTArtificial SequencehSG16.45 vK2
IMGT CDR1 182Ser Ser Val Ser Val Met Tyr1 5 1839PRTArtificial
SequencehSG16.45 vK2 IMGT CDR3 183His Gln Trp Ser Ser Asp Pro Pro
Thr1 5 18410PRTArtificial SequencehSG16.45 vK3 Kabat CDR1 184Arg
Ala Ser Ser Ser Val Ser Val Met Tyr1 5 10 1857PRTArtificial
SequencehSG16.45 vK3 Kabat CDR2 185Ser Thr Ser Ser Leu Ala Ser1 5
1869PRTArtificial SequencehSG16.45 vK3 Kabat CDR3 186His Gln Trp
Ser Ser Asp Pro Pro Thr1 5 1877PRTArtificial SequencehSG16.45 vK3
IMGT CDR1 187Ser Ser Val Ser Val Met Tyr1 5 1889PRTArtificial
SequencehSG16.45 vK3 IMGT CDR3 188His Gln Trp Ser Ser Asp Pro Pro
Thr1 5 18910PRTArtificial SequencehSG16.45 vK5 Kabat CDR1 189Leu
Ala Thr Ser Ser Val Ser Val Met Tyr1 5 10 1907PRTArtificial
SequencehSG16.45 vK5 Kabat CDR2 190Ser Thr Ser Ser Leu Ala Ser1 5
1919PRTArtificial SequencehSG16.45 vK5 Kabat CDR3 191His Gln Trp
Ser Ser Asp Pro Pro Thr1 5 1927PRTArtificial SequencehSG16.45 vK5
IMGT CDR1 192Ser Ser Val Ser Val Met Tyr1 5 1939PRTArtificial
SequencehSG16.45 vK5 IMGT CDR3 193His Gln Trp Ser Ser Asp Pro Pro
Thr1 5
* * * * *