U.S. patent application number 15/151449 was filed with the patent office on 2016-12-01 for compositions and methods of treating lupus nephritis.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Paul BRUNETTA.
Application Number | 20160346387 15/151449 |
Document ID | / |
Family ID | 56087522 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160346387 |
Kind Code |
A1 |
BRUNETTA; Paul |
December 1, 2016 |
COMPOSITIONS AND METHODS OF TREATING LUPUS NEPHRITIS
Abstract
The invention provides methods for treating or delaying
progression of lupus nephritis in an individual that has lupus. In
some embodiments, the methods comprise administering to the
individual an effective amount of a type II anti-CD20 antibody. The
invention also provides methods for treating or delaying
progression of rheumatoid arthritis (RA) or systemic lupus
erythematosus (SLE) in an individual. In some embodiments, the
methods comprise administering an effective amount of an anti-CD20
antibody.
Inventors: |
BRUNETTA; Paul; (South San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
56087522 |
Appl. No.: |
15/151449 |
Filed: |
May 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62300052 |
Feb 25, 2016 |
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62159876 |
May 11, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/138 20130101;
A61P 37/06 20180101; A61K 39/3955 20130101; C07K 2317/24 20130101;
A61K 45/06 20130101; C07K 2317/41 20130101; C07K 2317/77 20130101;
A61P 19/02 20180101; A61P 37/08 20180101; A61P 13/12 20180101; C07K
2317/71 20130101; A61K 31/5377 20130101; C07K 2317/70 20130101;
A61P 43/00 20180101; C07K 2317/73 20130101; C07K 16/2887 20130101;
A61K 31/167 20130101; A61K 31/573 20130101; C07K 2317/524 20130101;
A61P 37/02 20180101; C07K 2317/734 20130101; A61P 29/00 20180101;
A61K 2039/545 20130101; A61K 2039/507 20130101; A61P 9/12 20180101;
A61P 33/06 20180101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/167 20060101 A61K031/167; A61K 31/573 20060101
A61K031/573; A61K 31/138 20060101 A61K031/138; A61K 45/06 20060101
A61K045/06; A61K 31/5377 20060101 A61K031/5377 |
Claims
1. A method for treating or delaying progression of lupus nephritis
in an individual that has lupus, comprising administering to the
individual at least a first antibody exposure to a type II
anti-CD20 antibody and a second antibody exposure to the type II
anti-CD20 antibody, the second antibody exposure not being provided
until from about 18 weeks to about 26 weeks after the first
antibody exposure; wherein the first antibody exposure comprises
one or two doses of the type II anti-CD20 antibody, the first
antibody exposure comprising a total exposure of between about 1800
mg and about 2200 mg of the type II anti-CD20 antibody; wherein the
second antibody exposure comprises one or two doses of the type II
anti-CD20 antibody, the second antibody exposure comprising a total
exposure of between about 1800 mg and about 2200 mg of the type II
anti-CD20 antibody; and wherein the type II anti-CD20 antibody
comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:1,
HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3,
and a light chain comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2
sequence of SEQ ID NO:5, and HVR-L3 sequence of SEQ ID NO:6.
2. The method of claim 1, wherein the first antibody exposure
comprises a first dose of between about 900 mg and about 1100 mg of
the type II anti-CD20 antibody and a second dose of between about
900 mg and about 1100 mg of the type II anti-CD20 antibody.
3. The method of claim 1, wherein the first antibody exposure
comprises a first dose of the type II anti-CD20 antibody and a
second dose of the type II anti-CD20 antibody, and wherein the
second dose of the first antibody exposure is not provided until
from about 1.5 weeks to about 2.5 weeks after the first dose of the
first antibody exposure.
4. The method of claim 3, wherein the first antibody exposure
comprises a first dose of the type II anti-CD20 antibody and a
second dose of the type II anti-CD20 antibody, and wherein the
second dose of the first antibody exposure is not provided until
about 2 weeks after the first dose of the first antibody
exposure.
5. The method of claim 3, wherein the first dose of the first
antibody exposure is about 1000 mg of the type II anti-CD20
antibody.
6. The method of claim 3, wherein the second dose of the first
antibody exposure is about 1000 mg of the type II anti-CD20
antibody.
7. The method of claim 1, wherein the second antibody exposure
comprises a first dose of between about 900 mg and about 1100 mg of
the type II anti-CD20 antibody and a second dose of between about
900 mg and about 1100 mg of the type II anti-CD20 antibody.
8. The method of claim 1, wherein the second antibody exposure
comprises a first dose of the type II anti-CD20 antibody and a
second dose of the type II anti-CD20 antibody, and wherein the
second dose of the second antibody exposure is not provided until
from about 1.5 weeks to about 2.5 weeks after the first dose of the
second antibody exposure.
9. The method of claim 8, wherein the second antibody exposure
comprises a first dose of the type II anti-CD20 antibody and a
second dose of the type II anti-CD20 antibody, and wherein the
second dose of the second antibody exposure is not provided until
about 2 weeks after the first dose of the second antibody
exposure.
10. The method of claim 8, wherein the first dose of the second
antibody exposure is about 1000 mg of the type II anti-CD20
antibody.
11. The method of claim 8, wherein the second dose of the second
antibody exposure is about 1000 mg of the type II anti-CD20
antibody.
12. The method of claim 1, wherein the first antibody exposure and
the second antibody exposure are administered intravenously.
13. The method of claim 1, wherein the individual has class III or
class IV lupus nephritis.
14. The method of claim 1, wherein the individual is at risk for
developing class III or class IV lupus nephritis.
15. A method for treating or delaying progression of lupus
nephritis in an individual that has lupus, comprising administering
to the individual an effective amount of a type II anti-CD20
antibody; wherein the type II anti-CD20 antibody comprises a heavy
chain comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of
SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chain
comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ
ID NO:5, and HVR-L3 sequence of SEQ ID NO:6; and wherein the
individual has class III or class IV lupus nephritis.
16. The method of claim 14, wherein the type II anti-CD20 antibody
is administered intravenously.
17. The method of claim 1, wherein the individual does not have
class III (C) or class IV (C) lupus nephritis.
18. The method of claim 1, wherein the individual has class V lupus
nephritis.
19. The method of claim 1, further comprising administering to the
individual an effective amount of an immunosuppressive agent.
20. The method of claim 19, wherein the immunosuppressive agent
comprises mycophenolic acid, a derivative thereof, or a salt
thereof.
21. The method of claim 20, wherein the immunosuppressive agent
comprises mycophenolate mofetil.
22. The method of claim 1, further comprising administering to the
individual an effective amount of a glucocorticoid or
corticosteroid.
23. The method of claim 22, wherein the glucocorticoid or
corticosteroid comprises methylprednisolone.
24. The method of claim 22, wherein the glucocorticoid or
corticosteroid comprises prednisone.
25. The method of claim 1, further comprising administering to the
individual an effective amount of an antihistamine.
26. The method of claim 25, wherein the antihistamine comprises
diphenhydramine.
27. The method of claim 1, further comprising administering to the
individual an effective amount of a non-steroidal anti-inflammatory
drug (NSAID).
28. The method of claim 27, wherein the NSAID comprises
acetaminophen.
29. The method of claim 1, further comprising administering to the
individual an effective amount of an antihypertensive agent.
30. The method of claim 29, wherein the antihypertensive agent is
an angiotensin-converting enzyme (ACE) inhibitor or an
angiotensin-receptor blocker.
31. The method of claim 1, further comprising administering to the
individual a standard of care treatment.
32. The method of claim 31, wherein the standard of care treatment
comprises treatment with one or more of an angiotensin-converting
enzyme (ACE) inhibitor, an angiotensin-receptor blocker,
cyclophosphamide, mycophenolate mofetil, azathioprine, and a
glucocorticoid or corticosteroid.
33. The method of claim 1, wherein the method results in a complete
renal response (CRR) in the individual.
34. The method of claim 1, wherein the method results in a
depletion of circulating peripheral B cells in the individual.
35. The method of claim 34, wherein the circulating peripheral B
cells are CD19+ B cells.
36. The method of claim 1, wherein the type II anti-CD20 antibody
is a humanized or human antibody.
37. The method of claim 1, wherein the type II anti-CD20 antibody
is afucosylated.
38. The method of claim 1, wherein the heavy chain of the type II
anti-CD20 antibody comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:7.
39. The method of claim 1, wherein the light chain of the type II
anti-CD20 antibody comprises a light chain variable region
comprising the amino acid sequence of SEQ ID NO:8.
40. The method of claim 1, wherein the type II anti-CD20 antibody
is obinutuzumab.
41. The method of claim 1, wherein the individual is a human.
42. A kit for treating or delaying progression of lupus nephritis
in an individual that has lupus, comprising: (a) a container
comprising a type II anti-CD20 antibody, wherein the type II
anti-CD20 antibody comprises a heavy chain comprising HVR-H1
sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3
sequence of SEQ ID NO:3, and a light chain comprising HVR-L1
sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3
sequence of SEQ ID NO:6; and (b) a package insert with instructions
for treating or delaying progression of lupus nephritis in an
individual, wherein the instructions indicate that at least a first
antibody exposure to a type II anti-CD20 antibody and a second
antibody exposure to the type II anti-CD20 antibody are
administered to the individual, the second antibody exposure not
being provided until from about 18 weeks to about 26 weeks after
the first antibody exposure; wherein the first antibody exposure
comprises one or two doses of the type II anti-CD20 antibody, the
first antibody exposure comprising a total exposure of between
about 1800 mg and about 2200 mg of the type II anti-CD20 antibody;
and wherein the second antibody exposure comprises one or two doses
of the type II anti-CD20 antibody, the second antibody exposure
comprising a total exposure of between about 1800 mg and about 2200
mg of the type II anti-CD20 antibody.
43. The kit of claim 42, further comprising a container comprising:
(c) a second medicament, wherein the type II anti-CD20 antibody is
a first medicament; and (d) instructions on the package insert for
administering the second medicament to the subject.
44. The kit of claim 43, wherein the second medicament is an
immunosuppressive agent, a glucocorticoid, a corticosteroid, an
anti-malarial agent, a cytotoxic agent, an integrin antagonist, a
cytokine antagonist, or a hormone.
45. A kit for treating or delaying progression of lupus nephritis
in an individual that has lupus, comprising: (a) a container
comprising a type II anti-CD20 antibody, wherein the type II
anti-CD20 antibody comprises a heavy chain comprising HVR-H1
sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3
sequence of SEQ ID NO:3, and a light chain comprising HVR-L1
sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3
sequence of SEQ ID NO:6; and (b) a package insert with instructions
for treating or delaying progression of class III or class IV lupus
nephritis in an individual.
46. The kit of claim 45, further comprising a container comprising:
(c) a second medicament, wherein the type II anti-CD20 antibody is
a first medicament; and (d) instructions on the package insert for
administering the second medicament to the subject.
47. The kit of claim 46, wherein the second medicament is an
immunosuppressive agent, a glucocorticoid, a corticosteroid, an
anti-malarial agent, a cytotoxic agent, an integrin antagonist, a
cytokine antagonist, or a hormone.
48. A method for treating or delaying progression of rheumatoid
arthritis (RA) or systemic lupus erythematosus (SLE) in an
individual, comprising administering to the individual an effective
amount of an anti-CD20 antibody, wherein the antibody comprises a
heavy chain variable region comprising an HVR-H1 sequence of SEQ ID
NO:1, an HVR-H2 sequence of SEQ ID NO:2, and an HVR-H3 sequence of
SEQ ID NO:3, and a light chain variable region comprising an HVR-L1
sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an
HVR-L3 sequence of SEQ ID NO:6.
49. The method of claim 48, wherein the antibody is administered
intravenously.
50. The method of claim 48, wherein the method results in a
depletion of circulating peripheral B cells in the individual.
51. The method of claim 50, wherein the circulating peripheral B
cells are CD19+ B cells.
52. The method of claim 48, wherein the antibody is a humanized or
human antibody.
53. The method of claim 48, wherein the antibody is
afucosylated.
54. The method of claim 48, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:7.
55. The method of claim 48, wherein the light chain variable region
comprises the amino acid sequence of SEQ ID NO:8.
56. The method of claim 48, wherein the antibody is
obinutuzumab.
57. The method of claim 48, wherein the antibody comprises a
modified Fc region.
58. The method of claim 57, wherein the Fc region comprises a
modification for attenuating effector function.
59. The method of claim 57, wherein the Fc region is a human IgG1
Fc region.
60. The method of claim 59, wherein the Fc region comprises L234A,
L235A and P329G amino acid substitutions, numbering according to EU
index.
61. The method of claim 48, wherein the individual is a human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. No. 62/159,876, filed May 11, 2015;
and 62/300,052, filed Feb. 25, 2016; each of which is incorporated
herein by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392032200SeqList.txt, date recorded: May 5, 2016, size: 37
KB).
FIELD OF THE INVENTION
[0003] Provided herein are methods for treating or delaying
progression of lupus nephritis in an individual that has lupus by
administering a type II anti-CD20 antibody. Also provided herein
are methods for treating or delaying progression of rheumatoid
arthritis (RA) or systemic lupus erythematosus (SLE) in an
individual by administering an anti-CD20 antibody.
BACKGROUND
[0004] Lupus is an autoimmune disease involving antibodies that
attack connective tissue. The disease is estimated to affect nearly
1 million Americans, primarily women between the ages of 20-40. The
principal form of lupus is a systemic one (systemic lupus
erythematosus; SLE). SLE has an incidence of about 1 in 700 women
between the ages of 20 and 60. SLE can affect any organ system and
can cause severe tissue damage. Untreated lupus can be fatal as it
progresses from attack of skin and joints to internal organs,
including lung, heart, and kidneys, with renal disease, termed
lupus nephritis (LN), being the primary concern. Lupus mainly
appears as a series of flare-ups, with intervening periods of
little or no disease manifestation.
[0005] LN is one of the most acute areas of damage associated with
pathogenicity in SLE, and accounts for at least 50% of the
mortality and morbidity of the disease. Currently, there are no
really curative treatments for patients who have been diagnosed
with SLE or LN. From a practical standpoint, physicians generally
employ a number of powerful immunosuppressive drugs such as
high-dose corticosteroids, e.g., prednisone, or azathioprine or
cyclophosphamide, which are given during periods of flare-ups, but
may also be given persistently for those who have experienced
frequent flare-ups. Even with effective treatment, which reduces
symptoms and prolongs life, many of these drugs have potentially
harmful side effects to the patients being treated. As such, there
remains a need for more effective treatments against LN with fewer
harmful side effects.
[0006] Two anti-CD20 antibodies have been tested in clinical
studies for efficacy in treating lupus nephritis. Rituximab, a type
I anti-CD20 antibody, failed to meet its primary endpoint of
overall response (weighted toward complete renal response, or CRR)
but resulted in a 15.3% increase in partial renal response (PRR)
(Rovin, B. H. et al. (2012) Arthritis Rheum. 64:1215-1226).
Ocrelizumab, another type I anti-CD20 antibody, was terminated, in
part, because of an imbalance of serious infectious events (Mysler,
E. F. et al. (2013) Arthritis Rheum. 65:2368-2379).
[0007] Obinutuzumab, a type II anti-CD20 antibody, has been shown
to produce superior B cell depletion, as compared to rituximab.
Significantly greater B cell depletion was observed with
obinutuzumab treatment, compared to rituximab treatment, in
cynomolgous monkeys (Mossner, E. et al. (2010) Blood
115:4393-4402). Therefore, there remains a need for testing the
efficacy of type II anti-CD20 antibodies in treating or preventing
LN in patients with lupus.
[0008] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
SUMMARY
[0009] In certain aspects, provided herein are methods for treating
or delaying progression of lupus nephritis in an individual,
comprising administering to the individual at least a first
antibody exposure to a type II anti-CD20 antibody and a second
antibody exposure to the type II anti-CD20 antibody. In some
embodiments, the individual has lupus. In some embodiments, the
second antibody exposure is not provided until from about 18 weeks
to about 26 weeks after the first antibody exposure. In some
embodiments, the second antibody exposure is not provided until
from about 4.5 months to about 6.5 months after the first antibody
exposure. In some embodiments, the first antibody exposure
comprises one or two doses of the type II anti-CD20 antibody, the
first antibody exposure comprising a total exposure of between
about 1800 mg and about 2200 mg of the type II anti-CD20 antibody.
In some embodiments, the second antibody exposure comprises one or
two doses of the type II anti-CD20 antibody, the second antibody
exposure comprising a total exposure of between about 1800 mg and
about 2200 mg of the type II anti-CD20 antibody. In some
embodiments, the type II anti-CD20 antibody comprises a heavy chain
comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ
ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chain
comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ
ID NO:5, and HVR-L3 sequence of SEQ ID NO:6. In some embodiments,
the individual is at risk for developing class III or class IV
lupus nephritis. In some embodiments, the methods are for
preventing lupus nephritis in an individual that has lupus. In some
embodiments, the methods are for preventing lupus nephritis in an
individual that has SLE. In some embodiments, the methods are for
treating or delaying progression of lupus nephritis in an
individual that has SLE.
[0010] In some embodiments, the first antibody exposure comprises a
first dose of the type II anti-CD20 antibody and a second dose of
the type II anti-CD20 antibody, and the second dose of the first
antibody exposure is not provided until from about 1.5 weeks to
about 2.5 weeks after the first dose of the first antibody
exposure. In some embodiments, the first antibody exposure
comprises a first dose of the type II anti-CD20 antibody and a
second dose of the type II anti-CD20 antibody, and the second dose
of the first antibody exposure is not provided until about 2 weeks
after the first dose of the first antibody exposure. In some
embodiments, the first antibody exposure comprises a first dose of
the type II anti-CD20 antibody and a second dose of the type II
anti-CD20 antibody, and the second dose of the first antibody
exposure is not provided until from about 10 days to about 17 days
after the first dose of the first antibody exposure. In some
embodiments, the first antibody exposure comprises a first dose of
the type II anti-CD20 antibody and a second dose of the type II
anti-CD20 antibody, and the second dose of the first antibody
exposure is not provided until about 14 after the first dose of the
first antibody exposure. In some embodiments, the first dose of the
first antibody exposure is about 1000 mg of the type II anti-CD20
antibody. In some embodiments, the second dose of the first
antibody exposure is about 1000 mg of the type II anti-CD20
antibody. In some embodiments, the second antibody exposure
comprises a first dose of between about 900 mg and about 1100 mg of
the type II anti-CD20 antibody and a second dose of between about
900 mg and about 1100 mg of the type II anti-CD20 antibody. In some
embodiments, the second antibody exposure comprises a first dose of
the type II anti-CD20 antibody and a second dose of the type II
anti-CD20 antibody, and the second dose of the second antibody
exposure is not provided until from about 1.5 weeks to about 2.5
weeks after the first dose of the second antibody exposure. In some
embodiments, the second antibody exposure comprises a first dose of
the type II anti-CD20 antibody and a second dose of the type II
anti-CD20 antibody, and the second dose of the second antibody
exposure is not provided until about 2 weeks after the first dose
of the second antibody exposure. In some embodiments, the second
antibody exposure comprises a first dose of the type II anti-CD20
antibody and a second dose of the type II anti-CD20 antibody, and
the second dose of the second antibody exposure is not provided
until from about 10 days to about 17 days after the first dose of
the second antibody exposure. In some embodiments, the second
antibody exposure comprises a first dose of the type II anti-CD20
antibody and a second dose of the type II anti-CD20 antibody, and
the second dose of the second antibody exposure is not provided
until about 14 days after the first dose of the second antibody
exposure. In some embodiments, the first dose of the second
antibody exposure is about 1000 mg of the type II anti-CD20
antibody. In some embodiments, the second dose of the second
antibody exposure is about 1000 mg of the type II anti-CD20
antibody. In some embodiments, the first antibody exposure and the
second antibody exposure are administered intravenously. In some
embodiments, the individual has class III or class IV lupus
nephritis. In some embodiments, the individual is at risk for
developing class III or class IV lupus nephritis.
[0011] In certain aspects, provided herein are methods for treating
or delaying progression of lupus nephritis in an individual that
has lupus, comprising administering to the individual an effective
amount of a type II anti-CD20 antibody; wherein the type II
anti-CD20 antibody comprises a heavy chain comprising HVR-H1
sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3
sequence of SEQ ID NO:3, and a light chain comprising HVR-L1
sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3
sequence of SEQ ID NO:6; and wherein the individual has class III
or class IV lupus nephritis. In some embodiments, the individual is
at risk for developing class III or class IV lupus nephritis. In
some embodiments, the methods are for preventing lupus nephritis in
an individual that has lupus. In some embodiments, the methods are
for preventing lupus nephritis in an individual that has SLE. In
some embodiments, the methods are for treating or delaying
progression of lupus nephritis in an individual that has SLE.
[0012] In certain aspects, provided herein are methods for treating
or delaying progression of lupus nephritis in an individual that
has lupus, comprising administering to the individual a dose of
about 1000 mg of a type II anti-CD20 antibody, wherein the type II
anti-CD20 antibody comprises a heavy chain comprising HVR-H1
sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3
sequence of SEQ ID NO:3, and a light chain comprising HVR-L1
sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3
sequence of SEQ ID NO:6, and wherein the dose is administered to
the individual once on days 1, 15, 168, and 182. In certain
aspects, provided herein are methods for treating or delaying
progression of lupus nephritis in an individual that has lupus,
comprising administering to the individual a dose of about 1000 mg
of a type II anti-CD20 antibody, wherein the type II anti-CD20
antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ
ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ
ID NO:3, and a light chain comprising HVR-L1 sequence of SEQ ID
NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3 sequence of SEQ ID
NO:6, and wherein the dose is administered to the individual once
on weeks 0, 2, 24, and 26. In some embodiments, week 0 corresponds
to day 1. In some embodiments, the individual has class III or
class IV lupus nephritis. In some embodiments, the type II
anti-CD20 antibody is obinutuzumab.
[0013] In some embodiments of any of the above embodiments, the
type II anti-CD20 antibody is administered intravenously. In some
embodiments of any of the above embodiments, the individual does
not have class III (C) or class IV (C) lupus nephritis. In some
embodiments of any of the above embodiments, the individual has
class V lupus nephritis. In some embodiments of any of the above
embodiments, the methods further include administering to the
individual an effective amount of an immunosuppressive agent. In
some embodiments, the immunosuppressive agent comprises
mycophenolic acid, a derivative thereof, or a salt thereof. In some
embodiments, the immunosuppressive agent comprises mycophenolate
mofetil. In some embodiments of any of the above embodiments, the
methods further include administering to the individual an
effective amount of a glucocorticoid or corticosteroid. In some
embodiments, the glucocorticoid or corticosteroid comprises
methylprednisolone. In some embodiments, the glucocorticoid or
corticosteroid comprises prednisone. In some embodiments of any of
the above embodiments, the methods further include administering to
the individual an effective amount of an antihistamine. In some
embodiments, the antihistamine comprises diphenhydramine. In some
embodiments of any of the above embodiments, the methods further
include administering to the individual an effective amount of a
non-steroidal anti-inflammatory drug (NSAID). In some embodiments,
the NSAID comprises acetaminophen. In some embodiments of any of
the above embodiments, the methods further include administering to
the individual a standard of care treatment. In some embodiments,
the standard of care treatment comprises treatment with one or more
of an angiotensin-converting enzyme (ACE) inhibitor, an
angiotensin-receptor blocker, cyclophosphamide, mycophenolate
mofetil, azathioprine, and a glucocorticoid or corticosteroid. In
some embodiments, the standard of care treatment is administered
after the first antibody exposure to the type II anti-CD20 antibody
and/or after the second antibody exposure to the type II anti-CD20
antibody. In some embodiments of any of the above embodiments, the
methods further include administering to the individual an
effective amount of an antihypertensive agent. In some embodiments,
the antihypertensive agent is an angiotensin-converting enzyme
(ACE) inhibitor or an angiotensin-receptor blocker. In some
embodiments of any of the above embodiments, the method results in
a complete renal response (CRR) in the individual. In some
embodiments of any of the above embodiments, the method results in
a depletion of circulating peripheral B cells in the individual. In
some embodiments, the circulating peripheral B cells are CD19+ B
cells. In some embodiments of any of the above embodiments, the
type II anti-CD20 antibody is a humanized or human antibody. In
some embodiments of any of the above embodiments, the type II
anti-CD20 antibody is afucosylated. In some embodiments of any of
the above embodiments, the type II anti-CD20 antibody is
nonfucosylated (e.g., as described in U.S. Pat. No. 8,883,980). In
some embodiments of any of the above embodiments, the heavy chain
of the type II anti-CD20 antibody comprises a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO:7. In some
embodiments of any of the above embodiments, the light chain of the
type II anti-CD20 antibody comprises a light chain variable region
comprising the amino acid sequence of SEQ ID NO:8. In some
embodiments of any of the above embodiments, the type II anti-CD20
antibody is obinutuzumab. In some embodiments of any of the above
embodiments, the individual or patient is a human.
[0014] In certain aspects, provided herein are kits or articles of
manufacture for treating or delaying progression of lupus nephritis
in an individual that has lupus, comprising (a) a container
comprising a type II anti-CD20 antibody, wherein the type II
anti-CD20 antibody comprises a heavy chain comprising HVR-H1
sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3
sequence of SEQ ID NO:3, and a light chain comprising HVR-L1
sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3
sequence of SEQ ID NO:6; and (b) a package insert with instructions
for treating or delaying progression of lupus nephritis in an
individual, wherein the instructions indicate that at least a first
antibody exposure to a type II anti-CD20 antibody and a second
antibody exposure to the type II anti-CD20 antibody are
administered to the individual, the second antibody exposure not
being provided until from about 18 weeks to about 26 weeks after
the first antibody exposure; wherein the first antibody exposure
comprises one or two doses of the type II anti-CD20 antibody, the
first antibody exposure comprising a total exposure of between
about 1800 mg and about 2200 mg of the type II anti-CD20 antibody;
and wherein the second antibody exposure comprises one or two doses
of the type II anti-CD20 antibody, the second antibody exposure
comprising a total exposure of between about 1800 mg and about 2200
mg of the type II anti-CD20 antibody. In some embodiments, the kits
or articles of manufacture further include (c) a second medicament,
wherein the type II anti-CD20 antibody is a first medicament; and
(d) instructions on the package insert for administering the second
medicament to the subject. In some embodiments, the second
medicament is an immunosuppressive agent, a glucocorticoid, a
corticosteroid, an anti-malarial agent, a cytotoxic agent, an
integrin antagonist, a cytokine antagonist, or a hormone. In some
embodiments, the heavy chain of the type II anti-CD20 antibody
comprises a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:7. In some embodiments, the light chain of
the type II anti-CD20 antibody comprises a light chain variable
region comprising the amino acid sequence of SEQ ID NO:8. In some
embodiments, the type II anti-CD20 antibody is obinutuzumab. In
some embodiments, the kits or articles of manufacture are for
preventing lupus nephritis in an individual that has SLE. In some
embodiments, the kits or articles of manufacture are for treating
or delaying progression of lupus nephritis in an individual that
has SLE.
[0015] In certain aspects, provided herein are methods for treating
or delaying progression of rheumatoid arthritis (RA) or systemic
lupus erythematosus (SLE) in an individual, comprising
administering to the individual an effective amount of an anti-CD20
antibody, wherein the antibody comprises a heavy chain variable
region comprising an HVR-H1 sequence of SEQ ID NO:1, an HVR-H2
sequence of SEQ ID NO:2, and an HVR-H3 sequence of SEQ ID NO:3, and
a light chain variable region comprising an HVR-L1 sequence of SEQ
ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an HVR-L3 sequence
of SEQ ID NO:6. In some embodiments, the antibody is administered
intravenously. In some embodiments, the method results in a
depletion of circulating peripheral B cells in the individual. In
some embodiments, the circulating peripheral B cells are CD19+ B
cells. In some embodiments, the antibody is a humanized or human
antibody. In some embodiments, the antibody is afucosylated. In
some embodiments, the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO:7. In some embodiments, the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:8. In some embodiments, the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:7 and the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:8. In some embodiments, the antibody is obinutuzumab. In some
embodiments, the antibody comprises a modified Fc region. In some
embodiments, the Fc region comprises a modification for attenuating
effector function. In some embodiments, the Fc region is a human
IgG1 Fc region. In some embodiments, the human IgG1 Fc region
comprises L234A, L235A and P329G amino acid substitutions,
numbering according to EU index.
[0016] In certain aspects, provided herein are compositions for use
in treating or delaying progression of rheumatoid arthritis (RA) or
systemic lupus erythematosus (SLE) in an individual, the
compositions comprising an anti-CD20 antibody, wherein the antibody
comprises a heavy chain variable region comprising an HVR-H1
sequence of SEQ ID NO:1, an HVR-H2 sequence of SEQ ID NO:2, and an
HVR-H3 sequence of SEQ ID NO:3, and a light chain variable region
comprising an HVR-L1 sequence of SEQ ID NO:4, an HVR-L2 sequence of
SEQ ID NO:5, and an HVR-L3 sequence of SEQ ID NO:6. In some
embodiments, the composition is administered intravenously. In some
embodiments, administering the composition results in a depletion
of circulating peripheral B cells in the individual. In some
embodiments, the circulating peripheral B cells are CD19+ B cells.
In some embodiments, the antibody is a humanized or human antibody.
In some embodiments, the antibody is afucosylated. In some
embodiments, the heavy chain variable region comprises the amino
acid sequence of SEQ ID NO:7. In some embodiments, the light chain
variable region comprises the amino acid sequence of SEQ ID NO:8.
In some embodiments, the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO:7 and the light chain variable
region comprises the amino acid sequence of SEQ ID NO:8. In some
embodiments, the antibody is obinutuzumab. In some embodiments, the
antibody comprises a modified Fc region. In some embodiments, the
Fc region comprises a modification for attenuating effector
function. In some embodiments, the Fc region is a human IgG1 Fc
region. In some embodiments, the human IgG1 Fc region comprises
L234A, L235A and P329G amino acid substitutions, numbering
according to EU index. In some embodiments of any of the above
embodiments, the individual or patient is a human.
[0017] In certain aspects, provided herein is use of an anti-CD20
antibody for the manufacture of a medicament for use in treatment
of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE)
in an individual, wherein the antibody comprises a heavy chain
variable region comprising an HVR-H1 sequence of SEQ ID NO:1, an
HVR-H2 sequence of SEQ ID NO:2, and an HVR-H3 sequence of SEQ ID
NO:3, and a light chain variable region comprising an HVR-L1
sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an
HVR-L3 sequence of SEQ ID NO:6.
[0018] In certain aspects, provided herein are kits or articles of
manufacture for treating or delaying progression of rheumatoid
arthritis (RA) or systemic lupus erythematosus (SLE) in an
individual, comprising (a) a container comprising an anti-CD20
antibody, wherein the antibody comprises a heavy chain variable
region comprising an HVR-H1 sequence of SEQ ID NO:1, an HVR-H2
sequence of SEQ ID NO:2, and an HVR-H3 sequence of SEQ ID NO:3, and
a light chain variable region comprising an HVR-L1 sequence of SEQ
ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an HVR-L3 sequence
of SEQ ID NO:6; and (b) a package insert with instructions for
administering an effective amount of anti-CD20 antibody to treat or
delay progression of rheumatoid arthritis (RA) or systemic lupus
erythematosus (SLE) in an individual. In some embodiments, the
package insert includes instructions for administering the antibody
intravenously. In some embodiments, the antibody is a humanized or
human antibody. In some embodiments, the antibody is afucosylated.
In some embodiments, the heavy chain variable region comprises the
amino acid sequence of SEQ ID NO:7. In some embodiments, the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:8. In some embodiments, the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:7 and the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:8. In some embodiments, the antibody is obinutuzumab. In some
embodiments, the antibody comprises a modified Fc region. In some
embodiments, the Fc region comprises a modification for attenuating
effector function. In some embodiments, the Fc region is a human
IgG1 Fc region. In some embodiments, the human IgG1 Fc region
comprises L234A, L235A and P329G amino acid substitutions,
numbering according to EU index.
[0019] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art. These and other embodiments of the invention are
further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows the study design for a Phase II study examining
obinutuzumab+mycophenolate mofetil vs. placebo+mycophenolate
mofetil. EP=endpoint; MMF=mycophenolate mofetil.
[0021] FIG. 2A-2D show whole blood B-cell-depletion,
internalization and complement-dependent cellular cytotoxicity
elicited by Obinutuzumab or Rituximab in RA and SLE patient
samples. FIG. 2A shows whole blood samples from patients with RA
(n=31) and SLE (n=34). Samples were incubated with or without
anti-CD20 mAbs, RTX, OBZ.sub.Gly and OBZ for 24 hours before flow
cytometry to analyze B cell death. Values are the mean of
triplicate wells. The horizontal line in the box represents the
median, the box represents the interquartile range and the whiskers
represent the range. FIG. 2B shows the frequency of surface
accessible mAbs. The frequency was assessed by flow cytometry after
six hours of incubation with isolated B cells from patients with RA
(n=5) and SLE (n=8) with or without prior incubation with
anti-Fc.gamma.RII blocking mAb, AT10. The horizontal line
represents the median. FIG. 2C shows CDC induced by RTX and OBZ.
Isolated B cells (Healthy control (HC), n=2; RA, n=2 and SLE, n=3)
were incubated with RTX or OBZ for 30 minutes with normal healthy
serum (NHS) or heat inactivated serum (HIS) before analyzing for
the frequency of lysed CD19+Av+PI+ B cells. FIG. 2D shows the fold
increase in CD19+Av+PI+ cells in samples incubated with NHS vs HIS
representing the efficiency of CDC by mAbs. RTX, rituximab; OBZ,
Obinutuzumab; RA, rheumatoid arthritis; SLE, systemic lupus
erythematosus. HC, Healthy control * p<0.05; **, p<0.005;
***, p<0.0001 and ns, not significant.
[0022] FIG. 3A-3G show the flow cytometry-gating strategy to assess
NK cell degranulation, describing the relationship between NK cell
expression of CD107a and CD16. Whole blood samples were incubated
with or without mAbs for 24 hours before analyzing by flow
cytometry. NK cells were identified based on forward- and
side-scatter properties and expression of CD56 but not CD3. The
frequency of CD3-CD56+CD107a+ cells represented
activated/degranulated NK cells. FSC, forward-scatter; SSC,
side-scatter. FIG. 3A shows flow cytometry gating of forward
scatter vs. side scatter. FIG. 3B shows flow cytometry gating of
CD56 vs. CD3. FIG. 3C shows flow cytometry gating of forward
scatter vs. CD107a. FIG. 3D shows flow cytometry gating of forward
scatter vs. CD16. Three subpopulations of CD3-CD56+ NK cells were
identified based on the relative expression of CD16 (boxed as high,
medium, and low). The relative frequency of activated CD107a+NK
cells differed in these 3 subpopulations based on CD16 expression
in a hierarchical manner CD16++<CD16+<CD16-. FIG. 3E shows
flow cytometry gating of forward scatter vs. CD107a for the high
box. FIG. 3F shows flow cytometry gating of forward scatter vs.
CD107a for the medium box. FIG. 3G shows flow cytometry gating of
forward scatter vs. CD107a for the low box.
[0023] FIG. 4A-4D show that OBZ is more efficient than RTX at
activating NK cells in RA and SLE patient samples. NK cell
activation was assessed in whole blood samples from patients with
RA (n=18) and SLE (n=23) incubated for 24 hours in the presence or
absence of mAbs. *, p<0.05; **, p<0.005; ***, p<0.0001;
ns, not significant and Spearman correlation coefficient, r.sup.2
was considered significant when p was at least <0.05. FIG. 4A
shows the frequency of CD3-CD56+NK cells in the lymphocyte gate,
CD3-CD56+CD107a+NK cells and CD3-CD56-CD16+NK cells as a percentage
of total NK cells or CD19+ cells. Horizontal lines represent the
median. FIG. 4B shows the frequency of CD3-CD56+CD107a+NK cells and
the fold increase in the frequency of CD3-CD56+CD107a+NK cells and
the frequency of CD3-CD56+16+NK cells in samples incubated with RTX
and OBZ, from patients with RA and SLE. Horizontal lines represent
the median. FIG. 4C shows the relationship between the frequency of
CD3-CD56+CD107a+NK cells in samples incubated with or without RTX
and OBZ in samples from patients with RA (n=18). FIG. 4D shows the
relationship between the frequency of CD3-CD56+CD107a+NK cells in
samples incubated with or without RTX and OBZ in samples from
patients with SLE (n=23).
[0024] FIG. 5A-5D show that Obinutuzumab is more efficient than
Rituximab at evoking NK cell-mediated cellular cytotoxicity in RA
and SLE patient samples. FIG. 5A shows a whole blood B-cell
depletion assay showing the percentage B-cell depletion by RTX,
OBZ.sub.Gly and OBZ in samples from patients with RA (n=18) and SLE
(n=23). Box and whiskers represent the interquartile range and the
range, the horizontal line in the box represents the median. FIG.
5B shows the frequency of CD3-CD56+CD107a+NK cells in whole blood
samples from patients with RA and SLE after 24-hour incubation with
or without mAbs, analyzed by flow cytometry. FIG. 5C shows the
relative increase in the frequency of CD3-CD56+CD107a+NK cells in
whole blood samples incubated with or without mAbs, analyzed by
flow cytometry. FIG. 5D shows the frequency of CD3-CD56+CD16+NK
cells in whole blood samples from patients with RA (n=18) and SLE
(n=23) after 24 hour incubation with or without mAbs, analyzed by
flow cytometry. For the bar graphs, the error bars represent the
median and interquartile ranges. * p<0.05; **, p<0.005; ***,
p<0.0001; and ns, not significant.
[0025] FIG. 6A-6D show that Obinutuzumab is more efficient than
Rituximab at activating neutrophils in RA and SLE patient samples.
FIG. 6A shows the mean fluorescence intensity (MFI) of CD11b on
CD15+ neutrophils after 24 hour incubation of whole blood samples
from patients with RA (n=10) and SLE (n=22) incubated with or
without mAbs (1 .mu.g/ml). The Median and interquartile ranges are
represented by the error bars. FIG. 6B shows the relationship
between the MFI of CD11b on CD15+neutrophils in samples incubated
with or without mAbs in RA and SLE samples. FIG. 6C shows the MFI
of CD62L on CD15+neutrophils in samples incubated with or without
mAbs in RA and SLE samples. FIG. 6D shows the relationship between
the MFI of CD62L on CD15+ neutrophils, in samples incubated with or
without mAbs in RA (n=10) and SLE (n=22) samples. * p<0.05; **,
p<0.005; ***, p<0.0001. Spearman correlation coefficient,
r.sup.2 was considered significant when p was at least
<0.05.
[0026] FIG. 7A-7D show assessment of direct cell death,
internalization and expression of CD20 and Fc.gamma.RIIb in B-cell
subpopulations from RA and SLE samples. FIG. 7A shows the frequency
of Annexin V+ cells as a proportion of all CD19+ B cells and also
B-cell subpopulations based on the relative expression of IgD and
CD27: (IgD+CD27-naive cells; IgD+CD27+ unswitched memory cells;
IgD-CD27+ switched memory cells; and IgD-CD27-double negative
cells); in samples from patients with RA (n=5) and SLE (n=4)
incubated with or without mAbs. FIG. 7B shows the mean fluorescence
intensity (MFI) of CD20 on all CD19+ cells and B-cell
subpopulations in samples from patients with SLE (n=9). FIG. 7C
shows a surface fluorescence-quenching assay. The frequency of
surface accessible mAbs after 6 hours of incubation with isolated
B-cells, with or without prior incubation with anti-Fc.gamma.RII
mAb, AT10 from patients with SLE (n=9), in all CD19+ B-cells and
B-cell subpopulations. FIG. 7D shows the MFI of Fc.gamma.RIIb on
all CD19+ B-cells and B-cell subpopulations in samples from
patients with SLE (n=9). For the bar graphs, the error bars
represent the median and interquartile ranges. Box and whiskers
represent the interquartile range and the horizontal line in the
box represents the median. * p<0.05; **, p<0.005; ***,
p<0.0001.
[0027] FIG. 8 shows the gating strategy for the
complement-dependent cytotoxicity assay, and CDC by RTX and OBZ.
Isolated B cells were incubated with mAbs either with NHS or HIS
for 30 minutes at room temperature before analyzing by flow
cytometry. The frequency of An V+PI+ cells represented cell death.
HIS, heat inactivated serum; NHS, normal healthy serum; RTX,
rituximab; OBZ, Obinutuzumab; An V, Annexin V and PI, propidium
iodide.
[0028] FIG. 9 shows the flow cytometry-gating strategy to assess
neutrophil activation. After 24 hours of incubation, whole blood
samples were analysed by flow cytometry. Neutrophils were
identified by forward- and side-scatter and CD15 positively. The
mean fluorescence intensity of CD11b and CD62L was analyzed on
gated neutrophils positive for CD15.
[0029] FIG. 10 shows the flow cytometry-gating strategy to assess
direct cell death. After 6 hours of incubation with or without mAbs
at 37.degree. C. and 5% CO.sub.2, isolated B-cells were analyzed by
flow cytometry. CD19+ B-cells were categorized into naive
(IgD+CD27-), unswitched memory cells (IgD+CD27+), switched memory
cells (IgD-CD27+) and double negative cells (IgD-CD27-). The
frequency of Annexin V+ cells represented direct cell death.
[0030] FIG. 11 shows the inherent susceptibility to spontaneous
cell death in B-cell subpopulations. Isolated B-cells incubated in
RPMI supplemented with 10% foetal calf serum for 6 hours at at
37.degree. C. and 5% CO.sub.2 were analyzed by flow cytometry. The
frequency of Annexin V+ cells represented direct cell death in
CD19+ cells as a whole and also in B-cell subpopulations
categorized into naive (IgD+CD27-), unswitched memory cells
(IgD+CD27+), switched memory cells (IgD-CD27+) and double negative
cells (IgD-CD27-). Av, Annexin V; * p<0.05; ***, p<0.0001;
ns, not significant.
DETAILED DESCRIPTION
[0031] In one aspect, provided herein are methods for treating or
delaying progression of lupus nephritis in an individual, including
administering to the individual at least a first antibody exposure
to a type II anti-CD20 antibody and a second antibody exposure to
the type II anti-CD20 antibody. In some embodiments, the individual
has lupus. In some embodiments, the second antibody exposure is not
provided until from about 18 weeks to about 26 weeks after the
first antibody exposure. In some embodiments, the first antibody
exposure includes one or two doses of the type II anti-CD20
antibody, the first antibody exposure containing a total exposure
of between about 1800 mg and about 2200 mg of the type II anti-CD20
antibody. In some embodiments, the second antibody exposure
includes one or two doses of the type II anti-CD20 antibody, the
second antibody exposure containing a total exposure of between
about 1800 mg and about 2200 mg of the type II anti-CD20 antibody.
In some embodiments, the antibody comprises a heavy chain
comprising HVR-H1 sequence of SEQ ID NO:1, HVR-H2 sequence of SEQ
ID NO:2, and HVR-H3 sequence of SEQ ID NO:3, and a light chain
comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2 sequence of SEQ
ID NO:5, and HVR-L3 sequence of SEQ ID NO:6.
[0032] In another aspect, provided herein are methods for treating
or delaying progression of lupus nephritis in an individual that
has lupus, including administering to the individual an effective
amount of a type II anti-CD20 antibody. In some embodiments, the
antibody includes a heavy chain containing HVR-H1 sequence of SEQ
ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ
ID NO:3, and a light chain containing HVR-L1 sequence of SEQ ID
NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3 sequence of SEQ ID
NO:6. In some embodiments, the individual has class III or class IV
lupus nephritis.
[0033] In another aspect, provided herein are methods for treating
or delaying progression of rheumatoid arthritis (RA) or systemic
lupus erythematosus (SLE) in an individual, comprising
administering to the individual an effective amount of an anti-CD20
antibody. In some embodiments, the antibody comprises a heavy chain
variable region comprising an HVR-H1 sequence of SEQ ID NO:1, an
HVR-H2 sequence of SEQ ID NO:2, and an HVR-H3 sequence of SEQ ID
NO:3, and a light chain variable region comprising an HVR-L1
sequence of SEQ ID NO:4, an HVR-L2 sequence of SEQ ID NO:5, and an
HVR-L3 sequence of SEQ ID NO:6.
I. GENERAL TECHNIQUES
[0034] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current
Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,
(2003)); the series Methods in Enzymology (Academic Press, Inc.):
PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A
Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed.
(1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods
in Molecular Biology, Humana Press; Cell Biology: A Laboratory
Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell
Culture (R. I. Freshney), ed., 1987); Introduction to Cell and
Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;
Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons;
Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain
Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: A Practical Approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal Antibodies: A Practical Approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:
Principles and Practice of Oncology (V. T. DeVita et al., eds., J.
B. Lippincott Company, 1993).
II. DEFINITIONS
[0035] The term "lupus nephritis (LN)" refers to a manifestation of
lupus (e.g., systemic lupus erythematosus, drug-induced lupus,
neonatal lupus, or discoid lupus) in the kidney(s).
[0036] The term "antibody" includes monoclonal antibodies
(including full length antibodies which have an immunoglobulin Fc
region), antibody compositions with polyepitopic specificity,
multispecific antibodies (e.g., bispecific antibodies, diabodies,
and single-chain molecules, as well as antibody fragments (e.g.,
Fab, F(ab').sub.2, and Fv). The term "immunoglobulin" (Ig) is used
interchangeably with "antibody" herein.
[0037] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer units along with an additional polypeptide
called a J chain, and contains 10 antigen binding sites, while IgA
antibodies comprise from 2-5 of the basic 4-chain units which can
polymerize to form polyvalent assemblages in combination with the J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain at its
other end. The V.sub.L is aligned with the V.sub.H and the C.sub.L
is aligned with the first constant domain of the heavy chain
(C.sub.H1). Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The pairing of a V.sub.H and V.sub.L together forms a single
antigen-binding site. For the structure and properties of the
different classes of antibodies, see e.g., Basic and Clinical
Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram
G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page
71 and Chapter 6. The L chain from any vertebrate species can be
assigned to one of two clearly distinct types, called kappa and
lambda, based on the amino acid sequences of their constant
domains. Depending on the amino acid sequence of the constant
domain of their heavy chains (CH), immunoglobulins can be assigned
to different classes or isotypes. There are five classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains
designated .alpha., .delta., .epsilon., .gamma. and .mu.,
respectively. The .gamma. and .alpha. classes are further divided
into subclasses on the basis of relatively minor differences in the
CH sequence and function, e.g., humans express the following
subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
[0038] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domains of the heavy chain and light
chain may be referred to as "VH" and "VL", respectively. These
domains are generally the most variable parts of the antibody
(relative to other antibodies of the same class) and contain the
antigen binding sites.
[0039] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines the
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
entire span of the variable domains. Instead, it is concentrated in
three segments called hypervariable regions (HVRs) both in the
light-chain and the heavy chain variable domains. The more highly
conserved portions of variable domains are called the framework
regions (FR). The variable domains of native heavy and light chains
each comprise four FR regions, largely adopting a beta-sheet
configuration, connected by three HVRs, which form loops
connecting, and in some cases forming part of, the beta-sheet
structure. The HVRs in each chain are held together in close
proximity by the FR regions and, with the HVRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies (see Kabat et al., Sequences of Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in the binding of
antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity.
[0040] The term "monoclonal antibody" as used herein 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 and/or post-translation modifications (e.g.,
isomerizations, amidations) that may be present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigenic site. In contrast to polyclonal antibody
preparations which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. 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 a
variety of techniques, including, for example, the hybridoma method
(e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et
al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2.sup.nd
ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies
(see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et
al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132
(2004), and technologies for producing human or human-like
antibodies in animals that have parts or all of the human
immunoglobulin loci or genes encoding human immunoglobulin
sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735;
WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:
2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);
Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S.
Pat. No. 5,661,016; Marks et al., Bio/Technology 10: 779-783
(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,
Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:
845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and
Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0041] The term "naked antibody" refers to an antibody that is not
conjugated to a cytotoxic moiety or radiolabel.
[0042] The terms "full-length antibody," "intact antibody" or
"whole antibody" are used interchangeably to refer to an antibody
in its substantially intact form, as opposed to an antibody
fragment. Specifically whole antibodies include those with heavy
and light chains including an Fc region. The constant domains may
be native sequence constant domains (e.g., human native sequence
constant domains) or amino acid sequence variants thereof. In some
cases, the intact antibody may have one or more effector
functions.
[0043] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding and/or the variable region
of the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2 and Fv fragments; diabodies; linear antibodies
(see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein
Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules and
multispecific antibodies formed from antibody fragments. Papain
digestion of antibodies produced two identical antigen-binding
fragments, called "Fab" fragments, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. The Fab
fragment consists of an entire L chain along with the variable
region domain of the H chain (V.sub.H), and the first constant
domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having different antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having a few additional
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0044] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, the
region which is also recognized by Fc receptors (FcR) found on
certain types of cells.
[0045] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three HVRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0046] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of the sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0047] "Functional fragments" of the antibodies of the invention
comprise a portion of an intact antibody, generally including the
antigen binding or variable region of the intact antibody or the Fc
region of an antibody which retains or has modified FcR binding
capability. Examples of antibody fragments include linear antibody,
single-chain antibody molecules and multispecific antibodies formed
from antibody fragments.
[0048] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10) residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, thereby resulting in a bivalent
fragment, i.e., a fragment having two antigen-binding sites.
Bispecific diabodies are heterodimers of two "crossover" sFv
fragments in which the V.sub.H and V.sub.L domains of the two
antibodies are present on different polypeptide chains. Diabodies
are described in greater detail in, for example, EP 404,097; WO
93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993).
[0049] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is(are) identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include PRIMATIZED.RTM. antibodies
wherein the antigen-binding region of the antibody is derived from
an antibody produced by, e.g., immunizing macaque monkeys with an
antigen of interest. As used herein, "humanized antibody" is used a
subset of "chimeric antibodies."
[0050] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from an HVR (hereinafter defined) of the recipient are replaced by
residues from an HVR of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
specificity, affinity, and/or capacity. In some instances,
framework ("FR") residues of the human immunoglobulin are replaced
by corresponding non-human residues. Furthermore, humanized
antibodies may comprise residues that are not found in the
recipient antibody or in the donor antibody. These modifications
may be made to further refine antibody performance, such as binding
affinity. In general, a humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the hypervariable
loops correspond to those of a non-human immunoglobulin sequence,
and all or substantially all of the FR regions are those of a human
immunoglobulin sequence, although the FR regions may include one or
more individual FR residue substitutions that improve antibody
performance, such as binding affinity, isomerization,
immunogenicity, etc. The number of these amino acid substitutions
in the FR are typically no more than 6 in the H chain, and in the L
chain, no more than 3. The humanized antibody optionally will also
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see, e.g., Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.
Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos.
6,982,321 and 7,087,409.
[0051] A "human antibody" is an antibody that possesses an
amino-acid sequence corresponding to that of an antibody produced
by a human and/or has been made using any of the techniques for
making human antibodies as disclosed herein. This definition of a
human antibody specifically excludes a humanized antibody
comprising non-human antigen-binding residues. Human antibodies can
be produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562
(2006) regarding human antibodies generated via a human B-cell
hybridoma technology.
[0052] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0053] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0054] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0055] The expression "variable-domain residue-numbering as in
Kabat" or "amino-acid-position numbering as in Kabat," and
variations thereof, refers to the numbering system used for
heavy-chain variable domains or light-chain variable domains of the
compilation of antibodies in Kabat et al., supra. Using this
numbering system, the actual linear amino acid sequence may contain
fewer or additional amino acids corresponding to a shortening of,
or insertion into, a FR or HVR of the variable domain. For example,
a heavy-chain variable domain may include a single amino acid
insert (residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy-chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0056] "Framework" or "FR" residues are those variable-domain
residues other than the HVR residues as herein defined.
[0057] A "human consensus framework" or "acceptor human framework"
is a framework that represents the most commonly occurring amino
acid residues in a selection of human immunoglobulin VL or VH
framework sequences. Generally, the selection of human
immunoglobulin VL or VH sequences is from a subgroup of variable
domain sequences. Generally, the subgroup of sequences is a
subgroup as in Kabat et al., Sequences of Proteins of Immunological
Interest, 5.sup.th Ed. Public Health Service, National Institutes
of Health, Bethesda, Md. (1991). Examples include for the VL, the
subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV
as in Kabat et al., supra. Additionally, for the VH, the subgroup
may be subgroup I, subgroup II, or subgroup III as in Kabat et al.,
supra. Alternatively, a human consensus framework can be derived
from the above in which particular residues, such as when a human
framework residue is selected based on its homology to the donor
framework by aligning the donor framework sequence with a
collection of various human framework sequences. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain pre-existing amino acid sequence
changes. In some embodiments, the number of pre-existing amino acid
changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less,
5 or less, 4 or less, 3 or less, or 2 or less.
[0058] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al., supra. In one
embodiment, the VH subgroup III consensus framework amino acid
sequence comprises at least a portion or all of each of the
following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1)(SEQ ID
NO:35), WVRQAPGKGLEWV (HC-FR2), (SEQ ID NO:36),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:37),
WGQGTLVTVSA (HC-FR4), (SEQ ID NO:38).
[0059] A "VL kappa I consensus framework" comprises the consensus
sequence obtained from the amino acid sequences in variable light
kappa subgroup I of Kabat et al., supra. In one embodiment, the VH
subgroup I consensus framework amino acid sequence comprises at
least a portion or all of each of the following sequences:
DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQ ID NO:39), WYQQKPGKAPKLLIY
(LC-FR2) (SEQ ID NO:40), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(LC-FR3)(SEQ ID NO:41), FGQGTKVEIKR (LC-FR4)(SEQ ID NO:42).
[0060] An "amino-acid modification" at a specified position, e.g.
of the Fc region, refers to the substitution or deletion of the
specified residue, or the insertion of at least one amino acid
residue adjacent the specified residue. Insertion "adjacent" to a
specified residue means insertion within one to two residues
thereof. The insertion may be N-terminal or C-terminal to the
specified residue. The preferred amino acid modification herein is
a substitution.
[0061] An "affinity-matured" antibody is one with one or more
alterations in one or more HVRs thereof that result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody that does not possess those alteration(s). In
one embodiment, an affinity-matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity-matured
antibodies are produced by procedures known in the art. For
example, Marks et al., Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH- and VL-domain shuffling. Random
mutagenesis of HVR and/or framework residues is described by, for
example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813
(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.
Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.
154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896
(1992).
[0062] As use herein, the term "specifically binds to" or is
"specific for" refers to measurable and reproducible interactions
such as binding between a target and an antibody, which is
determinative of the presence of the target in the presence of a
heterogeneous population of molecules including biological
molecules. For example, an antibody that specifically binds to a
target (which can be an epitope) is an antibody that binds this
target with greater affinity, avidity, more readily, and/or with
greater duration than it binds to other targets. In one embodiment,
the extent of binding of an antibody to an unrelated target is less
than about 10% of the binding of the antibody to the target as
measured, e.g., by a radioimmunoassay (RIA). In certain
embodiments, an antibody that specifically binds to a target has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In certain
embodiments, an antibody specifically binds to an epitope on a
protein that is conserved among the protein from different species.
In another embodiment, specific binding can include, but does not
require exclusive binding.
[0063] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native-sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy-chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue. Suitable native-sequence Fc regions for
use in the antibodies of the invention include human IgG1, IgG2
(IgG2A, IgG2B), IgG3 and IgG4.
[0064] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors, Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see M. Daeron, Annu. Rev.
Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,
Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods
4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41
(1995). Other FcRs, including those to be identified in the future,
are encompassed by the term "FcR" herein.
[0065] The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and
Kim et al., J. Immunol. 24: 249 (1994). Methods of measuring
binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol.
Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology
15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8):
6213-6 (2004); WO 2004/92219 (Hinton et al.). Binding to FcRn in
vivo and serum half-life of human FcRn high-affinity binding
polypeptides can be assayed, e.g., in transgenic mice or
transfected human cell lines expressing human FcRn, or in primates
to which the polypeptides having a variant Fc region are
administered. WO 2004/42072 (Presta) describes antibody variants
which improved or diminished binding to FcRs. See also, e.g.,
Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).
[0066] The phrase "substantially reduced," or "substantially
different," as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with a molecule and the other associated with a
reference/comparator molecule) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values). The
difference between said two values is, for example, greater than
about 10%, greater than about 20%, greater than about 30%, greater
than about 40%, and/or greater than about 50% as a function of the
value for the reference/comparator molecule.
[0067] The term "substantially similar" or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two numeric values (for example, one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody), such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is, for
example, less than about 50%, less than about 40%, less than about
30%, less than about 20%, and/or less than about 10% as a function
of the reference/comparator value.
[0068] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0069] A "package insert" refers to instructions customarily
included in commercial packages of medicaments that contain
information about the indications customarily included in
commercial packages of medicaments that contain information about
the indications, usage, dosage, administration, contraindications,
other medicaments to be combined with the packaged product, and/or
warnings concerning the use of such medicaments, etc.
[0070] As used herein, the term "treatment" refers to clinical
intervention designed to alter the natural course of the individual
or cell being treated during the course of clinical pathology.
Desirable effects of treatment include decreasing the rate of
disease progression, ameliorating or palliating the disease state,
and remission or improved prognosis. For example, an individual is
successfully "treated" if one or more symptoms associated with
lupus nephritis are mitigated or eliminated, including, but are not
limited to, elevated serum creatinine, proteinuria, red cell casts,
reduced renal function, nephrotic syndrome, granular casts,
microhematuria, macrohematuria, hypertension, tubular
abnormalities, hyperkalemia, rapidly progressive glomerulonephritis
(RPGN), and acute renal failure (ARF).
[0071] As used herein, "delaying progression" of a disease (e.g.,
lupus nephritis) means to defer, hinder, slow, retard, stabilize,
and/or postpone development of the disease. This delay can be of
varying lengths of time, depending on the history of the disease
and/or individual being treated. As is evident to one skilled in
the art, a sufficient or significant delay can, in effect,
encompass prevention, in that the individual, e.g., an individual
at risk for developing the disease, does not develop the disease.
For example, the progression of SLE in an individual before the
onset of LN symptoms and/or pathology may be delayed such that the
development of LN is postponed or prevented.
[0072] As used herein, "complete renal response (CRR)" refers to a
response to treatment that includes a normalization of serum
creatinine, inactive urinary sediment, and a urinary protein to
creatinine ratio of less than 0.5.
[0073] As used herein, "partial renal response (PRR)" refers to a
response to treatment that is less than a CRR but still includes
mitigation of one or more symptoms including without limitation a
reduction in serum creatinine, reduced urinary sediment, and a
reduction in proteinuria.
[0074] An "effective amount" is at least the minimum concentration
required to effect a measurable improvement or prevention of a
particular disorder. An effective amount herein may vary according
to factors such as the disease state, age, sex, and weight of the
patient, and the ability of the antibody to elicit a desired
response in the individual. An effective amount is also one in
which any toxic or detrimental effects of the treatment are
outweighed by the therapeutically beneficial effects. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the onset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as decreasing one
or more symptoms resulting from the disease, increasing the quality
of life of those suffering from the disease, decreasing the dose of
other medications required to treat the disease, enhancing effect
of another medication such as via targeting, delaying the
progression of the disease, and/or prolonging survival. In the case
of lupus nephritis, an effective amount of the drug may have the
effect in and/or relieving to some extent one or more of the
symptoms associated with the disorder. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount of drug, compound, or pharmaceutical
composition is an amount sufficient to accomplish prophylactic or
therapeutic treatment either directly or indirectly. As is
understood in the clinical context, an effective amount of a drug,
compound, or pharmaceutical composition may or may not be achieved
in conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective amount" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0075] "CD20" as used herein refers to the human B-lymphocyte
antigen CD20 (also known as CD20, B-lymphocyte surface antigen B1,
Leu-16, Bp35, BM5, and LF5; the sequence is characterized by the
SwissProt database entry P11836) is a hydrophobic transmembrane
protein with a molecular weight of approximately 35 kD located on
pre-B and mature B lymphocytes. (Valentine, M. A., et al., J. Biol.
Chem. 264(19) (1989 11282-11287; Tedder, T. F., et al, Proc. Natl.
Acad. Sci. U.S.A. 85 (1988) 208-12; Stamenkovic, I., et al., J.
Exp. Med. 167 (1988) 1975-80; Einfeld, D. A., et al., EMBO J. 7
(1988) 711-7; Tedder, T. F., et al., J. Immunol. 142 (1989)
2560-8). The corresponding human gene is Membrane-spanning
4-domains, subfamily A, member 1, also known as MS4A1. This gene
encodes a member of the membrane-spanning 4A gene family. Members
of this nascent protein family are characterized by common
structural features and similar intron/exon splice boundaries and
display unique expression patterns among hematopoietic cells and
nonlymphoid tissues. This gene encodes the B-lymphocyte surface
molecule which plays a role in the development and differentiation
of B-cells into plasma cells. This family member is localized to
11q12, among a cluster of family members. Alternative splicing of
this gene results in two transcript variants which encode the same
protein.
[0076] The terms "CD20" and "CD20 antigen" are used interchangeably
herein, and include any variants, isoforms and species homologs of
human CD20 which are naturally expressed by cells or are expressed
on cells transfected with the CD20 gene. Binding of an antibody of
the invention to the CD20 antigen mediate the killing of cells
expressing CD20 (e.g., a tumor cell) by inactivating CD20. The
killing of the cells expressing CD20 may occur by one or more of
the following mechanisms: Cell death/apoptosis induction, ADCC and
CDC.
[0077] Synonyms of CD20, as recognized in the art, include
B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16,
Bp35, BM5, and LF5.
[0078] The term "anti-CD20 antibody" according to the invention is
an antibody that binds specifically to CD20 antigen. Depending on
binding properties and biological activities of anti-CD20
antibodies to the CD20 antigen, two types of anti-CD20 antibodies
(type I and type II anti-CD20 antibodies) can be distinguished
according to Cragg, M. S., et al., Blood 103 (2004) 2738-2743; and
Cragg, M. S., et al., Blood 101 (2003) 1045-1052, see Table 1
below.
TABLE-US-00002 TABLE 1 Properties of type I and type II anti-CD20
antibodies Type I anti-CD20 antibodies type II anti-CD20 antibodies
type I CD20 epitope type II CD20 epitope Localize CD20 to lipid
rafts Do not localize CD20 to lipid rafts Increased CDC (if IgG1
isotype) Decreased CDC (if IgG1 isotype) ADCC activity (if IgG1
isotype) ADCC activity (if IgG1 isotype) Full binding capacity
Reduced binding capacity Homotypic aggregation Stronger homotypic
aggregation Apoptosis induction upon Strong cell death induction
without cross-linking cross-linking
[0079] Examples of type II anti-CD20 antibodies include e.g.
humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody
as disclosed in WO 2005/044859), 11B8 IgG1 (as disclosed in WO
2004/035607), and AT80 IgG1. Typically type II anti-CD20 antibodies
of the IgG1 isotype show characteristic CDC properties. Type II
anti-CD20 antibodies have a decreased CDC (if IgG1 isotype)
compared to type I antibodies of the IgG1 isotype.
[0080] Examples of type I anti-CD20 antibodies include e.g.
rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in
WO 2005/103081), 2F2 IgG1 (as disclosed and WO 2004/035607 and WO
2005/103081) and 2H7 IgG1 (as disclosed in WO 2004/056312).
[0081] The afucosylated anti-CD20 antibodies according to the
invention are preferably type II anti-CD20 antibodies, more
preferably afucosylated humanized B-Lyl antibodies as described in
WO 2005/044859 and WO 2007/031875.
[0082] The "rituximab" antibody (reference antibody; example of a
type I anti-CD20 antibody) is a genetically engineered chimeric
human gamma 1 murine constant domain containing monoclonal antibody
directed against the human CD20 antigen. However this antibody is
not glycoengineered and not afocusylates and thus has an amount of
fucose of at least 85%. This chimeric antibody contains human gamma
1 constant domains and is identified by the name "C2B8" in U.S.
Pat. No. 5,736,137 (Andersen, et. al.) issued on Apr. 17, 1998,
assigned to IDEC Pharmaceuticals Corporation. Rituximab is approved
for the treatment of patients with relapsed or refracting low-grade
or follicular, CD20 positive, B cell non-Hodgkin's lymphoma. In
vitro mechanism of action studies have shown that rituximab
exhibits human complement-dependent cytotoxicity (CDC) (Reff, M.
E., et. al, Blood 83(2) (1994) 435-445). Additionally, it exhibits
activity in assays that measure antibody-dependent cellular
cytotoxicity (ADCC).
[0083] The term "GA101 antibody" as used herein refers to any one
of the following antibodies that bind human CD20: (1) an antibody
comprising an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2,
an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, an
HVR-L1 comprising the amino acid sequence of SEQ ID NO:4, an HVR-L2
comprising the amino acid sequence of SEQ ID NO:5, and an HVR-L3
comprising the amino acid sequence of SEQ ID NO:6; (2) an antibody
comprising a VH domain comprising the amino acid sequence of SEQ ID
NO:7 and a VL domain comprising the amino acid sequence of SEQ ID
NO:8, (3) an antibody comprising an amino acid sequence of SEQ ID
NO:9 and an amino acid sequence of SEQ ID NO: 10; (4) an antibody
known as obinutuzumab, or (5) an antibody that comprises an amino
acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence
identity with amino acid sequence of SEQ ID NO:9 and that comprises
an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99%
sequence identity with an amino acid sequence of SEQ ID NO:10. In
one embodiment, the GA101 antibody is an IgG1 isotype antibody. In
some embodiments, the anti-CD20 antibody is a humanized B-Ly1
antibody.
[0084] The term "humanized B-Ly1 antibody" refers to humanized
B-Ly1 antibody as disclosed in WO 2005/044859 and WO 2007/031875,
which were obtained from the murine monoclonal anti-CD20 antibody
B-Ly1 (variable region of the murine heavy chain (VH): SEQ ID NO:
11; variable region of the murine light chain (VL): SEQ ID NO:
12--see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987)
131-139) by chimerization with a human constant domain from IgG1
and following humanization (see WO 2005/044859 and WO 2007/031875).
These "humanized B-Ly1 antibodies" are disclosed in detail in WO
2005/044859 and WO 2007/031875.
[0085] Variable region of the murine monoclonal anti-CD20 antibody
B-Ly1 heavy chain (VH) (SEQ ID NO: 11)
TABLE-US-00003 Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys 1 5
10 Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr 15 20 Ser Trp
Met Asn Trp Val Lys Leu Arg Pro Gly Gln 25 30 35 Gly Leu Glu Trp
Ile Gly Arg Ile Phe Pro Gly Asp 40 45 Gly Asp Thr Asp Tyr Asn Gly
Lys Phe Lys Gly Lys 50 55 60 Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn Thr Ala 65 70 Tyr Met Gln Leu Thr Ser Leu Thr Ser Val Asp Ser
75 80 Ala Val Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly 85 90 95 Tyr
Trp Leu Val Tyr Trp Gly Gln Gly Thr Leu Val 100 105 Thr Val Ser Ala
110
[0086] Variable region of the murine monoclonal anti-CD20 antibody
B-Ly1 light chain (VL) (SEQ ID NO: 12)
TABLE-US-00004 Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser 1 5
10 Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly 15 20 Ile Thr
Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly 25 30 35 Gln Ser Pro Gln
Leu Leu Ile Tyr Gln Met Ser Asn 40 45 Leu Val Ser Gly Val Pro Asp
Arg Phe Ser Ser Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Arg
Ile Ser Arg 65 70 Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala
75 80 Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly 85 90 95 Thr
Lys Leu Glu Ile Lys Arg 100
[0087] In one embodiment, the "humanized B-Ly1 antibody" has
variable region of the heavy chain (VH) selected from group of SEQ
ID NO:7, 8, and 13 to 33 (corresponding to, inter alia, B-HH2 to
B-HH9 and B-HL8 to B-HL17 of WO 2005/044859 and WO 2007/031875). In
one specific embodiment, such variable domain is selected from the
group consisting of SEQ ID NOS:14, 15, 7, 19, 25, 27, and 29
(corresponding to B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 and
B-HL13 of WO 2005/044859 and WO 2007/031875). In one specific
embodiment, the "humanized B-Ly1 antibody" has variable region of
the light chain (VL) of SEQ ID NO:8 (corresponding to B-KV1 of WO
2005/044859 and WO 2007/031875). In one specific embodiment, the
"humanized B-Ly1 antibody" has a variable region of the heavy chain
(VH) of SEQ ID NO:7 (corresponding to B-HH6 of WO 2005/044859 and
WO 2007/031875) and a variable region of the light chain (VL) of
SEQ ID NO:8 (corresponding to B-KV1 of WO 2005/044859 and WO
2007/031875). Furthermore in one embodiment, the humanized B-Ly1
antibody is an IgG1 antibody. According to the invention such
afocusylated humanized B-Ly1 antibodies are glycoengineered (GE) in
the Fc region according to the procedures described in WO
2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al.,
Nature Biotechnol. 17 (1999) 176-180 and WO 99/154342. In one
embodiment, the afucosylated glyco-engineered humanized B-Ly1 is
B-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody is
obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No.
4, 2012, p. 453). As used herein, obinutuzumab is synonymous for
GA101 or RO5072759. This replaces all previous versions (e.g. Vol.
25, No. 1, 2011, p. 75-'76), and is formerly known as afutuzumab
(recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009, p.
176; Vol. 22, No. 2, 2008, p. 124). In some embodiments, the
humanized B-Ly1 antibody is an antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO:9 and a light chain
comprising the amino acid sequence of SEQ ID NO:10 or an
antigen-binding fragment thereof. In some embodiments, the
humanized B-Ly1 antibody comprises a heavy chain variable region
comprising the three heavy chain CDRs of SEQ ID NO:9 and a light
chain variable region comprising the three light chain CDRs of SEQ
ID NO:10.
TABLE-US-00005 Heavy chain (SEQ ID NO: 9) QVQLVQSGAE VKKPGSSVKV
SCKASGYAFS YSWINWVRQA PGQGLEWMGR 50 IFPGDGDTDY NGKFKGRVTI
TADKSTSTAY MELSSLRSED TAVYYCARNV 100 FDGYWLVYWG QGTLVTVSSA
STKGPSVFPL APSSKSTSGG TAALGCLVKD 150 YFPEPVTVSW NSGALTSGVH
TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200 ICNVNHKPSN TKVDKKVEPK
SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250 DTLMISRTPE VTCVVVDVSH
EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE
YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350 YTLPPSRDEL TKNQVSLTCL
VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400 DSDGSFFLYS KLTVDKSRWQ
QGNVFSCSVM HEALHNHYTQ KSLSLSPG 449 Light chain (SEQ ID NO: 10)
DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ 50
LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP 100
YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK 150
VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 200
VTHQGLSSPV TKSFNRGEC 219
[0088] In some embodiments, the humanized B-Ly1 antibody is an
afucosylated glyco-engineered humanized B-Ly1. Such glycoengineered
humanized B-Ly1 antibodies have an altered pattern of glycosylation
in the Fc region, preferably having a reduced level of fucose
residues. Preferably the amount of fucose is 60% or less of the
total amount of oligosaccharides at Asn297 (in one embodiment the
amount of fucose is between 40% and 60%, in another embodiment the
amount of fucose is 50% or less, and in still another embodiment
the amount of fucose is 30% or less). Furthermore the
oligosaccharides of the Fc region are preferably bisected. These
glycoengineered humanized B-Ly1 antibodies have an increased
ADCC.
[0089] The "ratio of the binding capacities to CD20 on Raji cells
(ATCC-No. CCL-86) of an anti-CD20 antibodies compared to rituximab"
is determined by direct immunofluorescence measurement (the mean
fluorescence intensities (MFI) is measured) using said anti-CD20
antibody conjugated with Cy5 and rituximab conjugated with Cy5 in a
FACSArray (Becton Dickinson) with Raji cells (ATCC-No. CCL-86), as
described in Example No. 2, and calculated as follows:
Ratio of the binding capacities to CD 20 on Raji cells ( ATCC - No
. CCL - 86 ) = MFI ( Cy 5 - anti - CD 20 antibody ) MFI ( Cy 5 -
rituximab ) .times. Cy 5 - labeling ratio ( Cy 5 - rituximab ) Cy 5
- labeling ratio ( Cy 5 - anti - CD 20 antibody ) ##EQU00001##
[0090] MFI is the mean fluorescent intensity. The "Cy5-labeling
ratio" as used herein means the number of Cy5-label molecules per
molecule antibody.
[0091] Typically said type II anti-CD20 antibody has a ratio of the
binding capacities to CD20 on Raji cells (ATCC-No. CCL-86) of said
second anti-CD20 antibody compared to rituximab of 0.3 to 0.6, and
in one embodiment, 0.35 to 0.55, and in yet another embodiment, 0.4
to 0.5.
[0092] In one embodiment said type II anti-CD20 antibody, e.g., a
GA101 antibody, has increased antibody dependent cellular
cytotoxicity (ADCC).
[0093] By "antibody having increased antibody dependent cellular
cytotoxicity (ADCC)", it is meant an antibody, as that term is
defined herein, having increased ADCC as determined by any suitable
method known to those of ordinary skill in the art. One accepted in
vitro ADCC assay is as follows: [0094] 1) the assay uses target
cells that are known to express the target antigen recognized by
the antigen-binding region of the antibody; [0095] 2) the assay
uses human peripheral blood mononuclear cells (PBMCs), isolated
from blood of a randomly chosen healthy donor, as effector cells;
[0096] 3) the assay is carried out according to following protocol:
[0097] i) the PBMCs are isolated using standard density
centrifugation procedures and are suspended at 5.times.10.sup.6
cells/ml in RPMI cell culture medium; [0098] ii) the target cells
are grown by standard tissue culture methods, harvested from the
exponential growth phase with a viability higher than 90%, washed
in RPMI cell culture medium, labeled with 100 micro-Curies of
.sup.51Cr, washed twice with cell culture medium, and resuspended
in cell culture medium at a density of 10.sup.5 cells/ml; [0099]
iii) 100 microliters of the final target cell suspension above are
transferred to each well of a 96-well microtiter plate; [0100] iv)
the antibody is serially-diluted from 4000 ng/ml to 0.04 ng/ml in
cell culture medium and 50 microliters of the resulting antibody
solutions are added to the target cells in the 96-well microtiter
plate, testing in triplicate various antibody concentrations
covering the whole concentration range above; [0101] v) for the
maximum release (MR) controls, 3 additional wells in the plate
containing the labeled target cells, receive 50 microliters of a 2%
(VN) aqueous solution of non-ionic detergent (Nonidet, Sigma, St.
Louis), instead of the antibody solution (point iv above); [0102]
vi) for the spontaneous release (SR) controls, 3 additional wells
in the plate containing the labeled target cells, receive 50
microliters of RPMI cell culture medium instead of the antibody
solution (point iv above); [0103] vii) the 96-well microtiter plate
is then centrifuged at 50.times.g for 1 minute and incubated for 1
hour at 4.degree. C.; [0104] viii) 50 microliters of the PBMC
suspension (point i above) are added to each well to yield an
effector:target cell ratio of 25:1 and the plates are placed in an
incubator under 5% CO2 atmosphere at 37.degree. C. for 4 hours;
[0105] ix) the cell-free supernatant from each well is harvested
and the experimentally released radioactivity (ER) is quantified
using a gamma counter; [0106] x) the percentage of specific lysis
is calculated for each antibody concentration according to the
formula (ER-MR)/(MR-SR).times.100, where ER is the average
radioactivity quantified (see point ix above) for that antibody
concentration, MR is the average radioactivity quantified (see
point ix above) for the MR controls (see point V above), and SR is
the average radioactivity quantified (see point ix above) for the
SR controls (see point vi above); [0107] 4) "increased ADCC" is
defined as either an increase in the maximum percentage of specific
lysis observed within the antibody concentration range tested
above, and/or a reduction in the concentration of antibody required
to achieve one half of the maximum percentage of specific lysis
observed within the antibody concentration range tested above. In
one embodiment, the increase in ADCC is relative to the ADCC,
measured with the above assay, mediated by the same antibody,
produced by the same type of host cells, using the same standard
production, purification, formulation and storage methods, which
are known to those skilled in the art, except that the comparator
antibody (lacking increased ADCC) has not been produced by host
cells engineered to overexpress GnTIII and/or engineered to have
reduced expression from the fucosyltransferase 8 (FUT8) gene (e.g.,
including, engineered for FUT8 knock out).
[0108] Said "increased ADCC" can be obtained by, for example,
mutating and/or glycoengineering of said antibodies. In one
embodiment, the antibody is glycoengineered to have a biantennary
oligosaccharide attached to the Fc region of the antibody that is
bisected by GlcNAc, e.g., in WO 2003/011878 (Jean-Mairet et al.);
U.S. Pat. No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et
al.), Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180). In
another embodiment, the antibody is glycoengineered to lack fucose
on the carbohydrate attached to the Fc region by expressing the
antibody in a host cell that is deficient in protein fucosylation
(e.g., Lec13 CHO cells or cells having an
alpha-1,6-fucosyltransferase gene (FUT8) deleted or the FUT gene
expression knocked down (see, e.g., Yamane-Ohnuki et al. Biotech.
Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,
94(4):680-688 (2006); and WO2003/085107). In yet another
embodiment, the antibody sequence has been engineered in its Fc
region to enhance ADCC (e.g., in one embodiment, such engineered
antibody variant comprises an Fc region with one or more amino acid
substitutions at positions 298, 333, and/or 334 of the Fc region
(EU numbering of residues)).
[0109] The term "complement-dependent cytotoxicity (CDC)" refers to
lysis of human tumor target cells by the antibody according to the
invention in the presence of complement. CDC can be measured by the
treatment of a preparation of CD20 expressing cells with an
anti-CD20 antibody according to the invention in the presence of
complement. CDC is found if the antibody induces at a concentration
of 100 nM the lysis (cell death) of 20% or more of the tumor cells
after 4 hours. In one embodiment, the assay is performed with
.sup.51Cr or Eu labeled tumor cells and measurement of released
.sup.51Cr or Eu. Controls include the incubation of the tumor
target cells with complement but without the antibody.
[0110] The term "expression of the CD20" antigen is intended to
indicate a significant level of expression of the CD20 antigen in a
cell, e.g., a T- or B-Cell. In one embodiment, patients to be
treated according to the methods of this invention express
significant levels of CD20 on a B-cell. CD20 expression on a B-cell
can be determined by standard assays known in the art. e.g., CD20
antigen expression is measured using immunohistochemical (IHC)
detection, FACS or via PCR-based detection of the corresponding
mRNA.
[0111] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a molecule" optionally includes a combination of two
or more such molecules, and the like.
[0112] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se.
[0113] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
III. METHODS
[0114] In one aspect, provided herein are methods for treating or
delaying progression of lupus nephritis in an individual that has
lupus by administering an effective amount of a type II anti-CD20
antibody. In some embodiments, the individual has or is at risk for
developing lupus nephritis. In some embodiments, the lupus
nephritis is class III or class IV lupus nephritis. In some
embodiments, the methods include administering to the individual at
least a first antibody exposure to a type II anti-CD20 antibody and
a second antibody exposure to the type II anti-CD20 antibody, the
second antibody exposure not being provided until from about 18
weeks to about 26 weeks after the first antibody exposure; wherein
the first antibody exposure comprises one or two doses of the type
II anti-CD20 antibody, the first antibody exposure comprising a
total exposure of between about 1800 mg and about 2200 mg of the
type II anti-CD20 antibody; and wherein the second antibody
exposure comprises one or two doses of the type II anti-CD20
antibody, the second antibody exposure comprising a total exposure
of between about 1800 mg and about 2200 mg of the type II anti-CD20
antibody. As described below, in some embodiments, the antibody
comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:1,
HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ ID NO:3,
and a light chain comprising HVR-L1 sequence of SEQ ID NO:4, HVR-L2
sequence of SEQ ID NO:5, and HVR-L3 sequence of SEQ ID NO:6. In
some embodiments, the antibody comprises a VH domain comprising the
amino acid sequence of SEQ ID NO:7 and a VL domain comprising the
amino acid sequence of SEQ ID NO:8. In some embodiments, the
antibody comprises an amino acid sequence of SEQ ID NO:9 and an
amino acid sequence of SEQ ID NO:10. In some embodiments, the
antibody comprises an antibody that comprises an amino acid
sequence that has at least 95%, 96%, 97%, 98% or 99% sequence
identity with amino acid sequence of SEQ ID NO:9 and that comprises
an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99%
sequence identity with an amino acid sequence of SEQ ID NO:10.
Anti-CD20 Antibodies
[0115] Certain aspects of the present disclosure relate to
anti-CD20 antibodies, e.g., for use in methods for treating or
preventing progression of lupus nephritis. In some embodiments, the
anti-CD20 antibody is a type II antibody. In some embodiments, the
anti-CD20 antibody is human or humanized. In some embodiments, the
anti-CD20 antibody is afucosylated. In some embodiments, the
anti-CD20 antibody is a GA101 antibody.
[0116] Examples of type II anti-CD20 antibodies include e.g.
humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody
as disclosed in WO 2005/044859), 11B8 IgG1 (as disclosed in WO
2004/035607), and AT80 IgG1. Typically type II anti-CD20 antibodies
of the IgG1 isotype show characteristic CDC properties. Type II
anti-CD20 antibodies have a decreased CDC (if IgG1 isotype)
compared to type I antibodies of the IgG1 isotype.
[0117] Examples of type I anti-CD20 antibodies include e.g.
rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in
WO 2005/103081), 2F2 IgG1 (as disclosed and WO 2004/035607 and WO
2005/103081) and 2H7 IgG1 (as disclosed in WO 2004/056312).
[0118] In some embodiments, the anti-CD20 antibody is a GA101
antibody described herein. In some embodiments, the anti-CD20 is
any one of the following antibodies that bind human CD20: (1) an
antibody comprising an HVR-H1 comprising the amino acid sequence of
GYAFSY (SEQ ID NO:1), an HVR-H2 comprising the amino acid sequence
of FPGDGDTD (SEQ ID NO:2), an HVR-H3 comprising the amino acid
sequence of NVFDGYWLVY (SEQ ID NO:3), an HVR-L1 comprising the
amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO:4), an HVR-L2
comprising the amino acid sequence of QMSNLVS (SEQ ID NO:5), and an
HVR-L3 comprising the amino acid sequence of AQNLELPYT (SEQ ID
NO:6); (2) an antibody comprising a VH domain comprising the amino
acid sequence of SEQ ID NO:7 and a VL domain comprising the amino
acid sequence of SEQ ID NO:8, (3) an antibody comprising an amino
acid sequence of SEQ ID NO:9 and an amino acid sequence of SEQ ID
NO:10; (4) an antibody known as obinutuzumab, or (5) an antibody
that comprises an amino acid sequence that has at least 95%, 96%,
97%, 98% or 99% sequence identity with amino acid sequence of SEQ
ID NO:9 and that comprises an amino acid sequence that has at least
95%, 96%, 97%, 98% or 99% sequence identity with an amino acid
sequence of SEQ ID NO:10. In one embodiment, the GA101 antibody is
an IgG1 isotype antibody. In some embodiments, the anti-CD20
antibody comprises an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and
HVR-L3 of any of the antibodies described herein, e.g., 3 HVRs from
SEQ ID NO:7 and 3 HVRs from SEQ ID NO:8, 3 HVRs from SEQ ID NO:9
and 3 HVRs from SEQ ID NO:10, or any HVRs of the amino acid
sequences provided in Table 2.
[0119] In some embodiments, the anti-CD20 antibody comprises a
heavy chain variable region (VH) comprising the amino acid sequence
of SEQ ID NO:7, and a light chain variable region (VL) comprising
the amino acid sequence of SEQ ID NO:8.
TABLE-US-00006 (SEQ ID NO: 7)
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGR
IFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNV
FDGYWLVYWGQGTLVTVSS (SEQ ID NO: 8)
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQ
LLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELP
YTFGGGTKVEIKRTV.
[0120] In some embodiments, the anti-CD20 antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:9, and
a light chain comprising the amino acid sequence of SEQ ID
NO:10.
TABLE-US-00007 (SEQ ID NO: 9) QVQLVQSGAEVKKPGSSVKVSCKAS
SWINWVRQAPGQGLEW MGRI YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAV YYCAR
WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10) DIVMTQTPLSLPVTPGEPASISC
WYLQKPGQ SPQLLIY GVPDRFSGSGSGTDFILKISRVEAEDVGVYYC
EGGGIKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS
KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0121] In some embodiments, the anti-CD20 antibody is a humanized
B-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody
comprises a heavy chain variable region comprising the three heavy
chain CDRs of SEQ ID NO:9 and a light chain variable region
comprising the three light chain CDRs of SEQ ID NO:10. In some
embodiments, the humanized B-Ly1 antibody comprises a heavy chain
comprising the sequence of SEQ ID NO:9 and a light chain comprising
the sequence of SEQ ID NO:10.
[0122] In some embodiments, the anti-CD20 antibody comprises an
amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to a polypeptide sequence listed in Table 2
below.
TABLE-US-00008 TABLE 2 Polypeptide sequences. SEQ ID CONSTRUCT
POLYPEPTIDE SEQUENCE NO B-HH1 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSYSWM 13
SWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRV
TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS B-HH2
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM 14
NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR
VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS B-HH3
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM 15
NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR
VTITADKSTSTAYMELSSLRSEDTAVYLCARNVFDG YWLVYWGQGTLVTVSS B-HH4
QVQLVQSGAEVKKPGASVKVSCKVSGYAFSYSWM 16
NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR
VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS B-HH5
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWM 17
SWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV
TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS B-HH6
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIN 7
WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HH7
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWIS 18
WVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HH8
QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWM 19
NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR
VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS B-HH9
QVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWM 20
NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGR
VTITADKSTSTAYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS B-HL1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWM 21
HWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGR
VTMTRDTSTSTVYMELSSLRSEDTAVYYCARNVFD GYWLVYWGQGTLVTVSS B-HL2
EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMH 22
WVQQAPGKGLEWMGRIFPGDGDTDYAEKFQGRVT
ITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGY WLVYWGQGTLVTVSS B-HL3
EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMN 23
WVQQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGY WLVYWGQGTLVTVSS B-HL4
QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYSWM 24
SWVRQAPGQGLEWMGRIFPGDGDTDYAQKFQGRV
TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDG YWLVYWGQGTLVTVSS B-HL8
EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN 25
WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL10
EVQLVESGGGLVKPGGSLRLSCAASGFAFSYSWMN 26
WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL11
QVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN 27
WVRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL12
EVQLVESGAGLVKPGGSLRLSCAASGFTFSYSWMN 28
WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL13
EVQLVESGGGVVKPGGSLRLSCAASGFTFSYSWMN 29
WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL14
EVQLVESGGGLKKPGGSLRLSCAASGFTFSYSWMN 30
WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL15
EVQLVESGGGLVKPGSSLRLSCAASGFTFSYSWMN 31
WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL16
EVQLVESGGGLVKPGGSLRVSCAASGFTFSYSWMN 32
WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS B-HL17
EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMN 33
WVRQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVT
ITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGY WLVYWGQGTLVTVSS VH Signal
MDWTWRILFLVAAATGAHS 34 Sequence B-KV1
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYL 8
YWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSG
TDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTK VEIKRTV VL Signal
MDMRVPAQLLGLLLLWFPGARC 43 Sequence
[0123] In some embodiments, the anti-CD20 antibody (e.g., a type II
anti-CD20 antibody) is an afucosylated glyco-engineered antibody.
Such glycoengineered antibodies have an altered pattern of
glycosylation in the Fc region, preferably having a reduced level
of fucose residues. Preferably the amount of fucose is 60% or less
of the total amount of oligosaccharides at Asn297 (in one
embodiment the amount of fucose is between 40% and 60%, in another
embodiment the amount of fucose is 50% or less, and in still
another embodiment the amount of fucose is 30% or less).
Furthermore the oligosaccharides of the Fc region are preferably
bisected. These glycoengineered humanized anti-CD20 (e.g., B-Ly1)
antibodies have an increased ADCC.
[0124] The oligosaccharide component can significantly affect
properties relevant to the efficacy of a therapeutic glycoprotein,
including physical stability, resistance to protease attack,
interactions with the immune system, pharmacokinetics, and specific
biological activity. Such properties may depend not only on the
presence or absence, but also on the specific structures, of
oligosaccharides. Some generalizations between oligosaccharide
structure and glycoprotein function can be made. For example,
certain oligosaccharide structures mediate rapid clearance of the
glycoprotein from the bloodstream through interactions with
specific carbohydrate binding proteins, while others can be bound
by antibodies and trigger undesired immune reactions. (Jenkins, N.,
et al., Nature Biotechnol. 14 (1996) 975-81).
[0125] Mammalian cells are the preferred hosts for production of
therapeutic glycoproteins, due to their capability to glycosylate
proteins in the most compatible form for human application.
(Cumming, D. A., et al., Glycobiology 1 (1991) 115-30; Jenkins, N.,
et al., Nature Biotechnol. 14 (1996) 975-81). Bacteria very rarely
glycosylate proteins, and like other types of common hosts, such as
yeasts, filamentous fungi, insect and plant cells, yield
glycosylation patterns associated with rapid clearance from the
blood stream, undesirable immune interactions, and in some specific
cases, reduced biological activity. Among mammalian cells, Chinese
hamster ovary (CHO) cells have been most commonly used during the
last two decades. In addition to giving suitable glycosylation
patterns, these cells allow consistent generation of genetically
stable, highly productive clonal cell lines. They can be cultured
to high densities in simple bioreactors using serum free media, and
permit the development of safe and reproducible bioprocesses. Other
commonly used animal cells include baby hamster kidney (BHK) cells,
NSO- and SP2/0-mouse myeloma cells. More recently, production from
transgenic animals has also been tested. (Jenkins, N., et al.,
Nature Biotechnol. 14 (1996) 975-981).
[0126] All antibodies contain carbohydrate structures at conserved
positions in the heavy chain constant regions, with each isotype
possessing a distinct array of N-linked carbohydrate structures,
which variably affect protein assembly, secretion or functional
activity. (Wright, A., and Morrison, S. L., Trends Biotech. 15
(1997) 26-32). The structure of the attached N-linked carbohydrate
varies considerably, depending on the degree of processing, and can
include high-mannose, multiply-branched as well as biantennary
complex oligosaccharides. (Wright, A., and Morrison, S. L., Trends
Biotech. 15 (1997) 26-32). Typically, there is heterogeneous
processing of the core oligosaccharide structures attached at a
particular glycosylation site such that even monoclonal antibodies
exist as multiple glycoforms. Likewise, it has been shown that
major differences in antibody glycosylation occur between cell
lines, and even minor differences are seen for a given cell line
grown under different culture conditions. (Lifely, M. R., et al.,
Glycobiology 5(8) (1995) 813-22).
[0127] One way to obtain large increases in potency, while
maintaining a simple production process and potentially avoiding
significant, undesirable side effects, is to enhance the natural,
cell-mediated effector functions of monoclonal antibodies by
engineering their oligosaccharide component as described in Umana,
P., et al., Nature Biotechnol. 17 (1999) 176-180 and U.S. Pat. No.
6,602,684. IgG1 type antibodies, the most commonly used antibodies
in cancer immunotherapy, are glycoproteins that have a conserved
N-linked glycosylation site at Asn297 in each CH2 domain. The two
complex biantennary oligosaccharides attached to Asn297 are buried
between the CH2 domains, forming extensive contacts with the
polypeptide backbone, and their presence is essential for the
antibody to mediate effector functions such as antibody dependent
cellular cytotoxicity (ADCC) (Lifely, M. R., et al., Glycobiology 5
(1995) 813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998)
59-76; Wright, A., and Morrison, S. L., Trends Biotechnol. 15
(1997) 26-32).
[0128] It was previously shown that overexpression in Chinese
hamster ovary (CHO) cells of
.beta.(1,4)-N-acetylglucosaminyltransferase I11 ("GnTII17y), a
glycosyltransferase catalyzing the formation of bisected
oligosaccharides, significantly increases the in vitro ADCC
activity of an antineuroblastoma chimeric monoclonal antibody
(chCE7) produced by the engineered CHO cells. (See Umana, P., et
al., Nature Biotechnol. 17 (1999) 176-180; and WO 99/154342, the
entire contents of which are hereby incorporated by reference). The
antibody chCE7 belongs to a large class of unconjugated monoclonal
antibodies which have high tumor affinity and specificity, but have
too little potency to be clinically useful when produced in
standard industrial cell lines lacking the GnTIII enzyme (Umana,
P., et al., Nature Biotechnol. 17 (1999) 176-180). That study was
the first to show that large increases of ADCC activity could be
obtained by engineering the antibody producing cells to express
GnTIII, which also led to an increase in the proportion of constant
region (Fc)-associated, bisected oligosaccharides, including
bisected, non-fucosylated oligosaccharides, above the levels found
in naturally-occurring antibodies.
[0129] In some embodiments, the anti-CD20 antibody (e.g., a type II
anti-CD20 antibody) comprises a human Fc region (e.g., a human IgG1
Fc region). In some embodiments, the Fc region comprises an
N-linked oligosaccharide that has been modified. In some
embodiments, the N-linked oligosaccharides of the Fc region have
reduced fucose residues as compared to an antibody with
non-modified N-linked oligosaccharides. In some embodiments, the
bisected oligosaccharide is a bisected complex oligosaccharide. In
some embodiments, the N-linked oligosaccharides have been modified
to have increased bisected, nonfucosylated oligosaccharides. In
some embodiments, the bisected, nonfucosylated oligosaccharides are
the hybrid type. In some embodiments, the bisected, nonfucosylated
oligosaccharides are the complex type. For more detailed
description, see, e.g., WO 2003/011878 (Jean-Mairet et al.); U.S.
Pat. No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.);
and U.S. Pat. No. 8,883,980 (Umana et al.).
[0130] In some embodiments, the anti-CD20 antibody (e.g., a type II
anti-CD20 antibody) is a multispecific antibody or a bispecific
antibody.
Antibody Preparation
[0131] An antibody according to any of the above embodiments (e.g.,
a type II anti-CD20 antibody of the present disclosure) may
incorporate any of the features, singly or in combination, as
described in Sections 1-7 below:
[0132] 1. Antibody Affinity
[0133] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8 M to
10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13 M).
[0134] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA). In one embodiment, an RIA is performed with
the Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of Fabs for antigen is measured
by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta et al.,
Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0135] According to another embodiment, Kd is measured using a
BIACORE.RTM. surface plasmon resonance assay. For example, an assay
using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
antigen CM5 chips at .about.10 response units (RU). In one
embodiment, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1
s-1 by the surface plasmon resonance assay above, then the on-rate
can be determined by using a fluorescent quenching technique that
measures the increase or decrease in fluorescence emission
intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at
25.degree. C. of a 20 nM anti-antigen antibody (Fab form) in PBS,
pH 7.2, in the presence of increasing concentrations of antigen as
measured in a spectrometer, such as a stop-flow equipped
spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO.TM.
spectrophotometer (ThermoSpectronic) with a stirred cuvette.
[0136] 2. Antibody Fragments
[0137] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0138] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0139] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0140] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0141] 3. Chimeric and Humanized Antibodies
[0142] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0143] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0144] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing specificity determining region (SDR)
grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing
"resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR shuffling"); and Osbourn et al., Methods 36:61-68
(2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)
(describing the "guided selection" approach to FR shuffling).
[0145] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0146] 4. Human Antibodies
[0147] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0148] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HuMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0149] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0150] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0151] 5. Library-Derived Antibodies
[0152] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0153] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0154] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0155] 6. Multispecific Antibodies
[0156] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for CD20 and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of CD20. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express CD20. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0157] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0158] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0159] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to CD20
as well as another, different antigen (see, US 2008/0069820, for
example).
[0160] 7. Antibody Variants
[0161] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0162] a) Substitution, Insertion, and Deletion Variants
[0163] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table A under the heading of "preferred
substitutions." More substantial changes are provided in Table A
under the heading of "exemplary substitutions," and as further
described below in reference to amino acid side chain classes.
Amino acid substitutions may be introduced into an antibody of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00009 TABLE A Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0164] Amino acids may be grouped according to common side-chain
properties:
[0165] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0166] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0167] (3) acidic: Asp, Glu;
[0168] (4) basic: His, Lys, Arg;
[0169] (5) residues that influence chain orientation: Gly, Pro;
[0170] (6) aromatic: Trp, Tyr, Phe.
[0171] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0172] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0173] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues
that contact antigen, with the resulting variant VH or VL being
tested for binding affinity. Affinity maturation by constructing
and reselecting from secondary libraries has been described, e.g.,
in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0174] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0175] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0176] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0177] b) Glycosylation Variants
[0178] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0179] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0180] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e.g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0181] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0182] c) Fc Region Variants
[0183] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0184] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc(RIII only, whereas monocytes express Fc(RI,
Fc(RII and Fc(RIII FcR expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337
(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0185] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0186] In certain embodiments, the Fc variants described herein
further comprise one or more amino acid modifications for
attenuating effector function (such as CDC and/or ADCC). In
exemplary embodiments, the modification to attenuate effector
function is a modification that does not alter the glycosylation
pattern of the Fc region. In certain embodiments, the modification
to attenuate effector function reduces or eliminates binding to
human effector cells, binding to one or more Fc receptors, and/or
binding to cells expressing an Fc receptor. In an exemplary
embodiment, the Fc variants described herein comprise the following
modifications: L234A, L235A and P329G in the Fc region of human
IgG1, that result in attenuated effector function. Substitutions
L234A, L235A, and P329G (the L234A/L235A/P329G triple variant is
referred to as LALAPG) have previously been shown to reduce binding
to Fc receptors and complement (see e.g., US Publication No.
2012/0251531).
[0187] In various embodiments, Fc variants having reduced effector
function refer to Fc variants that reduce effector function (e.g.,
CDC, ADCC, and/or binding to FcR, etc. activities) by at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more
as compared to the effector function achieved by a wild-type Fc
region (e.g., an Fc region not having a mutation to reduce effector
function, although it may have other mutations). In certain
embodiments, Fc variants having reduced effector function refer to
Fc variants that eliminate all detectable effector function as
compared to a wild-type Fc region. Assays for measuring effector
function are known in the art and described below.
[0188] In vitro and/or in vivo cytotoxicity assays can be conducted
to confirm the reduction/depletion of CDC and/or ADCC activities.
For example, Fc receptor (FcR) binding assays can be conducted to
ensure that the antibody lacks Fc.gamma.R binding (hence likely
lacking ADCC activity). The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII FcR expression on
hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337
(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)).
[0189] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0190] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0191] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0192] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0193] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0194] d) Cysteine engineered antibody variants
[0195] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0196] e) Antibody Derivatives
[0197] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0198] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0199] A. Recombinant Methods and Compositions
[0200] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-CD20 antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-CD20
antibody is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
[0201] For recombinant production of an anti-CD20 antibody, nucleic
acid encoding an antibody, e.g., as described above, is isolated
and inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0202] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0203] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0204] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0205] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0206] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0207] B. Assays
[0208] Anti-CD20 antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0209] 1. Binding assays and other assays
[0210] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc. CD20 binding may be determined using methods
known in the art and exemplary methods are disclosed herein. In one
embodiment, binding is measured using radioimmunoassay. An
exemplary radioimmunoassay is provided below. CD20 antibody is
iodinated, and competition reaction mixtures are prepared
containing a fixed concentration of iodinated antibody and
decreasing concentrations of serially diluted, unlabeled CD20
antibody. Cells expressing CD20 (e.g., BT474 cells stably
transfected with human CD20) are added to the reaction mixture.
Following an incubation, cells are washed to separate the free
iodinated CD20 antibody from the CD20 antibody bound to the cells.
Level of bound iodinated CD20 antibody is determined, e.g., by
counting radioactivity associated with cells, and binding affinity
determined using standard methods. In another embodiment, ability
of CD20 antibody to bind to surface-expressed CD20 (e.g., on B cell
subsets) is assessed using flow cytometry. Peripheral white blood
cells are obtained (e.g., from human, cynomolgus monkey, rat or
mouse) and cells are blocked with serum. Labeled CD20 antibody is
added in serial dilutions, and T cells are also stained to identify
T cell subsets (using methods known in the art). Following
incubation of the samples and washing, the cells are sorted using
flow cytometer, and data analyzed using methods well known in the
art. In another embodiment, CD20 binding may be analyzed using
surface plasmon resonance. An exemplary surface plasmon resonance
method is exemplified in the Examples.
[0211] In another aspect, competition assays may be used to
identify an antibody that competes with any of the anti-CD20
antibodies disclosed herein for binding to CD20. In certain
embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by any
of the anti-CD20 antibodies disclosed herein. Detailed exemplary
methods for mapping an epitope to which an antibody binds are
provided in Morris (1996) "Epitope Mapping Protocols," in Methods
in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).
[0212] In an exemplary competition assay, immobilized CD20 is
incubated in a solution comprising a first labeled antibody that
binds to CD20 (e.g., rituximab, a GA101 antibody, etc.) and a
second unlabeled antibody that is being tested for its ability to
compete with the first antibody for binding to CD20. The second
antibody may be present in a hybridoma supernatant. As a control,
immobilized CD20 is incubated in a solution comprising the first
labeled antibody but not the second unlabeled antibody. After
incubation under conditions permissive for binding of the first
antibody to CD20, excess unbound antibody is removed, and the
amount of label associated with immobilized CD20 is measured. If
the amount of label associated with immobilized CD20 is
substantially reduced in the test sample relative to the control
sample, then that indicates that the second antibody is competing
with the first antibody for binding to CD20. See Harlow and Lane
(1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0213] 2. Activity assays
[0214] Anti-CD20 antibodies of the present disclosure (e.g., a type
II antibody) may be identified and/or characterized by one or more
activity assays known in the art. For example, a
complement-dependent cytotoxicity (CDC) and/or antibody-dependent
cellular cytotoxicity (ADCC) may be used, as described herein.
[0215] It is understood that any of the above assays may be carried
out using an immunoconjugate of the invention in place of or in
addition to an anti-CD20 antibody.
[0216] It is understood that any of the above assays may be carried
out using anti-CD20 antibody and an additional therapeutic
agent.
[0217] C. Immunoconjugates
[0218] The invention also provides immunoconjugates comprising an
anti-CD20 antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0219] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0220] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0221] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or I123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0222] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0223] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
Methods for Treating or Delaying Progression of Lupus Nephritis
[0224] Certain aspects of the present disclosure relate to methods
for treating or delaying progression of lupus nephritis (LN) in an
individual that has lupus. In some embodiments, the individual or
patient is a human.
[0225] LN is known in the art as a manifestation of lupus (e.g.,
systemic lupus erythematosus, drug-induced lupus, neonatal lupus,
or discoid lupus) in the kidney(s). The most common type of lupus
that manifests in the kidneys is systemic lupus erythematosus
(SLE). It is thought that 25-50% of SLE patients have abnormalities
in the urine and/or renal function early in the course of their
disease, with up to 60% of adults and 80% of children eventually
developing LN (for more details, see Cameron, J. S. (1999) J. Am.
Soc. Nephrol. 10:413-424). LN is thought to account for at least
50% of the morbidity and mortality associated with SLE.
[0226] In addition, renal manifestations have also been noted in
other types of lupus, such as discoid (Roujeau, J. C. et al. (1984)
Acta Derm. Venereol. 64:160-163) and drug-induced lupus (Smith, P.
R. et al. (1999) Rheumatology (Oxford) 38:1017-1018). In some
embodiments, the individual has SLE, discoid lupus, or drug-induced
lupus.
[0227] Diagnosis of SLE may be according to current American
College of Rheumatology (ACR) criteria. Active disease may be
defined by one British Isles Lupus Activity Group's (BILAG) "A"
criteria or two BILAG "B" criteria; SLE Disease Activity Index
(SLEDAI); or systemic lupus erythematosus (SLE) responder index
(SRI) as noted in the Examples below and descrived in Furie et al.,
Arthritis Rheum. 61(9):1143-51 (2009). Some signs, symptoms, or
other indicators used to diagnose SLE adapted from: Tan et al. "The
Revised Criteria for the Classification of SLE" Arth Rheum 25
(1982) may be malar rash such as rash over the cheeks, discoid
rash, or red raised patches, photosensitivity such as reaction to
sunlight, resulting in the development of or increase in skin rash,
oral ulcers such as ulcers in the nose or mouth, usually painless,
arthritis, such as non-erosive arthritis involving two or more
peripheral joints (arthritis in which the bones around the joints
do not become destroyed), serositis, pleuritis or pericarditis,
renal disorder such as excessive protein in the urine (greater than
0.5 gm/day or 3+ on test sticks) and/or cellular casts (abnormal
elements derived from the urine and/or white cells and/or kidney
tubule cells), neurologic signs, symptoms, or other indicators,
seizures (convulsions), and/or psychosis in the absence of drugs or
metabolic disturbances that are known to cause such effects, and
hematologic signs, symptoms, or other indicators such as hemolytic
anemia or leukopenia (white blood count below 4,000 cells per cubic
millimeter) or lymphopenia (less than 1,500 lymphocytes per cubic
millimeter) or thrombocytopenia (less than 100,000 platelets per
cubic millimeter). The leukopenia and lymphopenia must be detected
on two or more occasions. The thrombocytopenia must be detected in
the absence of drugs known to induce it. The invention is not
limited to these signs, symptoms, or other indicators of lupus.
[0228] The presence of autoantibodies may be tested as an
indication for lupus. Autoantibodies may include without limitation
anti-dsDNA antibodies, anti-complement antibodies, and antinuclear
antibodies (e.g., an ENA panel). ENA refers to Extractable Nuclear
Antigens, i.e., a group of nuclear antigens including, e.g., RNP,
Ro/SS-A, La/SS-B, Sm, SCL-70, Jo-1, as described in McNeilage et
al., J., Clin. Lab. Immunol. 15:1-17 (1984); Whittingham, Ann.
Acad. Med. 17(2):195-200 (1988); Wallace and Hahn, DUBOIS' LUPUS
ERYTHEMATOSUS, 7.sup.TH ED. LIPPINCOTT (2007); Tang et al.,
Medicine 89(1): 62-67 (2010). Antibodies to ENA have been
correlated to lupus. McNeilage et al., 1984; Whittingham 1988;
Asherson et al., Medicine 68(6): 366-374 (1989); and Tang et al.,
2010. Reduced complement activity may also be associated with
lupus, e.g., as measured by C3 levels, C4 levels, and/or a CH50
assay.
[0229] As described above in reference to SLE, it is known in the
art that LN often manifests progressively in patients with lupus
(e.g., systemic lupus erythematosus, drug-induced lupus, neonatal
lupus, or discoid lupus). That is to say, a patient may be
diagnosed with lupus without a clinical or pathological
manifestation of one or more LN symptoms. Nonetheless, the patient
may still be considered to be at risk for developing LN due to the
high frequency of lupus patients that eventually develop LN.
Therefore, in some embodiments, the methods of the present
disclosure may find use in delaying progression of LN, or
preventing LN, in a patient with lupus. In some embodiments, the
methods of the present disclosure may find use in postponing or
preventing the onset of LN in a patient with lupus (e.g., a form of
lupus that lacks a manifestation in the kidney(s)).
[0230] LN pathology may be classified according to the
International Society of Nephrology/Renal Pathology Society
(ISN/RPS) 2003 classification system, as shown in the table below
(see Markowitz G S, D'Agati V D (2007) Kidney Int 71:491-495 and
Weening, J J (2004) Kidney Int 65:521-530 for further descriptions
and definitions of terms).
TABLE-US-00010 TABLE 3 ISN/RPS 2003 Classification of Lupus
Nephritis. Class Minimal mesangial LN I (Normal glomeruli by light
microscopy, but mesangial immune deposits by immunofluorescence)
Class Mesangial proliferative LN II (Purely mesangial
hypercellularity of any degree or mesangial matrix expansion by
light microscopy, with mesangial immune deposits. A few isolated
subepithelial or subendothelial deposits may be visible by
immunofluorescence or electron microscopy, but not by light
microscopy) Class Focal LN III (Active or inactive focal, segmental
or global endo-or extracapillary glomerulonephritis involving
<50% of all glomeruli, typically with focal subendothelial
immune deposits, with or without mesangial alterations) III (A):
active lesions (focal proliferative LN) III (A/C): active and
chronic lesions (focal proliferative and sclerosing LN) III (C):
chronic inactive lesions with glomerular scars (focal sclerosing
LN) Class Diffuse LN IV (Active or inactive diffuse, segmental or
global endo-or extracapillary glomerulonephritis involving
.gtoreq.50% of all glomeruli, typically with diffuse subendothelial
immune deposits, with or without mesangial alterations. This class
is divided into diffuse segmental (IV-S) LN when .gtoreq.50% of the
involved glomeruli have segmental lesions, and diffuse global
(IV-G) LN when .gtoreq.50% of the involved glomeruli have global
lesions. Segmental is defined as a glomerular lesion that involves
less than half of the glomerular tuft. This class includes cases
with diffuse wire loop deposits but with little or no glomerular
proliferation.) IV-S (A): active lesions (diffuse segmental
proliferative LN) IV-G (A): active lesions (diffuse global
proliferative LN) IV-S (A/C): active and chronic lesions (diffuse
segmental proliferative and sclerosing LN) IV-G (A/C): active and
chronic lesions (diffuse global proliferative and sclerosing LN)
IV-S (C): chronic inactive lesions with scars (diffuse segmental
sclerosing LN) IV-G (C): chronic inactive lesions with scars
(diffuse global sclerosing LN) Class Membranous LN V (Global or
segmental subepithelial immune deposits or their morphologic
sequelae by light microscopy and by immuno- fluorescence or
electron microscopy, with or without mesangial alterations.) Class
Advanced sclerotic LN VI 90% of glomeruli globally sclerosed
without residual activity) LN = lupus nephritis; A = active; C =
chronic; G = global; S = segmental. Note: Class V may occur in
combination with Class III or IV, in which case both will be
diagnosed. Class V LN may show advanced sclerosis.
[0231] In some embodiments, the patient has class III or class IV
LN. In some embodiments, the patient has class III LN. For example,
in some embodiments, the patient has class III(A) or class III(A/C)
LN. In some embodiments, the patient has class IV LN. For example,
in some embodiments, the patient has class IV-S(A), IV-G(A),
IV-S(A/C), or IV-G(A/C) LN. As shown in Table 3 above, class V LN
may also occur concomitantly with class III or class IV LN. In some
embodiments, the methods of the present disclosure are used to
treat a patient with class III or class IV LN and concomitant class
V LN.
[0232] As discussed above, a high frequency of patients with lupus
(e.g., SLE) eventually develop LN. In some embodiments, the patient
is at risk for developing LN. In some embodiments, the patient is
at risk for developing class III or class IV LN. In some
embodiments, the patient is at risk for developing class III or
class IV LN with concomitant class V LN.
[0233] In some embodiments, the patient does not have class III(C)
LN (e.g., as described in Table 3 above). In some embodiments, the
patient does not have class IV(C) LN, such as class IV-S(C) or
IV-G(C) LN (e.g., as described in Table 3 above).
[0234] Several lab tests known in the art may be used to diagnose
and/or monitor the presence, progression, and/or response to
treatment in lupus nephritis. In some embodiments, serum creatinine
may be measured. In some embodiments, the normal range for serum
creatinine may be from about 0.6 to about 1.3 mg/dL, with some
variation seen by age, between men and women, and from lab to lab.
In some embodiments, the presence of urinary sediment and/or casts
may be measured, e.g., by microscopic examination of urine. For
example, the number of red blood cells in a urine sample may be
assayed by microscopic examination. In some embodiments, a normal
value for urinary sediment may be about 4 red blood cells (RBC) or
less per high power field (HPF). Urinary casts may include without
limitation red blood cell casts, white blood cell casts, renal
tubular epithelial cell casts, waxy casts, hyaline casts, granular
casts, and fatty casts. In some embodiments, a urinary protein to
creatinine ratio (UPCR) may be measured. The presence of protein in
the urine (proteinuria) may also be assayed by tests including
without limitation a urine albumin to creatinine ratio (UACR) and
dipstick urinalysis. Other tests and/or measures that may be useful
for examining renal function include without limitation a renal
panel, creatinine clearance, sodium, potassium, chloride,
bicarbonate, phosphorus, calcium, albumin, blood urea nitrogen
(BUN), creatinine, glucose, estimated glomerular filtration rate
(eGFR), BUN/creatinine ratio, and anion gap, and may include a
measurement of the above parameters in the blood and/or urine,
where appropriate. For more detailed description, see, e.g., the
American College of Rheumatology Guidelines for Screening, Case
Definition, Treatment and Management of Lupus Nephritis (Hahn, B.
et al. (2012) Arthritis Care Res. 64:797-808).
[0235] In some embodiments, the methods of the present disclosure
include administering to the individual at least a first antibody
exposure to a type II anti-CD20 antibody of the present disclosure
and a second antibody exposure to the type II anti-CD20 antibody.
Any of the type II anti-CD20 antibodies described herein may be
used, e.g., a GA101 antibody such as obinutuzumab. In some
embodiments, the second antibody exposure is not provided until
from about 18 weeks to about 26 weeks after the first antibody
exposure. In some embodiments, the second antibody exposure is not
provided until about 18 weeks after the first antibody exposure,
about 19 weeks after the first antibody exposure, about 20 weeks
after the first antibody exposure, about 21 weeks after the first
antibody exposure, about 22 weeks after the first antibody
exposure, about 23 weeks after the first antibody exposure, about
24 weeks after the first antibody exposure, about 25 weeks after
the first antibody exposure, or about 26 weeks after the first
antibody exposure. In some embodiments, the second antibody
exposure is not provided until less than about any of the following
weeks after the first antibody exposure: 26, 25, 24, 23, 22, 21,
20, or 19. In some embodiments, the second antibody exposure is not
provided until greater than about any of the following weeks after
the first antibody exposure: 18, 19, 20, 21, 22, 23, 24, or 25.
That is, the second antibody exposure is not provided until any of
a range of weeks having an upper limit of 26, 25, 24, 23, 22, 21,
20, or 19 and an independently selected lower limit of 18, 19, 20,
21, 22, 23, 24, or 25, wherein the lower limit is less than the
upper limit.
[0236] The dosing regimens described herein use a consistent system
for tracking time between doses whereby the first dose is
administered to the patient on Day 1. As described herein, an
antibody exposure of the present disclosure may include one or two
doses. In cases where the antibody exposures contain one dose,
references to a second antibody exposure not provided until a
period of time has elapsed after a first antibody exposure (as
described herein) refer to the amount of time elapsed between the
dose of the first antibody exposure (e.g., Day 1) and the dose of
the second antibody exposure. If the first antibody exposure
includes two doses, the first dose of the first antibody exposure
is provided on Day 1. In cases where the antibody exposures contain
two doses, references to a second antibody exposure not provided
until a period of time has elapsed after a first antibody exposure
(as described herein) refer to the amount of time elapsed between
the first of the two doses of the first antibody exposure (e.g.,
Day 1) and the first dose of the two doses of the second antibody
exposure. For example, if a method of the present disclosure
includes a first antibody exposure with two doses and a second
antibody exposure with two doses, and the second antibody exposure
is not provided until about 22 weeks after the first antibody
exposure, then the interval between the first dose of the first
antibody exposure and the first dose of the second antibody
exposure is about 22 weeks.
[0237] In some embodiments, a first antibody exposure of the
present disclosure includes one or two doses of a type II anti-CD20
antibody of the present disclosure. In some embodiments, the first
antibody exposure contains a total exposure of between about 1800
mg and about 2200 mg of the type II anti-CD20 antibody. In some
embodiments, the first antibody exposure contains a total exposure
of about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or
about 2200 mg of the type II anti-CD20 antibody.
[0238] In some embodiments, the first antibody exposure includes
two doses. In some embodiments, the first antibody exposure
includes a first dose of between about 900 mg and about 1100 mg of
the type II anti-CD20 antibody and a second dose of between about
900 mg and about 1100 mg of the type II anti-CD20 antibody. In some
embodiments, the first dose of the first antibody exposure contains
about 1000 mg of the type II anti-CD20 antibody. In some
embodiments, the second dose of the first antibody exposure
contains about 1000 mg of the type II anti-CD20 antibody. In some
embodiments, the second dose of the first antibody exposure is not
provided until about 1.5 weeks to about 2.5 weeks after the first
dose of the first antibody exposure. In some embodiments, the
second dose of the first antibody exposure is not provided until
about 2 weeks after the first dose of the first antibody
exposure.
[0239] In some embodiments, a second antibody exposure of the
present disclosure includes one or two doses of a type II anti-CD20
antibody of the present disclosure. In some embodiments, the second
antibody exposure contains a total exposure of between about 1800
mg and about 2200 mg of the type II anti-CD20 antibody. In some
embodiments, the second antibody exposure contains a total exposure
of about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or
about 2200 mg of the type II anti-CD20 antibody.
[0240] In some embodiments, the second antibody exposure includes
two doses. In some embodiments, the second antibody exposure
includes a first dose of between about 900 mg and about 1100 mg of
the type II anti-CD20 antibody and a second dose of between about
900 mg and about 1100 mg of the type II anti-CD20 antibody. In some
embodiments, the first dose of the second antibody exposure
contains about 1000 mg of the type II anti-CD20 antibody. In some
embodiments, the second dose of the second antibody exposure
contains about 1000 mg of the type II anti-CD20 antibody. In some
embodiments, the second dose of the second antibody exposure is not
provided until about 1.5 weeks to about 2.5 weeks after the first
dose of the second antibody exposure. In some embodiments, the
second dose of the second antibody exposure is not provided until
about 2 weeks after the first dose of the second antibody
exposure.
[0241] In some embodiments, a type II anti-CD20 antibody of the
present disclosure is administered intravenously (e.g., by IV
infusion).
[0242] In some embodiments, the methods of the present disclosure
further include administering an effective amount of an
immunosuppressive agent (e.g., in conjunction with a type II
anti-CD20 antibody as described herein). Several classes of
immunosuppressive agents are known in the art, including without
limitation cytostatics (e.g., cytotoxic agents such as antibiotics,
alkylating agents (e.g., cyclophosphamide, also known as
cytophosphane), inosine monophosphate dehydrogenase inhibitors,
antimetabolites such as protein synthesis inhibitors, folic acid
analogs, purine analogs, pyrimidine analogs, and the like),
immunosuppressive antibodies, glucocorticoids, drugs targeting
immunophilins (e.g., tacrolimus, sirolimus, rapamycin and analogs
thereof, ciclosporin, and the like), mTOR active site inhibitors,
mycophenolic acid and derivatives or salts thereof, TNF binding
proteins, interferons, opiods, and other small molecules (e.g.,
fingolimod). In some embodiments, the immunosuppressive agent
includes mycophenolic acid, a derivative of mycophenolic acid, or a
salt of mycophenolic acid. In some embodiments, the
immunosuppressive agent includes mycophenolate mofetil. In some
embodiments, the immunosuppressive agent includes CellCept.RTM.
(Roche). In some embodiments, the immunosuppressive agent includes
Myfortic.RTM. (Novartis). Effective amounts of the
immunosuppressive agents of the present disclosure are known in the
art and readily ascertainable by standard assays. For example,
mycophenolate mofetil may be administered at 2.0-2.5 g/day as
illustrated in FIG. 1. In some embodiments, mycophenolate mofetil
may be administered starting at 1000 mg/day in divided doses (2
times/day) and titrating up to 2.0-2.5 g/day in divided doses (2
times/day) by week 4.
[0243] In some embodiments, an immunosuppressive agent may be
administered before, during, or after administration of a type II
anti-CD20 antibody of the present disclosure, e.g., as a treatment
for lupus. In some embodiments, an immunosuppressive agent may be
administered throughout the period of treatment with a type II
anti-CD20 antibody of the present disclosure. In some embodiments,
mycophenolate mofetil may be administered as described above
throughout the period of treatment with the type II anti-CD20
antibody.
[0244] In some embodiments, the methods of the present disclosure
further include administering an effective amount of a
glucocorticoid or corticosteroid (e.g., in conjunction with a type
II anti-CD20 antibody as described herein). A variety of naturally
occurring and synthetic glucocorticoids/corticosteroids are known
in the art, including without limitation beclometasone,
triamcinolone, dexamethasone, betamethasone, prednisone,
methylprednisolone, prednisolone, cortisone, and cortisol. In some
embodiments, the glucocorticoids/corticosteroid includes
methylprednisolone. In some embodiments, the
glucocorticoids/corticosteroid includes prednisone. Effective
amounts of the glucocorticoids/corticosteroids of the present
disclosure are known in the art and readily ascertainable by
standard assays. For example, methylprednisolone may be
administered at 750-1000 mg doses once daily by IV. As another
example, prednisone may be administered orally at 0.5 mg/kg and
optionally tapered to 7.5 mg/day.
[0245] In some embodiments, a glucocorticoid may be administered
before, during, or after administration of a type II anti-CD20
antibody of the present disclosure, e.g., to treat LN clinical
activity. In some embodiments, a glucocorticoid may be administered
prior to administration of a type II anti-CD20 antibody of the
present disclosure, e.g., 30-60 minutes before the type II
anti-CD20 antibody. In some embodiments, 80 mg methylprednisolone
may be administered by IV 30-60 minutes before administration of a
type II anti-CD20 antibody of the present disclosure. In some
embodiments, prednisone (e.g., orally administered) and/or methyl
prednisolone (e.g., IV administered) may be administered with
treatment, followed by a maintenance treatment (e.g., mycophenolate
mofetil or cyclophosphamide).
[0246] In some embodiments, the methods of the present disclosure
further include administering an effective amount of an
antihistamine (e.g., in conjunction with a type II anti-CD20
antibody as described herein). Antihistamines known in the art and
currently in clinical use include histamine H.sub.1-receptor and
histamine H.sub.2-receptor antagonists or inverse agonists. In some
embodiments, the antihistamine includes diphenhydramine. Effective
amounts of the antihistamines of the present disclosure are known
in the art and readily ascertainable by standard assays. For
example, diphenhydramine may be administered in 50 mg oral
doses.
[0247] In some embodiments, an antihistamine may be administered
before, during, or after administration of a type II anti-CD20
antibody of the present disclosure, e.g., as a prophylactic
treatment. In some embodiments, an antihistamine may be
administered prior to administration of a type II anti-CD20
antibody of the present disclosure, e.g., 30-60 minutes before the
type II anti-CD20 antibody. In some embodiments, 50 mg
diphenhydramine may be administered orally 30-60 minutes before
administration of a type II anti-CD20 antibody of the present
disclosure.
[0248] In some embodiments, the methods of the present disclosure
further include administering an effective amount of a
non-steroidal anti-inflammatory drug or NSAID (e.g., in conjunction
with a type II anti-CD20 antibody as described herein). NSAIDs
known in the art include acetic acid derivatives, propionic acid
derivatives, salicylates, enolic acid derivatives, anthranilic acid
derivatives, selective COX-2 inhibitors, sulfonanilides, and the
like. In some embodiments, the NSAID includes acetaminophen.
Effective amounts of the NSAIDs of the present disclosure are known
in the art and readily ascertainable by standard assays. For
example, acetaminophen may be administered in 650-1000 mg oral
doses.
[0249] In some embodiments, an NSAID may be administered before,
during, or after administration of a type II anti-CD20 antibody of
the present disclosure, e.g., as a prophylactic treatment. In some
embodiments, an NSAID may be administered prior to administration
of a type II anti-CD20 antibody of the present disclosure, e.g.,
30-60 minutes before the type II anti-CD20 antibody. In some
embodiments, 650-1000 mg acetaminophen may be administered orally
30-60 minutes before administration of a type II anti-CD20 antibody
of the present disclosure.
[0250] In some embodiments, the methods of the present disclosure
further include administering an effective amount of an
anti-malarial agent (e.g., in conjunction with a type II anti-CD20
antibody as described herein). Examples of anti-malarial agents
that may be used include without limitation hydroxychloroquine,
chloroquine, and quinacrine. In some embodiments, an anti-malarial
agent may be administered before, during, or after administration
of a type II anti-CD20 antibody of the present disclosure, e.g., as
a treatment for one or more symptoms of lupus.
[0251] In some embodiments, the methods of the present disclosure
further include administering an effective amount of an integrin
antagonist (e.g., in conjunction with a type II anti-CD20 antibody
as described herein). Examples of integrin antagonists that may be
used include without limitation an LFA-1 antibody, such as
efalizumab (RAPTI.GAMMA.VA.RTM.) commercially available from
Genentech, or an alpha 4 integrin antibody such as natalizumab
(ANTEGREN.RTM.) available from Biogen, or diazacyclic phenylalanine
derivatives, phenylalanine derivatives, phenylpropionic acid
derivatives, enamine derivatives, propanoic acid derivatives,
alkanoic acid derivatives, substituted phenyl derivatives, aromatic
amine derivatives, ADAM disintegrin domain polypeptides, antibodies
to alphavbeta3 integrin, aza-bridged bicyclic amino acid
derivatives, etc. In some embodiments, an integrin antagonist may
be administered before, during, or after administration of a type
II anti-CD20 antibody of the present disclosure, e.g., as a
treatment for one or more symptoms of lupus.
[0252] In some embodiments, the methods of the present disclosure
further include administering an effective amount of a cytokine
antagonist (e.g., in conjunction with a type II anti-CD20 antibody
as described herein). Examples of cytokine antagonists that may be
used include without limitation an antagonist (e.g., an antagonist
antibody) against IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-6, IL-7,
IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such as
TNF-.alpha. or TNF-.beta.; and other polypeptide factors including
LIF and kit ligand (KL). In some embodiments, a cytokine antagonist
may be administered before, during, or after administration of a
type II anti-CD20 antibody of the present disclosure, e.g., as a
treatment for one or more symptoms of lupus.
[0253] In some embodiments, the methods of the present disclosure
further include administering an effective amount of a hormone
(e.g., in conjunction with a type II anti-CD20 antibody as
described herein). In some embodiments, a hormone (e.g., for
hormone replacement therapy) may be administered before, during, or
after administration of a type II anti-CD20 antibody of the present
disclosure, e.g., for a medical treatment in a women with
lupus.
[0254] In some embodiments, the methods of the present disclosure
further include administering a standard of care treatment (e.g.,
in conjunction with a type II anti-CD20 antibody as described
herein). In some embodiments, a standard of care treatment may be
administered before, during, or after administration of a type II
anti-CD20 antibody of the present disclosure, e.g., for treating or
preventing one or more symptoms of lupus. In certain embodiments, a
standard of care treatment may be administered after a second
antibody exposure of the present disclosure. For example, a type II
anti-CD20 antibody of the present disclosure may be administered as
described herein to a patient as an induction therapy, then the
patient may be treated according to standard of care as a
maintenance therapy. Standard of care treatments for lupus are well
known in the art and include without limitation an
angiotensin-converting enzyme (ACE) inhibitor, an
angiotensin-receptor blocker, cyclophosphamide, mycophenolate
mofetil (e.g., at a dose as described herein, such as 2.0-2.5
g/day), azathioprine, and a glucocorticoid or corticosteroid (e.g.,
prednisone, such as a prednisone taper).
[0255] In some embodiments, the methods of the present disclosure
further include administering an anti-hypertensive agent (e.g., in
conjunction with a type II anti-CD20 antibody as described herein).
In some embodiments, an anti-hypertensive agent may be administered
before, during, or after administration of a type II anti-CD20
antibody of the present disclosure, e.g., for treating or
preventing hypertension. In some embodiments, anti-hypertensive
agents includes without limitation ACE inhibitors and
angiotensin-receptor blockers. In some embodiments, an
anti-hypertensive agent listed in Table 5 is administered, e.g., at
a dose within the ranges described in Table 5.
[0256] In some embodiments, the methods of the present disclosure
result in a complete renal response (CRR) in an individual. In some
embodiments, a CRR comprises all of the following: a normalization
of serum creatinine, an inactive urinary sediment, and a urinary
protein to creatinine ratio of <0.5. In some embodiments, a
normalization of serum creatinine is characterized by serum
creatinine less than or equal to the upper limit of normal (ULN)
range of central laboratory values, and/or serum creatinine 15%
above baseline and less than or equal to the ULN range of central
laboratory values if baseline (e.g., Day 1) serum creatinine is
within the normal range of the central laboratory values. In some
embodiments, an inactive urinary sediment is characterized by
<10 RBCs/high-power field (HPF) and/or the absence of red cell
casts. For more detailed discussion of CRR and partial renal
response (PRR) in LN, see, e.g., Chen, Y. E. et al. (2008) Clin. J.
Am. Soc. Nephrol. 3:46-53.
[0257] In some embodiments, the methods of the present disclosure
result in a complete renal response (CRR) or a partial renal
response (PRR) in an individual. In some embodiments, a PRR
comprises one or more of the following: a normalization of serum
creatinine, an inactive urinary sediment, and a urinary protein to
creatinine ratio of <0.5. In some embodiments, a PRR comprises
one or more of the following: mitigation of one or more symptoms
including without limitation a reduction in serum creatinine,
reduced urinary sediment, a reduction in proteinuria, and any other
improvement in renal function. In some embodiments, a CRR or PRR
comprises a reduction in one or more biomarkers of lupus activity,
including without limitation anti-dsDNA antibodies, antinuclear
antibodies/ENA, anti-complement antibodies, reduced levels of
complement C3 and/or C4, and reduced complement activity (e.g., as
measured by CH50 assay).
[0258] In some embodiments, the methods of the present disclosure
result in a depletion of circulating peripheral B cells in an
individual. In some embodiments, after administration of a type II
anti-CD20 antibody of the present disclosure (e.g., according to
any of the methods described herein), circulating peripheral B
cells are present in peripheral blood at about 10 cells/.mu.l, or
fewer, about 9 cells/.mu.l, or fewer, about 8 cells/.mu.l, or
fewer, about 7 cells/.mu.l, or fewer, about 6 cells/.mu.l, or
fewer, about 5 cells/.mu.l, or fewer, about 4 cells/.mu.l, or
fewer, about 3 cells/.mu.l, or fewer, about 2 cells/.mu.l, or
fewer, or about 1 cell/.mu.l, or fewer. In some embodiments,
circulating peripheral B cells in the individual are depleted by at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or about 100%. In some
embodiments, depletion of circulating peripheral B cells refers to
a measurement of circulating peripheral B cells taken after a first
antibody exposure (e.g., including 1 or 2 doses of an anti-CD20
antibody as described herein), after a second antibody exposure
(e.g., including 1 or 2 doses of an anti-CD20 antibody as described
herein), 3 months after treatment (e.g., after receiving a first
and/or a second antibody exposure as described herein), 6 months
after treatment (e.g., after receiving a first and/or a second
antibody exposure as described herein), 9 months after treatment
(e.g., after receiving a first and/or a second antibody exposure as
described herein), or 12 months after treatment (e.g., after
receiving a first and/or a second antibody exposure as described
herein), e.g., as compared to a corresponding measurement in the
same individual before treatment, or as compared to a corresponding
measurement in a control individual (e.g., an individual that has
not received treatment).
[0259] Methods for assaying depletion of circulating peripheral B
cells in an individual are known in the art, e.g., flow cytometry
using one or more antibodies that recognize a B cell marker. In
some embodiments, highly sensitive flow cytometry (HSFC) may be
used to assay depletion of circulating peripheral B cells (see,
e.g., Vital, E. M. et al. (2011) Arthritis Rheum. 63:3038-3047). In
some embodiments, the B cells are CD19+ B cells. In some
embodiments, the B cells are naive B cells (e.g., CD19+CD27- B
cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts
(e.g., CD19+CD27+CD38++ B cells).
IV. ARTICLES OF MANUFACTURE OR KITS
[0260] In another aspect of the invention, an article of
manufacture or kit containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture or kit comprises a container
and a label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody described herein (e.g., a
type II anti-CD20 antibody of the present disclosure). The label or
package insert indicates that the composition is used for treating
the condition of choice, e.g., according to any of the methods
described herein. Alternatively, or additionally, the article of
manufacture or kit may further comprise a second (or third)
container comprising a pharmaceutically-acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
[0261] In some embodiments, provided herein is a kit comprising a
container comprising a type II anti-CD20 antibody of the present
disclosure and an optional pharmaceutically acceptable carrier,
and, optionally, a package insert comprising instructions for
treating or delaying progression of lupus nephritis in an
individual, e.g., wherein the instructions indicate that at least a
first antibody exposure to a type II anti-CD20 antibody and a
second antibody exposure to the type II anti-CD20 antibody are
administered to the individual, the second antibody exposure not
being provided until from about 18 weeks to about 26 weeks after
the first antibody exposure; wherein the first antibody exposure
comprises one or two doses of the type II anti-CD20 antibody, the
first antibody exposure comprising a total exposure of between
about 1800 mg and about 2200 mg of the type II anti-CD20 antibody;
and wherein the second antibody exposure comprises one or two doses
of the type II anti-CD20 antibody, the second antibody exposure
comprising a total exposure of between about 1800 mg and about 2200
mg of the type II anti-CD20 antibody. In some embodiments, provided
herein is a kit comprising a container comprising a type II
anti-CD20 antibody of the present disclosure and an optional
pharmaceutically acceptable carrier, and, optionally, a package
insert comprising instructions for treating or delaying progression
of class III or class IV lupus nephritis in an individual. In some
embodiments of any of the above embodiments, the type II anti-CD20
antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ
ID NO:1, HVR-H2 sequence of SEQ ID NO:2, and HVR-H3 sequence of SEQ
ID NO:3, and a light chain comprising HVR-L1 sequence of SEQ ID
NO:4, HVR-L2 sequence of SEQ ID NO:5, and HVR-L3 sequence of SEQ ID
NO:6. In some embodiments of any of the above embodiments, the type
II anti-CD20 antibody is obinutuzumab.
[0262] The article of manufacture may still further comprise a
second or third container comprising a second medicament, wherein
the anti-CD20 antibody (e.g., a type II anti-CD20 antibody of the
present disclosure) is a first medicament, where the article
further comprises instructions on the package insert for treating
the subject with the second medicament. Exemplary second
medicaments include a chemotherapeutic agent, an immunosuppressive
agent, an anti-malarial agent, a cytotoxic agent, an integrin
antagonist, a cytokine antagonist, a hormone, and any of the
treatments that may be used in conjunction with a type II anti-CD20
antibody as described herein. The article of manufacture in these
embodiments may further comprise a package insert indicating that
the compositions can be used to treat a particular condition.
[0263] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an anti-CD20 antibody.
[0264] The specification is considered to be sufficient to enable
one skilled in the art to practice the invention. Various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description and fall within the scope of the
appended claims. All publications, patents, and patent applications
cited herein are hereby incorporated by reference in their entirety
for all purposes.
EXAMPLES
[0265] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1
A Pharmacology Study of Obinutuzumab Administered with
Mycophenolate Mofetil in Patients with Class III/IV Lupus
Nephritis
Study Design
[0266] This Phase II study is designed to assess the safety and
efficacy of obinutuzumab (i.e., a type II anti-CD20 antibody) as an
add-on therapy to mycophenolate mofetil (MMF) in patients with
active ISN/RPS Class III/IV lupus nephritis (LN). The Phase II
study is a parallel-group, double-blind, randomized,
placebo-controlled study comparing the efficacy and safety of
obinutuzumab plus MMF with placebo plus MMF in Class III and IV
patients with proliferative LN (FIG. 1).
[0267] The study is also a prospective, multicenter study. Patients
diagnosed with ISN/RPS Class III or IV LN, in some embodiments with
a diagnosis of SLE according to current ACR criteria (at least 4
criteria must be present, one of which must be a positive
anti-nuclear antibody), are enrolled in centers throughout the
world. The study includes standard-of-care therapy with
angiotensin-converting enzyme (ACE) inhibitors/angiotensin II
receptor blockers, MMF (dosed at 2.0-2.5 g/day), and a prednisone
taper.
[0268] As described in greater detail below, patients are 18-75
years of age and have ISN/RPS 2003 Class III or IV proliferative LN
(see Weening, J J (2004) J. Am. Soc. Nephrol. 15:241-250) as
evidenced by renal biopsy performed within 6 months prior to
screening and may have concomitant Class V disease (e.g., Class
III/V or Class IV/V). Patients with Class III (C) or Class IV (C)
disease are excluded because of the lower likelihood of response
within these categories.
[0269] Inclusion criteria for the study include:
(a) Signed Informed Consent Form;
[0270] (b) Age 18-75 years; (c) Ability to comply with study
protocol; (d) Diagnosis of systemic lupus erythematosus (SLE)
according to current ACR criteria (at least 4 criteria must be
present, one of which must be a positive anti-nuclear antibody);
(e) Diagnosis of ISN/RPS 2003 Class III or IV LN as evidenced by
renal biopsy performed within 6 months prior to screening (patients
may also co-exhibit Class V disease in addition to either Class III
or Class IV disease); (f) Demonstration of active urinary sediment
as evidenced by 10 RBCs/HPF or the presence of red cell casts; and
(g) Proteinuria (urine protein to creatinine ratio>1.0, based on
a 24-hour urine collection).
[0271] Key exclusion criteria include:
(a) Retinitis, poorly controlled seizure disorder, acute
confusional state, myelitis, stroke or stroke syndrome, cerebellar
ataxia, or dementia that is currently active and resulting from
SLE; (b) Presence of rapidly progressive glomerulonephritis
(defined by the presence of crescent formation in 50% of glomeruli
assessed on renal biopsy or the doubling of serum creatinine within
12 weeks of screening); (c) Severe renal impairment as defined by
estimated GFR<30 mL/min or the need for dialysis or renal
transplant; (d) Greater than 50% of glomeruli with sclerosis on
renal biopsy; (e) Treatment with cyclophosphamide or calcineurin
inhibitors within the 3 months prior to randomization; and (f)
Unstable disease with thrombocytopenia or at high risk for
developing clinically significant bleeding or organ dysfunction
requiring therapies such as plasmapheresis or acute blood or
platelet transfusions.
[0272] Patients receive an initial 1000 mg of methylprednisolone
intravenously (IV) prior to or during screening, and may receive up
to 3000 mg methylprednisolone IV prior to randomization for severe
clinical activity according to guidelines of routine care for these
patients. Patients receive 80 mg methylprednisolone (or
methylprednisolone placebo) IV on the day of the
obinutuzumab/placebo infusion to reduce infusion-related events.
The oral prednisone taper is 0.5 mg/kg and is reduced over 12
weeks. This modified taper is initiated in recognition that
prednisone doses above 10 mg/day are associated with significant
adverse events, including increased risk of cardiovascular events
(Bichile, T. and Petri, M. (2014) Presse Med. 43:e187-195). Prior
experience with rituximab suggests that it can potentially enable
complete and partial renal responses in the absence of oral
prednisone or a prednisone taper, thus allowing the use of lower
doses of corticosteroids (Condon, M. B. et al. (2013) Ann. Rheum.
Dis. 72:1280-1286).
[0273] Patients are followed for 12 months until the primary
endpoint evaluation, and an interim analysis at 6 months is
performed to evaluate early differences in CRR. All patients have
central reading of the renal biopsy histopathology and also receive
repeat renal biopsy as available on the basis of clinical status
and local practice. All patients are evaluated by high sensitivity
flow cytometry (HSFC) to evaluate the ability of obinutuzumab to
deplete circulating peripheral B cells, and an interim PD analysis
is performed to assess whether patients do not fully deplete
peripheral CD19+ B cells as anticipated.
Dosing and Non-Investigational Medicinal Products
[0274] The dosing regimen for the study is obinutuzumab
administered by IV infusion at a dose of 1000 mg on Days 1, 15,
168, and 182 (test group); or obinutuzumab placebo (e.g., saline
IV, corresponding to the obinutuzumab1000-mg dose) administered by
IV infusion on Days 1, 15, 168, and 182. The obinutuzumab/placebo
is administered in a hospital or clinic environment where full
resuscitation facilities are immediately available and under close
supervision of the investigator or designee. After the end of the
infusion, the IV line remains in place for at least 1 hour to
enable administration of IV drugs if necessary. If no adverse
events occur during this period of time, the IV line may be
removed.
[0275] After screening, patients who are not already receiving MMF
receive 1500 mg/day MMF in divided doses (2-3 times/day), and all
patient doses are titrated up to a target dose of 2.0-2.5 g/day in
divided doses (2-3 times/day) by Week 4, as tolerated. If
reductions in dose are necessary, decreases are allowed in 250-500
mg decrements. During screening or at randomization, if clinically
indicated, patients may receive 750-1000 mg methylprednisolone IV
once daily for up to three days to treat underlying LN clinical
activity. Patients receive 0.5 mg/kg oral prednisone during
screening or at randomization, tapering this prednisone dose, per
protocol, starting on Day 16 and reducing the prednisone dosage to
7.5 mg/day by Week 12. These treatments are described in further
detail below.
Concomitant Therapy and Clinical Practice
[0276] Patients who are not already taking vitamin D (400 IU/day)
and calcium supplements (1200 mg/day of calcium citrate or 1500
mg/day of calcium carbonate) begin taking these supplements at
randomization. All patients take either an angiotensin-converting
enzyme inhibitor or an angiotensin receptor blocker titrated to
adequate blood pressure control as recommended by the National
Kidney Foundation for chronic kidney disease. Other agents that
affect proteinuria are not allowed to be initiated during the
study, including but not limited to non-dihydropyridine calcium
antagonists, dihydropyridine calcium antagonists, aldosterone
antagonists, and direct renin antagonists.
Mycophenolate Mofetil (MMF)
[0277] All patients continue on or initiate use of MMF during
screening or no later than Day 1. The initial dosage is 1500 mg/day
by mouth, given in two or three divided doses and titrated upward
to 2.0-2.5 g/day in divided doses by Week 4. MMF may be increased
by 500 mg/week, as tolerated up to a maximum dosage of 2.5 g/day.
Reductions are allowed because of adverse effects.
[0278] Newly diagnosed patients with LN with no prior exposure to
MMF are recommended to initiate an induction agent (MMF or
cyclophosphamide) and then be re-assessed for eligibility. A
proportion of patients who are initially treated with MMF or
cyclophosphamide achieve CRR and therefore have minimal need for
added immunosuppression (Dall'Era, M. et al. (2011) Arthritis Care
Res. 63:351-357).
[0279] For those patients who enter the study already receiving a
dosage of MMF higher than 1500 mg/day, MMF will be titrated upward,
as tolerated, to a goal of 2.5 g/day, given in divided doses, by
Week 4. A patient's current dose of MMF is given in 2 or 3 divided
doses and increased by 500 mg/week as tolerated.
Corticosteroid Administration
[0280] All patients receive a combination of IV and oral
corticosteroids as part of their initial therapy for LN.
Methylprednisolone (e.g., Solu-Medrol.RTM.) is implemented for two
purposes: as part of the usual care for patients with active Class
III or IV LN and also to reduce infusion-related reactions (IRRs)
on the days of obinutuzumab/placebo infusions. Up to three doses of
IV methylprednisolone 1000 mg are given on the basis of
investigator judgement and local practice. Up to three 1000 mg
infusions may have been initiated prior to screening or during the
screening interval.
[0281] On Days 1, 15, 168, and 186, patients receive 80 mg IV
methylprednisolone or placebo 30-60 minutes prior to study drug
infusion to prevent IRRs. Additionally, oral prednisone may be
initiated before or during the screening interval, and a taper
commences on Day 2. From Days 2 to 16, 0.50 mg/kg/day oral
prednisone is given (maximum dose 60 mg), except on the day of IV
methylprednisolone/placebo infusions, and will continue until Day
16. From Day 16 onward, a prednisone taper commences.
[0282] All patients undergo a scheduled corticosteroid taper
commencing on Day 16. Patients fractionally reduce their prednisone
dose over 12 weeks until the dose is 7.5 mg/day by Week 12. After
14 weeks of tapering, patients continue on prednisone at 7.5 mg/day
or less. In patients whose disease is too clinically active for the
patient to make the first step in their prednisone taper, as
evidenced by active urinary sediment, rising serum creatinine, or
moderate-to-severe extra-renal symptoms, these patients may
continue to receive their initial prednisone dose for up to an
additional 28 days.
[0283] To maintain consistency in the treatment of renal flares,
retreatment with higher doses of corticosteroids is permitted if
judged clinically appropriate by the investigator and if patients
meet criteria for a renal flare. Patients may be treated with
prednisone (up to 0.5 mg/kg; not to exceed 60 mg/day) for 2 weeks.
Prednisone is then tapered to achieve 10 mg/day within 6 weeks
after initial corticosteroid increase. Patients are allowed to
receive corticosteroids for emergent illness (trauma, severe
asthma) or surgery, if clinically warranted; the corticosteroid use
is limited to a total of .ltoreq.7 days, if possible. Investigators
are then allowed to increase the prednisone dose by .ltoreq.2.5
mg/day to treat symptoms of adrenal insufficiency or corticosteroid
withdrawal, after the patient's dosage has been tapered to 10
mg/day.
[0284] Patients who experience a severe extra-renal SLE flare may
receive treatment with additional oral corticosteroids, if judged
clinically appropriate by the investigator. These patients may be
retreated with prednisone (up to 1.0 mg/kg) for up to 2 weeks on
the basis of the severity of disease and organ system involvement
and the dosage is tapered to 7.5 mg/day. Patients experiencing a
mild or moderate extra-renal flare may temporarily increase their
prednisone dose by up to 20 mg per day and taper this dose over 4
weeks, if judged clinically appropriate by the investigator. IV
corticosteroids in equivalent doses are allowed if gastrointestinal
involvement temporarily precludes treatment with oral
corticosteroids.
Anti-Malarial Medications
[0285] Patients taking anti-malarial medications at study entry
maintain a constant dosage throughout the study. Patients not
previously on anti-malarial medications may be enrolled in the
study but should not initiate anti-malarial medications unless
experiencing a disease flare that is unresponsive to
corticosteroids. Table 4 lists anti-malarial medications and dose
ranges expected to be used during the course of the study.
TABLE-US-00011 TABLE 4 Anti-malarial medications Anti-malarial
Medication Dose Range (oral) Hydroxychloroquine 200-400 mg daily
Chloroquine 500 mg every day or every other day Quinacrine 100 mg
every day
Antihypertensive Therapy
[0286] All patients who are not currently taking either an ACE
inhibitor or an angiotensin-receptor blocker should be started on
one at screening. Patients are on either an ACE inhibitor or
angiotensin-receptor blocker for at least 10 days prior to
randomization. Combination therapy with the two agents is not
allowed.
[0287] During screening, every effort is made to adequately control
patients' blood pressures. The dose of the ACE inhibitor or
angiotensin-receptor blocker may be titrated upward to the maximum
recommended dose in the current package insert to achieve adequate
blood pressure control as recommended by the Eighth Report of the
Joint National Committee on the Prevention, Detection, Evaluation,
and Treatment of High Blood Pressure (James, P. A. et al. (2014)
JAMA 311:507-520). If adequate blood pressure control is not
achieved, patients may be started on additional antihypertensive
agents but not on agents that affect proteinuria (e.g.,
nondihydropyridine calcium channel blockers, aldosterone
antagonists, direct renin antagonists). Additional agents that
specifically target the renin-angiotensin system are not initiated
during the study. Suggested dose ranges for specific ACE inhibitors
and angiotensin-receptor blockers are listed in Table 5. If
patients are intolerant to ACE inhibitors and angiotensin-receptor
blockers, they may use either a direct renin inhibitor or
aldosterone antagonists, but not in combination.
TABLE-US-00012 TABLE 5 Suggested Dose Ranges for ACE Inhibitors and
Angiotensin-Receptor Blockers ACE Inhibitor or Angiotensin- Dose
Range (Oral), Receptor Blocker mg/day ACE Inhibitors Benazepril
10-80 Ramipril 2.5-10 Lisinopril 10-80 Enalapril 10-40 Quinapril
10-80 Captopril 75-450 Perindopril 4-16 Trandolapril 1-8 Moexipril
7.5-30 Angiotensin-Receptor Blockers Eprosartan 400-600 Valsartan
80-320 Olmesartan 20-40 Candesartan 8-32 Telmisartan 20-80 Losartan
25-100 Irbesartan 75-300
Study Objectives and Outcome Measures
[0288] The primary objective of this proof-of-concept study is to
measure complete renal response (CRR) at 52 weeks with
administration of obinutuzumab. The ability of obinutuzumab plus
MMF to achieve a CRR at week 52 is compared to placebo plus MMF and
assessed by improvements in renal function, urinary sediment, and
proteinuria. Secondary objectives include evaluations of the safety
of obinutuzumab in this patient population, the ability of
obinutuzumab to induce an overall response (CRR+PRR) at Week 52,
the ability of obinutuzumab to improve time-to-response (CRR+PRR)
over the course of 52 weeks, and the ability of obinutuzumab to
improve biomarkers of LN disease activity (e.g., reduced anti-dsDNA
antibody levels, increased C3 and C4 levels; see Tew, G. W. et al.
(2010) Lupus 19:146-157).
[0289] The primary efficacy outcome measure is the proportion of
subjects who achieve a CRR, evaluated at 52 weeks. In this study,
CRR is defined by attainment of all of the following:
(a) Normalization of serum creatinine as evidenced by the
following: [0290] (i) Serum creatinine less than or equal to the
upper limit of normal (ULN) range of central laboratory values if
the baseline (Day 1) is not within the normal range of the central
laboratory values; and [0291] (ii) Serum creatinine.ltoreq.15%
above baseline and less than or equal to the ULN range of central
laboratory values if baseline (Day 1) serum creatinine is within
the normal range of the central laboratory values; (b) Inactive
urinary sediment (as evidenced by <10 RBCs/high-power field
(HPF) and the absence of red cell casts); and (c) Urinary protein
to creatinine ratio<0.5.
[0292] Any patient who switches to rescue medication prior to Week
52 is considered a non-responder. The proportions of patients
achieving CRR across treatment groups is compared using a
Cochrane-Mantel-Haenzel (CMH) test with race
(Afro-Caribbean/African-American versus others) and region (United
States versus non-United States) as stratification factors. If the
test result is in favor of the obinutuzumab group at
.alpha.<0.1-level (one-sided), a shift toward better renal
response associated with the obinutuzumab group is concluded.
[0293] The secondary efficacy outcome measures are the
following:
(a) Proportional analysis of patients who achieve an overall
response at Week 52 (CRR+PRR); (b) Time to overall response
(CRR+PRR) over the course of 52 weeks; (c) Percent reduction or
increase from baseline and mean and median assessments of
biomarkers of LN disease activity (e.g., reduction in anti-dsDNA
antibody levels, increased C3 and C4 levels); (d) Proportion of
patients that achieve a PRR at week 52 as defined by attainment of
all of the following:
[0294] (i) serum creatinine.ltoreq.15% above baseline levels;
[0295] (ii) RBCs/HPF.ltoreq.50% above baseline and no red cell
casts;
[0296] (iii) 50% improvement in the urine protein to creatinine
ratio, with one of the following conditions met: [0297] (A) if the
baseline urine protein to creatinine ratio is .ltoreq.3.0, then a
urine protein to creatine ratio of <1.0; [0298] (B) if the
baseline protein to creatinine ratio is >3.0, then a urine
protein to creatinine ratio of <3.0; (e) Proportion of patients
who achieve a CRR at Week 24; (f) Time to CRR, over the course of
52 weeks; (g) Proportion of patients that achieve a modified CRR
(mCRR1) at Week 52 employing the primary-efficacy measure
definition and removing the urinary sediment analysis criteria
[0299] mCRR1 refers to attainment of normalization of serum
creatinine as evidenced by the following: [0300] (i) Serum
creatinine less than or equal to the ULN range of central
laboratory values; [0301] (ii) Serum creatinine.ltoreq.15% above
baseline and less than or equal to the ULN range of central
laboratory values if baseline (Day 1) serum creatinine is within
the normal range of the central laboratory values; [0302] (iii)
Urinary protein to creatinine ratio<0.5; (h) Proportion of
patients that achieve a second CRR (mCRR2) at Week 52 as defined by
attainment of the following:
[0303] (i) Normalization of serum creatinine as evidenced by the
following: [0304] (A) Serum creatinine.ltoreq.the ULN range of
central laboratory values; [0305] (B) Serum creatinine.ltoreq.15%
above baseline if baseline (Day 1) serum creatinine is above the
normal range of the central laboratory values OR S.ltoreq.the ULN
range of central laboratory values if baseline (Day 1) serum
creatinine is within the normal range of the central laboratory
values;
[0306] (ii) Inactive urinary sediment (as evidenced by <10
RBCs/HPF and the absence of red cell casts);
[0307] (iii) Urinary protein to creatinine ratio<0.5.
[0308] The pharmacodynamic (PD) objective is to compare changes in
CD19+ B cells in the peripheral blood following treatment with
obinutuzumab versus placebo. Levels of circulating CD19+ B-cells
are measured at screening and Days 15, 28, 84, 168, 364, and
728.
[0309] The pharmacokinetic (PK) objectives are to characterize the
pharmacokinetics of obinutuzumab in the LN population and to assess
potential PK interactions between obinutuzumab and concomitant
medications, including mycophenolate mofetil (MMF). Non-linear
mixed-effects modeling (with software NONMEM) is used to analyze
the dose-concentration-time data of obinutuzumab. The PK profile
data is used to further develop a PK model, including the effect of
major covariates (e.g., sex, race/ethnicity, weight, biochemical
and hematological parameters at baseline, degree of underlying
disease), on the main parameters (e.g., clearance). Derivation of
individual measures of exposure, such as area under the
concentration-time curve (AUC) and maximum concentration observed
(C.sub.max) depend on the final PK model used. Serum obinutuzumab
is summarized (mean, minimum, maximum, SD, and geometric mean) and
reported.
[0310] The exploratory objectives for the study include evaluation
of pre-dose levels of exploratory biomarkers (including but not
limited to B-cell subsets and levels of protein and/or mRNA in
serum, blood, and urine) and potential associations with outcome,
evaluation of changes in exploratory biomarkers (including but not
limited to B-cell subsets and levels of protein and/or mRNA in
serum, blood, and urine) over time in patients dosed with
obinutuzumab versus placebo, evaluation of the occurrence of
extrarenal flares, evaluation of the impact of therapy on patient
and physician-reported outcomes, and assessment of renal biopsy
histopathology (e.g., for the presence of CD19+ B cells at the
screening and/or subsequent biopsies). The exploratory outcome
measures include:
(a) levels of circulating B-cell subsets at Screen and Days 15, 28,
84, 168, 364, and 728; (b) levels of exploratory biomarkers
(including but not limited to B-cell subsets and levels of protein
and/or mRNA in serum, blood, and urine) at Screen and Days 1, 15,
28, 84, 168, 252, 364, 532, and 728; (c) proportion of patients
experiencing a Systemic Lupus Erythematosus Disease Activity Index
(SLEDAI)-2K flare; (d) proportion of patients experiencing a renal
flare over 52 weeks and 104 weeks; (e) proportion of patients
achieving CRR, mCRR1, mCRR2 at additional timepoints (including
Week 12 and 36); (f) Physician's Global Assessment (visual analog
scale captured in screening, at the baseline visit, and at several
timepoints during study conduct); and (g) renal biopsy
evaluations.
Laboratory, Biomarker, and Other Biological Samples
[0311] The following laboratory assessments are recorded during the
study:
(a) Hematology: hemoglobin, hematocrit, RBC, mean corpuscular
volume, mean corpuscular hemoglobin, WBC (absolute and
differential), and quantitative platelet count; (b) Blood
chemistry: AST/SGOT, ALT/SGPT, alkaline phosphatase, amylase,
lipase, total protein, albumin, cholesterol, total bilirubin, urea,
uric acid, creatinine, random glucose, potassium, sodium, chloride,
calcium, phosphorus, lactic dehydrogenase, CPK, and triglycerides;
(c) Urinalysis: dipstick for blood, nitrate, protein, and glucose
and urine microscopy; (d) 24-hour urine collection (analyzed for
total protein, total creatinine, and creatinine clearance) to be
performed at randomization and at Months 3, 6, 9, and 12; (e) Flow
cytometry: B cell (including CD19, CD27, CD38, and IgD), T cell
(CD3, CD4, CD8), and NK cells (CD16, CD56); (f) Autoantibody
profile: anti-nuclear antibody (ANA), anti-dsDNA, anti-Sm,
anti-RNP, anti-Ro, anti-La, and anti-C1q; (g) Anti-dsDNA antibody:
measured by ELISA at all visits as part of SLEDAI-2K assessment;
(h) Quantitative immunoglobulin: total Ig levels including IgG,
IgM, and IgA isotypes;
(i) Complement: C3, C4, and CH50;
[0312] (j) Antibody titers: antibody titers to common antigens
(rubella, tetanus, influenza, S. pneumoniae); and (k) Pregnancy
test: urine pregnancy test performed at screening and prior to each
study drug infusion. Infusion is not administered unless test is
negative. At all other timepoints, urine pregnancy test is
performed on the basis of menstrual history and pregnancy risk.
[0313] The following samples are sent for analysis: cells from
blood and urine for B-cell and lupus-related biomarkers (including
but not limited to CD19+ B cells and mRNA associated with B-cell
activity), serum and urine for B-cell and lupus-related biomarkers
(including but not limited to B-cell activating factor or BAFF),
and renal biopsy slides for immunohistopathology assessment.
Infusions
[0314] Prior to each infusion of either study drug or placebo,
patients receive prophylactic treatment with acetaminophen
(650-1000 mg) and diphenhydramine (50 mg; or equivalent dose of a
similar agent) by mouth, given 30-60 minutes before the start of
the infusion period. The patients who are receiving obinutuzumab
receive 80 mg methylprednisolone IV and patients who are receiving
placebo receive placebo-methylprednisolone IV given 30-60 minutes
before the start of the obinutuzumab/placebo infusion. If a patient
experiences a mild infusion-related reaction (IRR) that is deemed
by the investigator to be clinically significant, the infusion rate
should be reduced to half of the initial infusion rate (in
compliance with non-Hodgkin's lymphoma protocol infusion rates and
schedules). After the reaction has resolved, the infusion should be
kept at the reduced rate for an additional 30 minutes. If the
reduced rate is tolerated, then the infusion rate may be increased
to the next closest rate on the infusion schedule. Patients who
experience a severe IRR should have their infusion interrupted
immediately and should receive aggressive symptomatic treatment.
The infusion should not be restarted until all of the symptoms have
disappeared. Upon restarting the infusion, the rate should be half
of that which precipitated the reaction. Instructions for
administration of obinutuzumab infusions are provided in Table 6
below.
TABLE-US-00013 TABLE 6 Administration of obinutuzumab infusions.
First Infusion (Day 1) Subsequent Infusions Begin infusion at an
initial rate of 50 mg/hr. If a patient experienced an infusion
reaction If no infusion reaction occurs, increase the during the
prior infusion, start at the same infusion rate in 50-mg/hr
increments every rate as the first infusion (50 mg/hr) and 30
minutes to a maximum of 400 mg/hr. follow those directions as
noted. If an infusion reaction develops, stop or slow If the
patient tolerated the prior infusion the infusion. Administer
infusion-reaction well, begin infusion at a rate of 100 mg/hr.
medications and supportive care in If no infusion reaction occurs,
increase the accordance with institutional protocol. infusion rate
in 100-mg/hr increments every Resume the infusion at a 50%
reduction in 30 minutes to a maximum of 400 mg/hr. rate (the rate
being used at the time that the If an infusion reaction develops,
stop or slow hypersensitivity or infusion-related reaction the
infusion. Administer infusion-reaction occurred) if the reaction
has resolved, medications and supportive care in accordance with
institutional protocol. Resume the infusion at a 50% reduction in
rate (the rate being used at the time that the hypersensitivity or
infusion-related reaction occurred) if the reaction has
resolved.
[0315] All renal biopsies and reports that are obtained as part of
study entry are photomicrographed and sent to an online central
reading portal for oversight of the histopathologic assessment
performed by the local renal histopathologist. An expert panel
assesses these biopsies and adjudicates a final interpretation.
Every effort is made to complete this process while patients are in
screening but is not mandatory for completion of screening
activities. All new biopsies obtained during screening or during
the study are processed in a manner to enable immunohistochemical
staining of the tubulointerstitium for the presence of B cells. The
study encourages but does not mandate the performance of repeat
renal biopsies for patients that have not achieved a CRR, and seeks
to enrich for study centers that perform repeat renal biopsies.
Example 2
Obinutuzumab Outperforms Rituximab at Inducing B-Cell Cytotoxicity
in Rheumatoid Arthritis and Systemic Lupus Erythematosus Patient
Samples Through Fc Gamma Receptor-Dependent and Independent
Effector Mechanisms
[0316] A proportion of Rheumatoid Arthritis (RA) and Systemic Lupus
Erythematosus (SLE) patients treated with standard doses of
rituximab (RTX) display inefficient B cell deletion and poor
clinical responses which can be augmented by delivering higher
doses, indicating that standard-dose RTX is a sub-optimal therapy
in these patients. To investigate whether better responses could be
achieved with other anti-CD20 mAbs, a comparison was made between
RTX with Obinutuzumab (OBZ), a new-generation, glycoengineered type
II anti-CD20 mAb in a series of in vitro assays measuring B cell
cytotoxicity in SLE and RA samples. It was found that OBZ was at
least 2-fold more efficient than RTX at inducing B-cell
cytotoxicity in in-vitro whole blood assays. Dissecting this
difference it was found that RTX elicited more potent
complement-dependent cellular cytotoxicity (CDC) than OBZ. In
contrast, OBZ was more effective at evoking Fc gamma receptor
(Fc.gamma.R)-mediated effector mechanisms including activation of
NK cells and neutrophils. OBZ was also more efficient at inducing
direct cell death. This was true for all CD19+ B-cells as a whole
and in naive (IgD+CD27-); and switched (IgD-CD27+) memory B-cells
specifically, a higher frequency of which is associated with poor
clinical response after RTX.
Materials and Methods
Patients
[0317] All participants of this study provided consent according to
the declaration of Helsinki approved by the local research ethics
committee. All patients with RA satisfied the American College of
Rheumatology (ACR)/European League Against Rheumatism
classification criteria (Aletaha D. et al. 2010 Ann Rheum Dis.
2010; 69(9):1580-8) and all patients with SLE met the ACR
classification criteria (Petri M. et al. Arthritis Rheum. 2012;
64(8):2677-86).
Antibodies and Reagents
[0318] Anti-CD20 mAbs used in the studies included RTX, OBZ and
non-glycoengineered, wild type glycosylated OBZ (OBZ.sub.Gly) and
in some experiments OBZ with a mutated Fc portion (P329G LALA) that
does not engage any Fc related effector functions (Herter S. et al.
Cancer Research. 2015; 75(15 Supplement):2460), OBZ-PG LALA. Roche
Innovation Center Zurich, Switzerland generated all anti-CD20 mAbs
except RTX, which was a kind gift from the pharmacy of University
College Hospital, U.K. AT10, an Fc.gamma.RII antagonist (Greenman
J. et al. Mol Immunol. 1991; 28(11):1243-54), was produced
in-house.
Flow Cytometry and B Cell Isolation
[0319] Fluorochrome-conjugated mAb were procured from Becton
Dickinson biosciences or Biolegend, U.K.): CD3 (phycoerythrin
[PE]-Cy 7), CD15 (fluorescein isothiocyanate, FITC): CD16
(Allophycoyanin, APC), CD19 (Alexa Fluor 700), CD45 (PE), CD56
(PE), CD 107a (Brilliant Violet 421), CD11b (PE), CD62L (APC),
propidium iodide and Annexin V (FITC). Flow cytometry was performer
using a Becton Dickinson LSR Fortessa cell analyzer. Lymphocytes
were identified based on forward- and side-scatter characteristics.
B cells were identified as CD19+ or CD20+, T cells as CD3+ and NK
cells as CD3-56+. Neutrophils were identified based on forward- and
side-scatter characteristics and CD15 positively. The mean
fluorescent intensity (MFI) of CD11b and CD62L in samples incubated
with mAbs was compared with that in samples incubated without
antibodies.
[0320] In all experiments, peripheral blood mononuclear cells
(PBMC) were separated from whole blood samples by Ficoll-Hypaque
density gradient and B-cells were isolated from PBMC using
EasySep.TM. Human B Cell Enrichment Kit (Cambridge, U.K.).
Whole Blood B-Cell Depletion Assays
[0321] Briefly, 300 .mu.l of freshly drawn whole blood
anticoagulated with heparin was incubated with or without mAbs at 1
.mu.g/mL for 24 hours at 37.degree. C. and 5% CO.sub.2. Samples
were then stained with anti-CD3, anti-CD19 and anti-CD45 before
lysing red cells and analyzing on flow cytometer, as described
previously (Reddy V. et al. Arthritis & rheumatology. 2015;
67(8):2046-55). The % B-cell depletion was calculated from the
proportion of B cells to T cells remaining after treatment and
defined as the cytotoxicity index (CTI) as described previously
(Mossner E. et al. Blood. 2010; 115(22):4393-402, and Reddy V. et
al. Arthritis & rheumatology. 2015; 67(8):2046-55).
Surface Fluorescence-Quenching Assays
[0322] Surface fluorescence-quenching assays were performed as
described previously (Beers S. A. et al. Blood. 2008;
112(10):4170-7, and Reddy V. et al. Arthritis & rheumatology.
2015; 67(8):2046-55) to assess internalization of mAbs by B-cells.
Isolated B-cells were incubated for 6 hours with Alexa-488
conjugated mAbs at a concentration of 5 .mu.g/mL before analyzing
by flow cytometry.
Complement-dependent Cellular Cytotoxicity Assays
[0323] CDC assays were performed as previously described (Cragg M.
S. et al. Blood. 2004; 103(7):2738-43). Isolated B cells were
incubated with mAbs at a concentration of 10 .mu.g/mL for 30
minutes at 37.degree. C. and 5% CO.sub.2. Samples were stained with
fluorescence conjugated anti-CD19 antibodies, Annexin V (Av) and
propidium iodide (PI) and the frequency of CD19+Av+PI+ cells
assessed by flow cytometry. Freshly collected normal healthy human
serum was used as a source of complement. To define the activity
relating to complement, part of the serum was heat inactivated
(HIS) at 56.degree. C. for 30 minutes. The ability of mAbs to
activate complement and lyse target cells was assessed by the
relative frequency of CD19+Av+PI+ cells in samples incubated either
with normal healthy serum or HIS.
Direct Cell Death
[0324] Isolated B-cells were incubated in RPMI supplemented with
10% heat inactivated foetal calf serum with or without mAbs at a
concentration of 10 .mu.g/mL for 6 hours at 37.degree. C. and 5%
CO.sub.2. The frequency of CD19+Av+cells in samples with mAbs
compared with that in samples without mAbs represented the ability
of mAbs to induce direct cell death.
NK Cell Degranulation Assays
[0325] NK cell degranulation was assessed using samples from the
whole blood B-cell depletion assay by measuring the expression of
CD107a or LAMP-1 (a lysosome associated membrane protein 1), which
is up regulated upon activation of NK cells and correlates with NK
cell mediated ADCC (Alter G. et al. J Immunol Methods. 2004;
294(1-2):15-22, and Aktas E. et al. Cell Immunol. 2009;
254(2):149-54). Therefore, the frequency of CD3-56+107a+ NK cells
in samples with mAbs were compared with that in samples incubated
without mAbs. Activation of NK cells is associated with an
increased activity of metalloproteinase, which cleaves CD16
reducing its expression upon NK cell activation (Romee R. et al.
Blood. 2013; 121(18):3599-608). Therefore the extent of CD16 loss
was also used as an indirect measure of NK cell activation
(Grzywacz B. et al. Leukemia. 2007; 21(2):356-9; author reply 9,
and Bowles J. A. et al. Blood. 2006; 108(8):2648-54).
Neutrophil Activation Assays
[0326] Neutrophil activation was assessed in the whole blood assay
by measuring increases in CD11b or decreases in CD62L on
CD15+neutrophils by flow cytometry (Golay J. et al. Blood. 2013;
122(20):3482-91, and Wittmann S. et al. Cytometry A. 2004;
57(1):53-62). The ability of mAbs to induce neutrophil activation
was assessed by comparing the mean fluorescent intensity (MFI) of
CD11b and CD62L on CD15+neutrophils in samples incubated with or
without mAbs.
Statistical Analysis
[0327] Data was analyzed using Graph Pad Prism Software version
5.0. Mann Whitney test or Wilcoxon matched-pairs signed rank test
were used to compare between groups as appropriate. Spearman
correlation coefficient was used to analyze for correlation.
Results
[0328] Type II mAbs are More Efficient than Type I at Inducing
B-Cell Cytotoxicity
[0329] To assess the effect of Type I and II mAbs on B cell
cytotoxicity in RA and SLE samples, whole blood B-cell depletion
assays were performed as described previously (Reddy V. et al.
Arthritis & rheumatology. 2015; 67(8):2046-55). OBZ was
>2-fold more efficient than RTX at deleting B-cells from
patients with RA (n=31) and SLE (n=34) with both non-glycomodified
OBZ.sub.Gly and OBZ more efficient than RTX in all samples tested
(FIG. 2A). In both RA and SLE, the median CTI of OBZ was
significantly greater than the CTI of OBZ.sub.Gly and RTX and the
CTI of OBZ.sub.Gly was significantly higher than the CTI of RTX in
both RA and SLE. In RA, the median (interquartile range) CTI of
RTX, OBZ.sub.Gly and OBZ was 29 (13-50), 60 (47-70) and 67 (60-77),
respectively and in SLE was 19 (11-39), 40 (31-53) and 59 (52-70),
respectively. Thus, in both RA and SLE, there was a hierarchy of
mAb-induced B cell depletion: RTX<OBZ.sub.Gly<OBZ. The
remarkable inter-sample variability in B-cell depletion,
particularly with RTX was also noted. The superior efficiency of
OBZ.sub.Gly (having a non-glycomodified Fc similar to RTX) suggests
that its type II nature accounts for the difference between the two
types of mAbs in the efficiency of B-cell depletion in the whole
blood assay; whereas the increased efficiency of OBZ compared to
OBZ.sub.Gly is attributable to afucosylation of the Fc portion.
B-Cells Internalize RTX More Rapidly than OBZ
[0330] Next, it was investigated whether the superior efficiency of
type II mAbs in B cell depletion was consistent with their type II
nature, and so it assessed whether B-cells from patients with RA
and SLE internalized RTX to a greater extent than OBZ. It was found
that RTX internalized more extensively than OBZ after 6 hours of
incubation with a median (range) percentage of surface accessible
RTX vs OBZ of: 55 (51-57) versus 83 (81-84), respectively in RA
(n=5); and 60 (49-77) versus 76 (70-80), respectively in SLE (n=8)
(FIG. 2B). To assess the role of Fc.gamma.RIIb in this
internalization, experiments were performed in the presence of the
Fc.gamma.RII-blocking mAb AT10, which partially inhibited
internalization of RTX and to a smaller extent, OBZ (FIG. 2B),
similar to previous observations using a non-glycomodified Type II
antibody variant of OBZ (Reddy V. et al. Arthritis &
rheumatology. 2015; 67(8):2046-55).
RTX is More Efficient than OBZ at Inducing Complement-Dependent
Cellular Cytotoxicity
[0331] The ability of these mAbs to elicit CDC was also
investigated. It was found that the frequency of lysed B cells
(CD19+Av+PI+) was significantly greater in samples incubated with
RTX in the presence of normal healthy serum (NHS) compared to heat
inactivated serum (HIS) with a median (range) difference of 10.9%
(8.1-21) whereas the difference for OBZ was 4.8% (0.9-6.5) (FIG.
2C). The mean.+-.SD fold increase in lysed cells in samples
incubated with NHS vs HIS was 1.9.+-.0.5 and 1.2.+-.0.2 for RTX and
OBZ, respectively (FIG. 2D). Thus, the data suggests that RTX was
superior to OBZ at evoking CDC.
OBZ is More Efficient than RTX at Activating NK Cells
[0332] These CDC results were consistent with the type I and II
nature of the mAbs but at odds with the superior efficiency of type
II mAbs in the whole blood assay. Next, the ability of the mAbs to
elicit Fc.gamma.R-mediated effector mechanisms was investigated;
first assessing NK activation in the whole blood B-cell depletion
assay. Gating as indicated in FIG. 3E, allowed assessment of NK
degranulation (CD107a increase) relative to expression of CD16. The
highest proportion of CD107a+NK (CD3-CD56+) cells was seen in the
CD56+CD16- fraction (FIG. 3A-3G) suggesting that degranulating NK
cells had down regulated CD16 as previously reported (Grzywacz B.
et al. Leukemia. 2007; 21(2):356-9; author reply 9).
[0333] Having established these parameters, equivalent assays were
performed comparing RTX and OBZ. After 24 hours of incubation in
the absence of mAbs there was no significant difference in the
frequency of NK cells, CD107a+NK cells, CD16+ NK cells or B-cells
between patients with RA (n=18) and SLE (n=23) (FIG. 4A). However,
the median (range) frequency of CD3-CD56+CD107a+ activated NK cells
was significantly higher in samples incubated with OBZ compared to
RTX 5.1% (1.9-22) vs 2.8% (0.3-14) and 5.5% (0.6-12) vs 4.3%
(1.2-8.9), respectively) but there was significantly lower median
(range) frequency of CD16+NK cells, in both RA and SLE 69 (36-94)
vs 89 (83-97) and 66 (42-91) vs 84 (61-95), respectively (FIG. 4B).
Also, there was a significantly higher fold-increase in the
frequency of CD3-CD56+CD107a+ NK cells in samples incubated with
OBZ compared to those incubated with RTX in SLE (FIG. 4B).
Furthermore, it was found that NK cell activation, as assessed by
either gain of CD107a or loss of CD16; or the fold increase in the
frequency of CD3-CD56+CD107a+ NK cells, was greater in RA compared
to SLE (FIG. 4B). NK cell activation, as assessed by the frequency
of CD3-CD56+CD107a+NK cells by RTX and OBZ, correlated
significantly with that in samples incubated without mAbs with
r.sup.2=0.89, p<0.05; r.sup.2=0.78, p<0.05, respectively, in
RA (FIG. 4C) and r.sup.2=0.52, p<0.05; r.sup.2=0.36, p<0.05,
respectively, in SLE (FIG. 4D). However, correlations were stronger
in RA compared to SLE. Taken together, these data suggest disease
related defects in the activation of NK cells in SLE may contribute
to inefficient B-cell depletion noted in whole blood depletion
assays (FIG. 2A) and that baseline activation status of NK cells
may influence response to activation by mAbs in both RA and SLE
(FIGS. 4B and 4C).
[0334] It was next investigated whether differential activation of
NK cells by RTX and OBZ was due to type I and type II
characteristics and/or due to the effect of Fc engineering using
either OBZ with wild-type glycosylation (OBZ.sub.Gly) or completely
lacking Fc.gamma.R engagement (OBZ-PG LALA), consequently,
less/in-efficient at inducing ADCC and CDC (Hessell A. J. et al.
Nature. 2007; 449(7158):101-4). Significant differences were not
found in the frequency of CD3-CD56+CD107a+ or CD3-CD56+CD16+NK
cells in samples incubated without mAbs compared to samples
incubated with OBZ-PG LALA in both RA (n=6) and SLE (n=12) showing
that Fc.gamma.R-engagement is essential as expected. In these
samples also OBZ was more efficient than OBZ.sub.Gly and RTX at
depleting B cells in the whole blood assay in both RA (n=18) and
SLE (n=23) (FIG. 5A), but an increasing hierarchy was noted in the
frequency of, and fold-increase in, CD3-CD56+CD107a+ NK cells as
follows: no mAbs=OBZ-PG LALA<RTX<OBZ (FIGS. 5B and 5C). The
frequency of CD3-CD56+CD16+NK cells was significantly lower in
samples incubated with OBZ when compared with other samples (FIG.
5D). The frequency of CD3-CD56+CD16+NK cells was also lower in
samples incubated with OBZ.sub.Gly compared to RTX in RA, but not
SLE (FIG. 5D).
[0335] Thus, the ability of mAbs to up-regulate the expression of
CD107a on CD3-CD56+ NK cells was greater in RA compared with SLE,
such that the mean fold difference in samples incubated with RTX,
OBZ-PG LALA, OBZ.sub.Gly and OBZ when compared with samples
incubated without mAbs was 1.2, 1.5, 1.9 and 3.1, respectively, in
RA and 1.5, 0.8, 1.4 and 1.8, respectively, in SLE (FIG. 5C).
Although the pattern of B-cell depletion achieved by mAbs in RA and
SLE (FIG. 5A) was similar to the pattern of NK cell activation by
mAbs (FIGS. 5B and 5D) there was no direct correlation between %
B-cell depletion achieved by mAbs and the frequency of
CD3-CD56+CD107a+ NK cells in individual samples (data not
shown).
OBZ is More Efficient than RTX at Activating Neutrophils
[0336] In addition to NK cells, neutrophils have also been proposed
as mAb effector cells (Golay J. et al. Blood. 2013;
122(20):3482-91). Therefore, next, the ability of mAbs to induce
neutrophil activation was assessed by measuring the expression of
CD11b and CD62L, as described previously (Wittmann S. et al.
Cytometry A. 2004; 57(1):53-62) and shown in FIG. 9. CD11b forms
part of the .beta. integrin (Mac-1) complex and several genetic
variants of this complex have been associated with lupus-related
phagocytic defects (Bologna L. et al. J Immunol. 2011;
186(6):3762-9). Upon neutrophil activation the surface expression
of CD11b is up regulated whereas the expression of the adhesion
molecule CD62L is down regulated (Golay J. et al. Blood. 2013;
122(20):3482-91, and Wittmann S. et al. Cytometry A. 2004;
57(1):53-62). It was found that the MFI of CD11b in samples
incubated with mAbs was significantly higher in both RA (n=10) and
SLE (n=22) (FIG. 6A) when compared with samples incubated without
mAbs. In both RA and SLE, significant correlations were noted
between the MFI of CD11b in samples incubated in the absence of
mAbs and that in samples incubated with RTX (r.sup.2=0.81, 0.82,
respectively) whereas significant correlation for OBZ was noted in
SLE (r.sup.2=0.81), but not RA (FIG. 6B). A hierarchy was also
noted in the ability of mAbs to up-regulate CD11b such that the MFI
of CD11b was lower in samples incubated with
RTX<OBZ.sub.Gly<OBZ, as in the case of NK cell activation.
Also, the MFI of CD62L was greater in samples incubated with
RTX>OBZ.sub.Gly>OBZ (FIG. 6C). In both RA and SLE,
significant correlations were noted between the MFI of CD62L in
samples incubated in the absence of mAbs and that in samples
incubated with RTX (r.sup.2=0.93, 0.91, respectively) and OBZ
(r.sup.2=0.64, 0.71, respectively) (FIG. 6D). Thus, the hierarchy
of mAbs in their ability to activate neutrophils was
OBZ>OBZ.sub.Gly>RTX. Taken together, these data suggested
that type II mAbs are superior to RTX in activating neutrophils in
the whole blood assay for both RA and SLE samples. OBZ-PG LALA did
not elicit significant changes for either marker in both RA (n=7)
and SLE (n=12) compared to samples incubated in the absence of
mAbs.
OBZ is More Efficient than RTX at Inducing Direct Cell Death
[0337] Next, direct cell death (DCD) was assessed, using the
Annexin V assay as shown in FIG. 10. The ability of OBZ to induce
direct cell death was greater than that of RTX for both CD19+ cells
as a whole and also B-cell subpopulations; IgD+CD27- naive cells
and IgD-CD27+ switched memory cells, FIG. 7A (RA, n=5 and SLE,
n=4). The proportion of Annexin V+ cells was highest for DN
cells>IgD+CD27+ unswitched memory cells>IgD-CD27+ switched
memory cells>IgD+CD27- naive cells, even in samples incubated
without mAbs. Nonetheless, OBZ was superior to RTX at inducing
DCD.
B-Cell Subpopulations: Expression of CD20, Fc.gamma.RIIb and
Internalization of mAbs
[0338] It was next investigated whether differences between B-cell
subpopulations in the expression of CD20, Fc.gamma.RIIb and/or
their ability to internalize mAbs provided explanations for the
differential sensitivity to mAb-induced DCD. B-cell subpopulations
displayed varying ability to internalize mAbs such that IgD-CD27+
switched memory cells internalized mAbs less than other B-cell
subpopulations; and IgD+CD27+ unswitched memory cells internalized
mAbs to a greater extent than other B-cell subpopulations.
Antagonizing the effects of Fc.gamma.RIIb with AT10 significantly
reduced internalization in both cases. When compared to naive and
IgD-CD27+ switched memory cells, IgD+CD27+ unswitched memory cells
had significantly greater expression of CD20 and Fc.gamma.RIIb and
displayed significantly greater ability to internalize mAbs whereas
naive and IgD-CD27+ switched memory cells had significantly lower
expression of CD20 and Fc.gamma.RIIb and displayed significantly
lower levels of internalization. DN cells had remarkably variable
levels of expression of CD20 and Fc.gamma.RIIb, but internalized
RTX to a significantly greater extent than IgD-CD27+ switched
memory cells. B cells from both RA and SLE samples consistently
displayed low levels of OBZ internalization. Taking these data
together, there was no clear relationship between the
susceptibility of B-cell subpopulations to mAb-induced DCD and the
ability to internalize mAbs or to express CD20 or
Fc.gamma.RIIb.
[0339] Here, it was shown that Obinutuzumab, a type II anti-CD20
mAb with a glycomodified Fc demonstrated at least 2-fold greater
potency at deleting B-cells from whole blood samples of patients
with both RA and SLE compared to the RTX. This increased activity
of OBZ was affected predominantly through Fc gamma receptor
(Fc.gamma.R)-mediated effector mechanisms and DCD. In contrast, RTX
recruited complement more efficiently for CDC, but was rapidly
internalized and significantly less efficient at evoking ADCC and
DCD. The subsequent analysis revealed that the expression of the
CD20 target molecule was less on IgD-CD27+ switched memory and DN
cells; perhaps accounting for their relative resistance to removal
by RTX.
Sequence CWU 1
1
4316PRTArtificial SequenceSynthetic Construct 1Gly Tyr Ala Phe Ser
Tyr1 5 28PRTArtificial SequenceSynthetic Construct 2Phe Pro Gly Asp
Gly Asp Thr Asp1 5 310PRTArtificial SequenceSynthetic Construct
3Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr1 5 10 416PRTArtificial
SequenceSynthetic Construct 4Arg Ser Ser Lys Ser Leu Leu His Ser
Asn Gly Ile Thr Tyr Leu Tyr1 5 10 15 57PRTArtificial
SequenceSynthetic Construct 5Gln Met Ser Asn Leu Val Ser1 5
69PRTArtificial SequenceSynthetic Construct 6Ala Gln Asn Leu Glu
Leu Pro Tyr Thr1 5 7119PRTArtificial SequenceSynthetic Construct
7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 8115PRTArtificial SequenceSynthetic Construct
8Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His
Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu
Val Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val
115 9448PRTArtificial SequenceSynthetic Construct 9Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30
Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala 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 Asn Val Phe Asp Gly Tyr Trp
Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165
170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro225 230 235 240 Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400 Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 445 10219PRTArtificial SequenceSynthetic Construct
10Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His
Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu
Val Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215 11112PRTMus musculus 11Gly Pro Glu Leu
Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys1 5 10 15 Ala Ser
Gly Tyr Ala Phe Ser Tyr Ser Trp Met Asn Trp Val Lys Leu 20 25 30
Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Phe Pro Gly Asp 35
40 45 Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
Thr 50 55 60 Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met Gln Leu Thr
Ser Leu Thr65 70 75 80 Ser Val Asp Ser Ala Val Tyr Leu Cys Ala Arg
Asn Val Phe Asp Gly 85 90 95 Tyr Trp Leu Val Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ala 100 105 110 12103PRTMus musculus 12Asn
Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser1 5 10
15 Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu
20 25 30 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met
Ser Asn 35 40 45 Leu Val Ser Gly Val Pro Asp Arg Phe Ser Ser Ser
Gly Ser Gly Thr 50 55 60 Asp Phe Thr Leu Arg Ile Ser Arg Val Glu
Ala Glu Asp Val Gly Val65 70 75 80 Tyr Tyr Cys Ala Gln Asn Leu Glu
Leu Pro Tyr Thr Phe Gly Gly Gly 85 90 95 Thr Lys Leu Glu Ile Lys
Arg 100 13119PRTArtificial SequenceSynthetic Construct 13Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr Ser 20
25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser
Thr Ser Thr Ala 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 Asn Val Phe Asp Gly
Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 14119PRTArtificial SequenceSynthetic Construct 14Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser
20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr
Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala 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 Asn Val Phe Asp
Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 15119PRTArtificial SequenceSynthetic Construct
15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 16119PRTArtificial SequenceSynthetic Construct
16Gln 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 Val Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 17119PRTArtificial SequenceSynthetic Construct
17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 18119PRTArtificial SequenceSynthetic Construct
18Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 19119PRTArtificial SequenceSynthetic Construct
19Gln 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 Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 20119PRTArtificial SequenceSynthetic Construct
20Gln 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 Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 21119PRTArtificial SequenceSynthetic Construct
21Gln 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 Tyr
Ser 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 22119PRTArtificial
SequenceSynthetic Construct 22Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val Lys Ile Ser Cys Lys
Val Ser Gly Tyr Thr Phe Thr Tyr Ser 20 25 30 Trp Met His Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile
Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Glu Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Thr Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly
Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
23119PRTArtificial SequenceSynthetic Construct 23Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Thr Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Ser 20 25 30
Trp Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Val Phe Asp Gly Tyr Trp
Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 24119PRTArtificial SequenceSynthetic Construct 24Gln Met Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr
Ser Thr Ala 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 Asn Val Phe Asp Gly Tyr
Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 25119PRTArtificial SequenceSynthetic Construct 25Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20
25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn
Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser
Thr Ser Thr Ala 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 Asn Val Phe Asp Gly
Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 26119PRTArtificial SequenceSynthetic Construct 26Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Tyr Ser
20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr
Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala 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 Asn Val Phe Asp
Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 27119PRTArtificial SequenceSynthetic Construct
27Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 28119PRTArtificial SequenceSynthetic Construct
28Glu Val Gln Leu Val Glu Ser Gly Ala Gly Leu Val Lys Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 29119PRTArtificial SequenceSynthetic Construct
29Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 30119PRTArtificial SequenceSynthetic Construct
30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Lys Lys Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 31119PRTArtificial SequenceSynthetic Construct
31Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ser1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 32119PRTArtificial SequenceSynthetic Construct
32Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 33119PRTArtificial SequenceSynthetic Construct
33Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala 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 Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 3419PRTArtificial SequenceSynthetic Construct
34Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1
5 10 15 Ala His Ser3525PRTArtificial SequenceSynthetic Construct
35Glu 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 20 25 3613PRTArtificial
SequenceSynthetic Construct 36Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val1 5 10 3732PRTArtificial SequenceSynthetic Construct
37Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln1
5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg 20 25 30 3811PRTArtificial SequenceSynthetic Construct 38Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ala1 5 10 3923PRTArtificial
SequenceSynthetic Construct 39Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
4015PRTArtificial SequenceSynthetic Construct 40Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10 15 4132PRTArtificial
SequenceSynthetic Construct 41Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr1 5 10 15 Leu Thr Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 4211PRTArtificial
SequenceSynthetic Construct 42Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg1 5 10 4322PRTArtificial SequenceSynthetic Construct 43Met
Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10
15 Phe Pro Gly Ala Arg Cys 20
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