U.S. patent application number 17/395366 was filed with the patent office on 2022-07-07 for combination therapy of a type ii anti-cd20 antibody with a selective bcl-2 inhibitor.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is ABBVIE INC., GENENTECH, INC., HOFFMANN-LA ROCHE INC.. Invention is credited to Sari L. Heitner Enschede, Wayne John Fairbrother, Rod A. Humerickhouse, Christian Klein, Andrew W. Roberts, Deepak Sampath, John F. Seymour.
Application Number | 20220211696 17/395366 |
Document ID | / |
Family ID | 1000006227188 |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220211696 |
Kind Code |
A1 |
Sampath; Deepak ; et
al. |
July 7, 2022 |
COMBINATION THERAPY OF A TYPE II ANTI-CD20 ANTIBODY WITH A
SELECTIVE BCL-2 INHIBITOR
Abstract
The present invention is directed to a combination therapy
involving a type II anti-CD20 antibody and a selective Bcl-2
inhibitor for the treatment of a patient suffering from cancer,
particularly, a CD20-expressing cancer.
Inventors: |
Sampath; Deepak; (South San
Francisco, CA) ; Klein; Christian; (Iffeldorf,
DE) ; Fairbrother; Wayne John; (South San Francisco,
CA) ; Enschede; Sari L. Heitner; (River Forest,
IL) ; Humerickhouse; Rod A.; (Highland Park, IL)
; Roberts; Andrew W.; (Melbourne, Victoria, AU) ;
Seymour; John F.; (Melbourne, Victoria, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC.
HOFFMANN-LA ROCHE INC.
ABBVIE INC. |
South San Francisco
Little Falls
North Chicago |
CA
NJ
IL |
US
US
US |
|
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
HOFFMANN-LA ROCHE INC.
Little Falls
NJ
ABBVIE INC.
North Chicago
IL
|
Family ID: |
1000006227188 |
Appl. No.: |
17/395366 |
Filed: |
August 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17187468 |
Feb 26, 2021 |
11110087 |
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17395366 |
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16827650 |
Mar 23, 2020 |
10993942 |
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17187468 |
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15365595 |
Nov 30, 2016 |
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16827650 |
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14020761 |
Sep 6, 2013 |
9539251 |
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15365595 |
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61698379 |
Sep 7, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/496
20130101 |
International
Class: |
A61K 31/496 20060101
A61K031/496 |
Claims
1. A method for the treatment of a cancer in a human in need
thereof comprising administering to said human an effective amount
of a GA101 antibody or
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyr-
an-4-yl)methyl)amino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods, followed by
co-administering an effective amount of said GA101 antibody and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyr-
an-4-yl)methyl)amino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods.
2.-29. (canceled)
Description
[0001] This application claims priority to U.S. Provisional No.
61/698,379, filed Sep. 7, 2012, the content of which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention is directed to a combination therapy
involving a type II anti-CD20 antibody and a selective Bcl-2
inhibitor for the treatment of a patient suffering from cancer,
particularly a CD20-expressing cancer.
BACKGROUND
[0003] The CD20 molecule (also called human B-lymphocyte-restricted
differentiation antigen or Bp35) 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; and Einfield, D. A., et al. EMBO
J. 7(3) (1988) 711-717). CD20 is found on the surface of greater
than 90% of B cells from peripheral blood or lymphoid organs and is
expressed during early pre-B cell development and remains until
plasma cell differentiation. CD20 is present on both normal B cells
as well as malignant B cells. In particular, CD20 is expressed on
greater than 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson,
K. C., et al., Blood 63(6) (1984) 1424-1433) but is not found on
hematopoietic stem cells, pro-B cells, normal plasma cells, or
other normal tissues (Tedder, T. F., et al., J. Immunol. 135(2)
(1985) 973-979).
[0004] The 85 amino acid carboxyl-terminal region of the CD20
protein is located within the cytoplasm. The length of this region
contrasts with that of other B cell-specific surface structures
such as IgM, IgD, and IgG heavy chains or histocompatibility
antigens class 11 a or chains, which have relatively short
intracytoplasmic regions of 3, 3, 28, 15, and 16 amino acids,
respectively (Komaromy, M., et al., NAR 11 (1983) 6775-6785). Of
the last 61 carboxyl-terminal amino acids, 21 are acidic residues,
whereas only 2 are basic, indicating that this region has a strong
net negative charge. The GenBank Accession No. is NP-690605. It is
thought that CD20 might be involved in regulating an early step(s)
in the activation and differentiation process of B cells (Tedder,
T. F., et al., Eur. J. Immunol. 16 (1986) 881-887) and could
function as a calcium ion channel (Tedder. T. F., et al., J. Cell.
Biochem. 14D (1990) 195).
[0005] There exist two different types of anti-CD20 antibodies
which differ significantly in their mode of CD20 binding and
biological activities (Cragg, M. S., et al., Blood 103 (2004)
2738-2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052).
Type I antibodies, as e.g. rituximab, are potent in complement
mediated cytotoxicity, whereas type 11 antibodies, as e.g.
Tositumomab (B1), 11B8, AT80 or humanized B-Ly1 antibodies,
effectively initiate target cell death via caspase-independent
apoptosis with concomitant phosphatidylserine exposure.
[0006] The shared common features of type I and type 11 anti-CD20
antibodies are summarized in Table 1 below.
TABLE-US-00001 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 lgG1
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
[0007] The Bcl-2 family of proteins regulates programmed cell death
triggered by developmental cues and in response to multiple Stress
signals (Cory. S., and Adams, J. M., Nature Reviews Cancer 2 (2002)
647-656; Adams, Genes und Development 17 (2003) 2481-2495; Danial,
N. N., and Korsmeyer, S. J., Cell 116 (2004) 205-219). Whereas cell
survival is promoted by Bcl-2 itself and several close relatives
(Bcl-xL, Bcl-W, Mcl-1 and Al), which bear three or four conserved
Bcl-2 homology (BH) regions, apoptosis is driven by two other
sub-families. The initial signal for cell death is conveyed by the
diverse group of B13-only proteins, including Bad, Bid, Bim, Puma
and Noxa, which have in common only the small BH3 interaction
domain (Huang and Strasser, Cell 103 (2000) 839-842). However, Bax
or Bak, multi-domain proteins containing BH1-BH3, are required for
commitment to cell death (Cheng, et al., Molecular Cell 8 (2001)
705-711; Wei, M. C., et al., Science 292 (2001) 727-730; Zong, W.
X., et al., Genes and Development 15 148 (2001) 1-1486). When
activated, they can permeabilize the outer membrane of mitochondria
and release pro-apoptogenic factors (e.g. cytochrome C) needed to
activate the caspases that dismantle the cell (Wang, K., Genes and
Development 15 (2001) 2922-2933; (Adams, 2003 supra); Green, D. R.,
and Kroemer, G., Science 305 (2004) 626-629).
[0008] Interactions between members of these three factions of the
Bcl-2 family dictate whether a cell lives or dies. When BH3-only
proteins have been activated, for example, in response to DNA
damage, they can bind via their BH3 domain to a groove on their
pro-survival relatives (Sattler, et al., Science 275 (1997)
983-986). How the BH3-only and Bcl-2-like proteins control the
activation of Bax and Bak, however, remains poorly understood
(Adams, 2003 supra). Most attention has focused on Bax. This
soluble monomeric protein (Hsu, Y. T., et al., Journal of
Biological Chemistry 272 (1997) 13289-1 3834; Wolter, K. G., et
al., Journal of Cell Biology 139 (1997) 1281-92) normally has its
membrane targeting domain inserted into its groove, probably
accounting for its cytosolic localization (Nechushtan, A., et al.,
EMBO Journal 18 (1999) 2330-2341; Suzuki, et al., Cell 103 (2000)
645-654; Schinzel, A., et al., J Cell Biol 164 (2004) 1021-1032).
Several unrelated peptides/proteins have been proposed to modulate
Bax activity, reviewed in Lucken-Ardjomande, S., and Martinou, J.
C., J Cell Sci 118 (2005) 473-483, but their physiological
relevance remains to be established. Alternatively, Bax may be
activated via direct engagement by certain BH3-only proteins
(Lucken-Ardjomande, S., and Martinou, J. C, 2005 supra), the best
documented being a truncated form of Bid, tBid (Wei, M. C., et al.,
Genes und Development 14 (2000) 2060-2071; Kuwana, T., et al., Cell
111 (2002) 331-342; Roucou, X., et al., Biochemical Journal 368
(2002) 915-921; Cartron, P. F., et al., Mol Cell 16 (2004)
807-818). As discussed elsewhere (Adams 2003 supra), the oldest
model, in which Bcl-2 directly engages Bax (Oltvai, Z. N., et al.,
Cell 74 (1993) 609-619), has become problematic because Bcl-2 is
membrane bound while Bax is cytosolic, and their interaction seems
highly dependent on the detergents used for cell lysis (Hsu, Y. T.,
and Youle, 1997 supra). Nevertheless, it is well established that
the BH3 region of Bax can mediate association with Bcl-2 (Zha, H.,
and Reed, J., Journal of Biological Chemistry 272 (1997) 31482-88;
Wang, K., et al., Molecular und Cellular Biology 18 (1998)
6083-6089) and that Bcl-2 prevents the oligomerization of Bax, even
though no heterodimers can be detected (Mikhailov, V., et al.,
Journal of Biological Chemistry 276 (2001) 18361-18374). Thus,
whether the pro-survival proteins restrain Bax activation directly
or indirectly remains uncertain.
[0009] Although Bax and Bak seem in most circumstances to be
functionally equivalent (Lindsten, T., et al., Molecular Cell 6
(2000) 1389-1399; Wei, M. C., et al., 2001 supra), substantial
differences in their regulation would be expected from their
distinct localization in healthy cells. Unlike Bax, which is
largely cytosolic, Bak resides in complexes on the outer membrane
of mitochondria and on the endoplasmic reticulum of healthy cells
(Wei, M. C., et al., 2000 supra; Zong, W. X., et al., Journal of
Cell Biology 162 (2003) 59-69). Nevertheless, on receipt of
cytotoxic signals, both Bax and Bak change conformation, and Bax
translocates to the organellar membranes, where both Bax and Bak
then form homo-oligomers that can associate, leading to membrane
permeabilization (Hsu. Y. T., et al., PNAS 94 (1997) 3668-3672;
Wolter, K. G., et al., 1997 supra; Antonsson, B., et al., Journal
of Biological Chemistry 276 (2001) 11615-11623; Nechushtan, A., et
al., Journal of Cell Biology 153 (2001) 1265-1276; Wei, M. C., et
al., 2001 supra; Mikhailov, V., et al., Journal of Biological
Chemistry 278 (2003) 5367-5376).
[0010] There exist various Bcl-2 inhibitors, which all have the
same property of inhibiting prosurvival members of the Bcl-2 family
of proteins and are therefore promising candidates for the
treatment of cancer. Such Bcl-2 inhibitors are e.g. Oblimersen,
SPC-2996, RTA-402, Gossypol, AT-101, Obatoclax mesylate, A-371191,
A-385358, A-438744, ABT-737, ABT-263, AT-101, BL-11, BL-193,
GX-15-003, 2-Methoxyantimycin A.sub.3, HA-14-1, KF-67544,
Purpurogallin, TP-TW-37, YC-137 and Z-24, and are described e.g. in
Zhai, D., et al., Cell Death and Differentiation 13 (2006)
1419-1421.
[0011] Smith, M. R., et al, Molecular Cancer Therapeutics 3(12)
(2004) 1693-1699 and Ramanarayanan, J. et al., British Journal of
Haematology 127(5) (2004) 519-530, refer to a combination of a type
I anti-CD20 antibody (rituximab) with antisense Bcl-2
oligonucleotides (Oblimersen).
SUMMARY OF THE INVENTION
[0012] Provided herein are methods for the treatment of a patient
suffering from cancer, comprising co-administering, to a patient in
need of such treatment, a type 11 anti-CD20 antibody and a
selective Bcl-2 inhibitor. The co-administration may be
simultaneous or sequential in either order.
[0013] An example of the type II anti-CD20 antibody for use in the
present invention is a GA101 antibody.
[0014] In an embodiment, the type II anti-CD20 antibody has
increased antibody dependent cellular cytotoxicity (ADCC).
[0015] In an embodiment, at least 40% of the oligosaccharides of
the Fc region of said type II anti-CD20 antibody are
non-fucosylated.
[0016] In an embodiment, the selective Bcl-2 inhibitor is GDC-0199
(also known as ABT-199), or a pharmaceutically acceptable salt
thereof.
[0017] In an embodiment, the cancer is a non-solid tumor.
[0018] In certain embodiments, methods are provided for the
treatment of a cancer in a human in need thereof comprising
administering to said human a GA101 antibody and/or GDC-0199 in
multiple dosing cycles. In an embodiment, each dosing cycle of the
multiple dosing cycle is for at least 1 week. In an embodiment,
each dosing cycle of the multiple dosing cycle is for at 2, for at
least 3, for at least 4, for at least 5, or for at least 6
weeks.
[0019] In an embodiment wherein the GA101 antibody and GDC-0199 are
administered to the human in multiple dosing cycles, GA101 antibody
can, for example, be administered once per dosing cycle for one or
more dosing cycles of the multiple dosing cycles. The amount of
GA101 administered per dose can, for example, be between about 300
mg to about 3000 mg, or between about 500 mg to about 3000 mg, or
about 500 mg to about 1200 mg.
[0020] In an embodiment wherein the GA101 antibody and GDC-0199 are
administered to the human in multiple dosing cycles, GDC-0199 can,
for example, be administered each day in a dosing cycle for one or
more dosing cycles of the multiple dosing cycles. In an embodiment,
GDC-0199 is administered in fewer than all of the days of the
initial dosing cycle, and is administered each day of the dosing
cycles of the multiple dosing cycles that follow the initial dosing
cycle. The amount of GDC-0199 administered per day can be between
about 10 mg to about 1,000 mg, about 20 mg to about 800 mg, about
20 mg to about 500 mg, or between about 50 mg to about 300 mg.
[0021] In an embodiment, both the GA101 antibody and GDC-0199 are
administered to the patient in at least 2, 3, 4, 5, 6, 7, 8, or
more than 8, dosing cycles of the multiple dosing cycles.
[0022] In certain embodiments of the methods provided for the
treatment of a cancer in a human in need thereof comprising
administering to said human both a GA101 antibody and GDC-0199 in
multiple dosing cycles, following the last dosing cycle of multiple
dosing cycles, GDC-0199 alone can be administered to the human in
the absence of the GA101 antibody, or the GA101 antibody alone can
be administered to the patient in the absence of GDC-0199. For
instance, when GDC-0199 is administered alone to the human (e.g.,
following the last cycle of multiple dosing cycles wherein both
GDC-0199 and the GA101 antibody are administered to the human),
GDC-0199 can be administered to the human for at least 3, 4, 5, 6,
7, 8 or 9 days, or for 10 or more days, for 20 or more days, or for
30 or more days.
[0023] In yet another embodiment of the methods provided wherein a
GA101 antibody and GDC-0199 are administered to the patient in
multiple dosing cycles, the multiple dosing cycles comprise an
escalating dosing cycle in which GDC-0199 is administered to the
patient in escalating daily dose amounts during the escalating
dosing cycle.
DESCRIPTION OF THE FIGURES
[0024] FIG. 1. Antitumor activity of combined treatment of a type
II anti-CD20 antibody (GA101 antibody, in this case, obinutuzumab)
with a Bcl-2 inhibitor (ABT-199, a.k.a. GDC-0199). Arrows and line
under the x-axis indicates the days of dosing of GA101 and
GDC-0199, respectively.
[0025] FIG. 2. Exemplary dosing schedule for administering GDC-199
with obinutuzumab.
[0026] FIG. 3. Exemplary dosing schedule for administering GDC-199
with obinutuzumab.
[0027] FIG. 4. Antitumor activities of a type II anti-CD20 antibody
(obinutuzumab. a.k.a. RO5072759) used alone or in combination with
GDC-0199, and of a type I anti-CD20 antibody (rituximab) used alone
or in combination with GDC-0199 on human Z138 mantle cell lymphoma
cells.
[0028] FIG. 5. Results from xenograft model of aggressive lymphoma
demonstrating that single agent treatment with GDC-0199 following
combination of GDC-0199 with type II anti-CD20 antibody (GA101
antibody, in this case, obinutuzumab) delays tumor regrowth.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention relates to the method described
above.
[0030] The present invention also relates to a method for the
treatment of a human in need thereof comprising administering to
said human an effective amount of a GA101 antibody or
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods, followed by
co-administering an effective amount of said GA101antibody and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods.
[0031] The present invention also relates to a method for the
treatment of a human in need thereof comprising administering to
said human an effective amount of a GA101antibody or
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide for 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, or 14 days, followed by
co-administering an effective amount of said GA101antibody and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods.
[0032] The present invention also relates to a method for the
treatment of a human in need thereof comprising administering an
effective amount of said GA101antibody for 1, 2, 3, 4, 5, 6, or 7
days, followed by co-administering an effective amount of said
GA101antibody antibody and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods.
[0033] The present invention also relates to a method for the
treatment of a human in need thereof comprising administering an
effective amount of
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for 1, 2, 3, 4, 5, 6, or 7 days, followed
by co-administering an effective amount of said GA101antibody
antibody and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof for one or more dosing periods.
[0034] The present invention also relates to a method for the
treatment of a human in need thereof comprising administering an
effective amount of said GA101antibody once every dosing period for
1, 2, 3, 4, 5 or 6 cycles, followed by co-administering an
effective amount of said GA101antibody antibody once every dosing
period and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof one, two or three times a day for one or
more dosing periods.
[0035] The present invention also relates to a method for the
treatment of a human in need thereof comprising administering an
effective amount of
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof one, two or three times a day for 1, 2, 3,
4, 5 or 6 dosing periods, followed by co-administering an effective
amount of said GA101antibody once every dosing period and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof one, two or three times a day for one or
more dosing periods.
[0036] The present invention also relates to any one of the above
methods, wherein the effective amount of said GA101antibody is 500,
600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,
2900, or 3000 mg and the effective amount of
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,
500, 510, 520, 530 540, 550, 560, 570, 580, 590, 600, 610, 620,
630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,
890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000
mg.
[0037] The present invention also relates to any one of the above
methods, wherein the effective amount of said GA101antibody is 800,
900, 1000, 1100, 1200, 1300, 1400 or 1500 mg, and the effective
amount of
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof is 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290 or 300 mg.
[0038] The present invention also relates to any one of the above
methods, wherein when said cancer is NHL, the effective amount of
said GA101antibody is 800, 900, 1000, 1100, 1200, 1300, 1400 or
1500 mg, and the effective amount of
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof is 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,
530 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,
660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,
790, and 800 mg.
[0039] The present invention also relates to any one of the above
methods, wherein when said cancer is AML, the effective amount of
said GA101antibody is 800.900, 1000, 1100, 1200, 1300, 1400 or 1500
mg, and the effective amount of
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-21-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof is 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,
530 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,
660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,
790, and 800 mg.
[0040] The present invention also relates to any one of the above
methods, wherein said GA101antibody and
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyra-
n-4-yl)methylamino)phenylsulfonyl)benzamide or a pharmaceutically
acceptable salt thereof were co-administered sequentially during
each dosing period, and each dosing period is 5, 6, 7, 8, 9, 10,
11, 12, 13, or 14 days.
[0041] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0042] 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 and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, 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 but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0043] In one embodiment, said type II anti-CD20 antibody is a
monoclonal antibody.
[0044] The term "chimeric antibody" refers to a monoclonal antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are especially preferred. Such
murine/human chimeric antibodies are the product of expressed
immunoglobulin genes comprising DNA segments encoding murine
immunoglobulin variable regions and DNA segments encoding human
immunoglobulin constant regions. Other forms of "chimeric
antibodies" encompassed by the present invention are those in which
the class or subclass has been modified or changed from that of the
original antibody. Such "chimeric" antibodies are also referred to
as "class-switched antibodies." Methods for producing chimeric
antibodies involve conventional recombinant DNA and gene
transfection techniques now well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[0045] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann. L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric and bifunctional
antibodies.
[0046] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374). Based on
such technology, human antibodies against a great variety of
targets can be produced. Examples of human antibodies are for
example described in Kellermann, S. A., et al., Curr Opin
Biotechnol. 13 (2002) 593-597.
[0047] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NS0 or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions derived from
human germline immunoglobulin sequences in a rearranged form. The
recombinant human antibodies according to the invention have been
subjected to in vivo somatic hypermutation. Thus, the amino acid
sequences of the VH and VL regions of the recombinant antibodies
are sequences that, while derived from and related to human
germline VH and VL sequences, may not naturally exist within the
human antibody germline repertoire in vivo.
[0048] As used herein, "specifically binding" or "binds
specifically to" refers a binding that is sufficiently selective to
a target as to distinguish it from a binding to unwanted or
nonspecific targets (e.g., an antibody that specifically binds to a
human CD20). In one embodiment, a GA101 antibody of this invention
has a binding affinity for human CD20 (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.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M). In yet another embodiment, the KD is 10.sup.-10
mol/l or lower (e.g. 10.sup.-12 mol/l). The binding affinity is
determined with a standard binding assay, such as Scatchard plot
analysis on CD20 expressing cells.
[0049] The term "nucleic acid molecule", as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded. In one
embodiment, it is double-stranded DNA.
[0050] The "constant domains" are not involved directly in binding
the antibody to an antigen but are involved in the effector
functions (ADCC, complement binding, and CDC).
[0051] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91
(2007).)
[0052] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops
("hypervariable loops") and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). Exemplary HVRs herein include:
[0053] (a) hypervariable loops occurring at amino acid residues
26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and
96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987));
[0054] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56
(L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991));
[0055] (c) antigen contacts occurring at amino acid residues 27c-36
(L1), 46-55 (L2), 89-96 (L3), 30-35b (H1). 47-58 (112), and 93-101
(H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
[0056] (d) combinations of (a), (b), and/or (c), including HVR
amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),
26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102
(H3).
[0057] Synonyms of CD20, as recognized in the art, include
B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1. Leu-16,
Bp35, BM5, and LF5.
[0058] 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 2.
TABLE-US-00002 TABLE 2 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
[0059] One property of type I and type II anti-CD20 antibodies is
their mode of binding. Type I and type II anti-CD20 antibodies can
be classified by the ratio of the binding capacities to CD20 on
Raji cells (ATCC-No. CCL-86) of said anti-CD20 antibody compared to
rituximab.
[0060] The type II anti-CD20 antibodies have a ratio of the binding
capacities to CD20 on Raji cells (ATCC-No. CCL-86) of said
anti-CD20 antibody compared to rituximab of 0.3 to 0.6, and in one
embodiment of 0.35 to 0.55, and in another embodiment, 0.4 to 0.5.
Examples of such type II anti-CD20 antibodies include e.g.
tositumomab (B1 IgG2a), GA101antibody IgG1 (a chimeric humanized
IgG1 antibody as disclosed in WO 2005/044859), 11B8 IgG1 (as
disclosed in WO 2004/035607), and AT80 IgG1. In one embodiment,
said type II anti-CD20 antibody is a monoclonal antibody that binds
to the same epitope as GA101antibody (as disclosed in WO
2005/044859).
[0061] 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 .times. .times. of .times. .times. the .times. .times.
binding .times. .times. capacities .times. .times. to .times.
.times. CD .times. .times. 20 .times. .times. on .times. .times.
Raji .times. .times. cells .function. ( ATCC .times. - .times. No .
.times. CCL .times. - .times. 86 ) = MFI .function. ( Cy .times.
.times. 5 - anti - CD .times. .times. 20 .times. .times. antibody )
MFI ( Cy .times. .times. 5 - rituximab .times. Cy .times. .times. 5
- labeling .times. .times. ratio ( Cy .times. .times. 5 - rituximab
Cy .times. .times. 5 - labeling .times. .times. ratio .function. (
Cy .times. .times. 5 - anti - CD .times. .times. 20 .times. .times.
antibody ) ##EQU00001##
[0062] MFI is the mean fluorescent intensity. The "Cy5-labeling
ratio" as used herein means the number of Cy5-label molecules per
molecule antibody.
[0063] 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.
[0064] In one embodiment said type II anti-CD20 antibody, e.g., a
GA101antibody, has increased antibody dependent cellular
cytotoxicity (ADCC).
[0065] 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: [0066] 1) the assay uses target
cells that are known to express the target antigen recognized by
the antigen-binding region of the antibody; [0067] 2) the assay
uses human peripheral blood mononuclear cells (PBMCs), isolated
from blood of a randomly chosen healthy donor, as effector cells;
[0068] 3) the assay is carried out according to following protocol:
[0069] 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; [0070] 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; [0071]
iii) 100 microliters of the final target cell suspension above are
transferred to each well of a 96-well microtiter plate; [0072] 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; [0073] 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); [0074]
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); [0075] 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.; [0076] 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;
[0077] ix) the cell-free supernatant from each well is harvested
and the experimentally released radioactivity (ER) is quantified
using a gamma counter; [0078] 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): [0079] 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).
[0080] 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 Fe 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)).
[0081] 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.
[0082] 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 VII 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
[0083] 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) 975-981).
[0084] 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) 115-130; Jenkins,
N., et al., Nature Biotechnol. 14 (1996) 975-981). 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).
[0085] 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 Monison, 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 (1995) 813-822).
[0086] 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).
[0087] It was previously shown that overexpression in Chinese
hamster ovary (CHO) cells of B(1,4)-N-acetylglucosaminyltransferase
III ("GnTIII), 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.
[0088] In one embodiment, a composition comprising a GA101 antibody
of this invention comprises GA101 antibodies engineered to have
increased ADCC activity.
[0089] The term "Bcl-2" as used herein refers to the Bcl-2 protein
(Swiss Prot ID No. P10415), a member of the Bcl-2 family of
proteins (Cory, S., and Adams, J. M., Nature Reviews Cancer 2
(2002) 647-656; Adams, Genes und Development 17 (2003) 2481-2495;
Danial, N. N., and Korsmeyer, S. J., Cell 116 (2004) 205-219;
Petros, A. M., Biochim Biophys Acta 1644 (2004) 83-94).
[0090] The term "selective Bcl-2 inhibitors" as used herein refers
to
2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimeth-
ylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-(((tetrahydro-2H-pyr-
an-4-yl)methyl)amino)phenylsulfonyl)benzamide, (a.k.a. ABT-199 or
GDC-0199), a Bcl-2 inhibitor of formula I, which is described in
International Publication No. WO2010/138588 and in US publication
NO. US2010/0305122, which are incorporated by reference herein.
##STR00001##
[0091] The term "expression of the CD20" antigen is intended to
indicate an 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 of the methods of this invention express
significant levels of CD20 on a B-cell tumor or cancer. Patients
having a "CD20 expressing cancer" can be determined by standard
assays known in the art. e.g., CD20 antigen expression is measured
using immunohistochemical (RHC) detection, FACS or via PCR-based
detection of the corresponding mRNA.
[0092] The term "CD20 expressing cancer" as used herein refers to
all cancers in which the cancer cells show an expression of the
CD20 antigen. Such CD20 expressing cancer may be, for example,
lymphomas, lymphocytic leukemias, lung cancer, non small cell lung
(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,
pancreatic cancer, skin cancer, cancer of the head or neck,
cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,
rectal cancer, cancer of the anal region, stomach cancer, gastric
cancer, colon cancer, breast cancer, uterine cancer, carcinoma of
the fallopian tubes, carcinoma of the endometrium, carcinoma of the
cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's
Disease, cancer of the esophagus, cancer of the small intestine,
cancer of the endocrine system, cancer of the thyroid gland, cancer
of the parathyroid gland, cancer of the adrenal gland, sarcoma of
soft tissue, cancer of the urethra, cancer of the penis, prostate
cancer, cancer of the bladder, cancer of the kidney or ureter,
renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,
hepatocellular cancer, biliary cancer, neoplasms of the central
nervous system (CNS), spinal axis tumors, brain stem glioma,
glioblastoma multiforme, astrocytomas, schwanomas, ependymonas,
medulloblastomas, meningiomas, squamous cell carcinomas, pituitary
adenoma, including refractory versions of any of the above cancers,
or a combination of one or more of the above cancers.
[0093] In one embodiment, CD20 expressing cancer as used herein
refers to lymphomas (e.g., B-Cell Non-Hodgkin's lymphomas (NHL))
and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias
include e.g. a) follicular lymphomas, b) Small Non-Cleaved Cell
Lymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma,
sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma) c) marginal
zone lymphomas (including extranodal marginal zone B cell lymphoma
(Mucosa-associated lymphatic tissue lymphomas. MALT), nodal
marginal zone B cell lymphoma and splenic marginal zone lymphoma),
d) Mantle cell lymphoma (MCL), e) Large Cell Lymphoma (including
B-cell diffuse large cell lymphoma (DLCL), Diffuse Mixed Cell
Lymphoma, Immunoblastic Lymphoma, Primary Mediastinal B-Cell
Lymphoma, Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma) f) hairy
cell leukemia, g) lymphocytic lymphoma, waldenstrom's
macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic
lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell
prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell
myeloma, multiple myeloma, plasmacytoma, j) Hodgkin's disease. k)
acute myeloid leukemia (AML); among other types of lymphomas and
lymphocyctic leukemias.
[0094] In one embodiment, the CD20 expressing cancer is a B-Cell
Non-Hodgkin's lymphomas (NHL). In another embodiment, the CD20
expressing cancer is a Mantle cell lymphoma (MCL), acute
lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL),
B-cell diffuse large cell lymphoma (DLCL), acute myeloid leukemia
(AML). Burkitt's lymphoma, hairy cell leukemia, follicular
lymphoma, multiple myeloma, marginal zone lymphoma, post transplant
lymphoproliferative disorder (PTLD), HIV associated lymphoma,
waldenstrom's macroglobulinemia, or primary CNS lymphoma.
[0095] "Relapsed or Refractory" CLL as used herein includes CLL
patients who have received at least 1 prior chemotherapy containing
treatment regimen. Relapsed patients generally have developed
progressive disease following a response to the prior
chemotherapy-containing treatment regimen. Refractory patients have
generally failed to respond or relapsed within 6 months to the last
prior chemotherapy-containing regimen.
[0096] "Previously untreated" CLL as used herein includes patients
diagnosed with CLL, but who have, in general, received no prior
chemotherapy or immunotherapy. Patients with a history of
emergency, loco-regional radiotherapy (e.g., for relief of
compressive signs or symptoms) or corticosteroids can still be
considered previously untreated.
[0097] The term "treating" as used herein, unless otherwise
indicated, means reversing, alleviating, inhibiting the progress
of, or preventing, either partially or completely, the growth of
tumors, tumor metastases, or other cancer-causing or neoplastic
cells in a patient. The term "treatment" as used herein, unless
otherwise indicated, refers to the act of treating.
[0098] The term "a method of treating" or its equivalent, when
applied to. for example, cancer refers to a procedure or course of
action that is designed to reduce or eliminate the number of cancer
cells in a patient, or to alleviate the symptoms of a cancer. "A
method of treating" cancer or another proliferative disorder does
not necessarily mean that the cancer cells or other disorder will,
in fact, be eliminated, that the number of cells or disorder will,
in fact, be reduced, or that the symptoms of a cancer or other
disorder will, in fact, be alleviated. Often, a method of treating
cancer will be performed even with a low likelihood of success. but
which, given the medical history and estimated survival expectancy
of a patient, is nevertheless deemed to induce an overall
beneficial course of action. The terms "co-administration" or
"co-administering" refer to the administration of said type II
anti-CD20 antibody and said selective Bcl-2 inhibitor as two
separate formulations. The co-administration can be simultaneous or
sequential in either order. In one further embodiment, there is a
time period while both (or all) active agents simultaneously exert
their biological activities. Said type II anti-CD20 antibody and
said selective Bcl-2 inhibitor are co-administered either
simultaneously or sequentially (e.g. via an intravenous (i.v.)
through a continuous infusion (one for the antibody and eventually
one for the Bcl-2 inhibitor; or the Bcl-2 inhibitor is administered
orally). When both therapeutic agents are co-administered
sequentially the agents are administered in two separate
administrations that are separated by a "specific period of time".
The term specific period of time is meant any where from I hour to
15 days. For example, one of the agents can be administered within
about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or
24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4, 3, 2 or 1 hour from the administration of the other
agent, and, in one embodiment, the specific period time is 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1 day, or 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour.
[0099] The term "simultaneously" means at the same time or within a
short period of time, usually less than 1 hour.
[0100] A dosing period as used herein is meant a period of time,
during which each therapeutic agent has been administered at least
once. A dosing cycle is usually about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 days, and, in one embodiment, 6, 7, 8, 9, 10, 11,
12, 13, or 14 days, for example, 7 or 14 days.
[0101] In certain embodiments, a dosing period is a dosing
cycle.
[0102] It is self-evident that the antibodies are administered to
the patient in a "therapeutically effective amount" (or simply
"effective amount") which is the amount of the respective compound
or combination that will elicit the biological or medical response
of a tissue, system, animal or human that is being sought by the
researcher, veterinarian, medical doctor or other clinician. The
administration of an effective amount of a therapeutically agent
can be a single administration or split dose administration. "split
dose administration" is meant an effective amount is a split into
multiple doses, preferably 2, and administered within 1 or 2 days.
For example, if 100 mg of a selective BCL-2 inhibitor is deemed
effective, it can be administered in one 100 mg administration or
two 50 mg administrations. Split dose administration is sometimes
desirable at the beginning of a dosing period to reduce side
effects. When an effective amount is administered in split dosing,
it is still considered one administration of an effective amount.
For example, when 100 mg is the effective amount of a selective
Bcl-2 inhibitor and that amount is administered in two 50 mg doses
over a period of time, e.g. 2 days, only one effective amount is
administered during that period of time.
[0103] The amount of co-administration of said type 11 anti-CD20
antibody and said Bcl-2 inhibitor and the timing of
co-administration will depend on the type (species, gender, age,
weight, etc.) and condition of the patient being treated and the
severity of the disease or condition being treated. Said type II
anti-CD20 antibody and said Bcl-2 inhibitor are suitably
co-administered to the patient at one time or over a series of
treatments. Depending on the type and severity of the disease,
about 1 .mu.g/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of said type II
anti-CD20 antibody, and 0.1 mg/kg to 200 mg/kg (e.g. 10-150 mg/kg)
of said selective Bcl-2 inhibitor is an initial candidate dosage
for co-administration of both drugs to the patient. If the
administration is intravenous the initial infusion time for said
type II anti-CD20 antibody or said Bel-2 inhibitor may be longer
than subsequent infusion times, for instance approximately 90
minutes for the initial infusion, and approximately 30 minutes for
subsequent infusions (if the initial infusion is well
tolerated).
[0104] In one embodiment, the preferred dosage of said type II
anti-CD20 antibody will be in the range from about 0.05 mg/kg to
about 30 mg/kg, preferably 1 mg/kg to 30 mg/kg; or 500 mg-3000 mg
flat dose. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,
4.0 mg/kg, 10 mg/kg or 30 mg/kg or 500 mg-3000 mg flat dose (or any
combination thereof) may be co-administered to the patient. The
preferred dosage of said Bcl-2 inhibitor will be in the range from
20 mg/kg to about 150 mg/kg, preferably 1 mg/kg to 10 mg/kg.
Depending on the on the type (species, gender, age, weight, etc.)
and condition of the patient and on the type of anti-CD20 antibody
and Bcl-2 inhibitor, the dosage and the administration schedule of
said anti-CD20 antibody can differ from the dosage of Bcl-2
inhibitor. E.g. the said anti-CD20 antibody may be administered
e.g. every one to three weeks and said Bcl-2 inhibitor may be
administered daily or every 2 to 7 days. An initial higher loading
dose, followed by one or more lower doses may also be
administered.
[0105] The present invention relates in part to a composition
comprising a type II anti-CD20 antibody and a selective Bcl-2
inhibitora selective Bcl-2 inhibitor.
[0106] In a preferred embodiment, the composition of the present
invention is useful for preventing or reducing metastasis or
further dissemination in such a patient suffering from CD20
expressing cancer. The composition is useful for increasing the
duration of survival of such a patient, increasing the progression
free survival of such a patient, increasing the duration of
response, resulting in a statistically significant and clinically
meaningful improvement of the treated patient as measured by the
duration of survival, progression free survival, response rate or
duration of response. In a preferred embodiment, the composition is
useful for increasing the response rate in a group of patients.
[0107] In the context of this invention, additional other
cytotoxic, chemotherapeutic or anti-cancer agents, or compounds
that enhance the effects of such agents (e.g. cytokines) may be
used in the type II anti-CD20 antibody and Bcl-2 inhibitor
combination treatment of CD20 expressing cancer. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended. Preferably the type II anti-CD20 antibody and
Bcl-2 inhibitor combination treatment is used without such
additional cytotoxic, chemotherapeutic or anti-cancer agents, or
compounds that enhance the effects of such agents.
[0108] Such agents include, for example: alkylating agents or
agents with an alkylating action, such as cyclophosphamide (CTX;
e.g. CYTOXAN.RTM.), chlorambucil (CHL; e.g. LEUKERAN.RTM.),
cisplatin (CisP: e.g. PLATINOL.RTM.) busulfan (e.g. MYLERAN.RTM.),
melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine
(TEM), mitomycin C, and the like; anti-metabolites, such as
methotrexate (MTX), etoposide (VP16: e.g. VEPESID.RTM.),
6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C),
5-fluorouracil (5-FU), capecitabine (e.g. XELODA.RTM.), dacarbazine
(DTIC), and the like; antibiotics, such as actinomycin D,
doxorubicin (DXR; e.g. ADRIAMYCIN.RTM.), daunorubicin (daunomycin),
bleomycin, mithramycin and the like; alkaloids, such as vinca
alkaloids such as vincristine (VCR), vinblastine, and the like; and
other antitumor agents, such as paclitaxel (e.g. TAXOL.RTM.) and
paclitaxel derivatives, the cytostatic agents, glucocorticoids such
as dexamethasone (DEX; e.g. DECADRON.RTM.) and corticosteroids such
as prednisone, nucleoside enzyme inhibitors such as hydroxyurea,
amino acid depleting enzymes such as asparaginase, leucovorin and
other folic acid derivatives, and similar, diverse antitumor
agents. The following agents may also be used as additional agents:
arnifostine (e.g. ETHYOL.RTM.), dactinomycin. mechlorethamine
(nitrogen mustard), streptozocin, cyclophosphamide, lomustine
(CCNU), doxorubicin lipo (e.g. DOXIL.RTM.), gemcitabine (e.g.
GEMZAR.RTM.), daunorubicin lipo (e.g. DAUNOXOME.RTM.),
procarbazine, mitomycin, docetaxel (e.g. TAXOTERE.RTM.),
aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin,
CPT 11 (irinotecan). 10-hydroxy 7-ethyl-camptothecin (SN38),
floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon
beta, interferon alpha, mitoxantrone. topotecan, leuprolide,
megestrol, melphalan, mercaptopurine. plicamycin, mitotane,
pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen,
teniposide, testolactone, thioguanine, thiotepa, uracil mustard,
vinorelbine, chlorambucil. Preferably the type II anti-CD20
antibody and Bcl-2 inhibitor combination treatment is used without
such additional agents.
[0109] The use of the cytotoxic and anticancer agents described
above as well as antiproliferative target-specific anticancer drugs
like protein kinase inhibitors in chemotherapeutic regimens is
generally well characterized in the cancer therapy arts, and their
use herein falls under the same considerations for monitoring
tolerance and effectiveness and for controlling administration
routes and dosages, with some adjustments. For example, the actual
dosages of the cytotoxic agents may vary depending upon the
patient's cultured cell response determined by using histoculture
methods. Generally, the dosage will be reduced compared to the
amount used in the absence of additional other agents.
[0110] Typical dosages of an effective cytotoxic agent can be in
the ranges recommended by the manufacturer, and where indicated by
in vitro responses or responses in animal models, can be reduced by
up to about one order of magnitude concentration or amount. Thus,
the actual dosage will depend upon the judgment of the physician,
the condition of the patient, and the effectiveness of the
therapeutic method based on the in vitro responsiveness of the
primary cultured malignant cells or histocultured tissue sample, or
the responses observed in the appropriate animal models.
[0111] In the context of this invention, an effective amount of
ionizing radiation may be carried out and/or a radiopharmaceutical
may be used in addition to the type II anti-CD20 antibody and Bcl-2
inhibitor combination treatment of CD20 expressing cancer. The
source of radiation can be either external or internal to the
patient being treated. When the source is external to the patient,
the therapy is known as external beam radiation therapy (EBRT).
When the source of radiation is internal to the patient, the
treatment is called brachytherapy (BT). Radioactive atoms for use
in the context of this invention can be selected from the group
including, but not limited to, radium, cesium-137, iridium-192,
americium-241, gold-198, cobalt-57, copper-67, technetium-99,
iodine-123. iodine-131, and indium-111. Is also possible to label
the antibody with such radioactive isotopes. Preferably the type II
anti-CD20 antibody and Bcl-2 inhibitor combination treatment is
used without such ionizing radiation.
[0112] Radiation therapy is a standard treatment for controlling
unresectable or inoperable tumors and/or tumor metastases. Improved
results have been seen when radiation therapy has been combined
with chemotherapy. Radiation therapy is based on the principle that
high-dose radiation delivered to a target area will result in the
death of reproductive cells in both tumor and normal tissues. The
radiation dosage regimen is generally defined in terms of radiation
absorbed dose (Gy), time and fractionation, and must be carefully
defined by the oncologist. The amount of radiation a patient
receives will depend on various considerations, but the two most
important are the location of the tumor in relation to other
critical structures or organs of the body, and the extent to which
the tumor has spread. A typical course of treatment for a patient
undergoing radiation therapy will be a treatment schedule over a 1
to 6 week period, with a total dose of between 10 and 80 Gy
administered to the patient in a single daily fraction of about 1.8
to 2.0 Gy, 5 days a week. In a preferred embodiment of this
invention there is synergy when tumors in human patients are
treated with the combination treatment of the invention and
radiation. In other words, the inhibition of tumor growth by means
of the agents comprising the combination of the invention is
enhanced when combined with radiation, optionally with additional
chemotherapeutic or anticancer agents. Parameters of adjuvant
radiation therapies are, for example, contained in WO 99/60023.
[0113] The type II anti-CD20 antibodies are administered to a
patient according to known methods. by intravenous administration
as a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, or intrathecal routes. Intravenous
or subcutaneous administration of the antibodies is preferred.
[0114] The Bcl-2 inhibitors are administered to a patient according
to known methods, e.g. by intravenous administration as a bolus or
by continuous infusion over a period of time, by intramuscular,
intraperitoneal. intracerobrospinal, subcutaneous. intra-articular,
intrasynovial, intrathecal, or peroral routes. Intravenous,
subcutaneous or oral administration of the Bcl-2 inhibitors is
preferred.
[0115] The invention also relates to a kit comprising a type II
anti-CD20 antibody and a selective Bcl-2 inhibitor for the
combination treatment of a patient suffering from a CD20 expressing
cancer.
[0116] In an embodiment of the present invention, the kit further
comprises a pharmaceutically acceptable carrier. The kit may
further include a sterile diluent, which is preferably stored in a
separate additional container. The kit may further include a
package insert comprising printed instructions directing the use of
the combined treatment as a method for a CD20 expressing cancer
disease. preferably a B-Cell Non-Hodgkin's lymphoma (NHL).
[0117] The term "package insert" refers to instructions customarily
included in commercial packages of therapeutic products, which may
include information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the
use of such therapeutic products.
[0118] In a preferred embodiment, the article of manufacture
containers may further include a pharmaceutically acceptable
carrier. The article of manufacture may further include a sterile
diluent, which is preferably stored in a separate additional
container.
[0119] As used herein, a "pharmaceutically acceptable carrier" is
intended to include any and all material compatible with
pharmaceutical administration including solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and other materials and compounds
compatible with pharmaceutical administration. Except insofar as
any conventional media or agent is incompatible with the active
compound. use thereof in the compositions of the invention is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
Pharmaceutical Compositions and Methods
[0120] Pharmaceutical compositions can be obtained by processing
the type II anti-CD20 antibody or the anti-Bcl-2 active agent
according to this invention with pharmaceutically acceptable,
inorganic or organic carriers. Lactose, corn starch or derivatives
thereof, talc, stearic acids or it's salts and the like can be
used, for example, as such carriers for tablets, coated tablets,
dragees and hard gelatine capsules. Suitable carriers for soft
gelatine capsules are, for example, vegetable oils, waxes, fats,
semi-solid and liquid polyols and the like. Depending on the nature
of the active substance no carriers are, however. usually required
in the case of soft gelatine capsules. Suitable carriers for the
production of solutions and syrups are, for example, water,
polyols, glycerol. vegetable oil and the like. Suitable carriers
for suppositories are, for example, natural or hardened oils,
waxes, fats, semi-liquid or liquid polyols and the like.
[0121] The pharmaceutical compositions can, moreover, contain
preservatives, solubilizers, stabilizers, wetting agents,
emulsifiers, sweeteners, colorants, flavorants, salts for varying
the osmotic pressure. buffers, masking agents or antioxidants. They
can also contain still other therapeutically valuable
substances.
[0122] Said pharmaceutical composition may further comprise one or
more pharmaceutically acceptable carriers.
[0123] The present invention further provides a pharmaceutical
composition, in particular for use in cancer, comprising (i) an
effective first amount of a type II anti-CD20 antibody, or (ii) an
effective second amount of a selective Bcl-2 inhibitor. Such
composition optionally comprises pharmaceutically acceptable
carriers and/or excipients.
[0124] Pharmaceutical compositions of the type II anti-CD20
antibody alone used in accordance with the present invention are
prepared for storage by mixing an antibody having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0125] Pharmaceutical compositions of the anti-Bcl-2 active agent
alone, e.g. the Bcl-2 inhibitor, depend on their pharmaceutical
properties; e.g. for small chemical compounds such as e.g. ABT-737,
ABT-199 or ABT-263, one formulation could be e.g. the
following:
TABLE-US-00003 a) Tablet Formulation (Wet Granulation): Item
Ingredients mg/tablet 1. Compound of formula (I) 5 25 100 500 2.
Lactose Anhydrous DTG 125 105 30 150 3. Sta-Rx 1500 6 6 6 30 4.
Microcrystalline Cellulose 30 30 30 150 5. Magnesium Stearate 1 1 1
1 Total 167 167 167 831
Manufacturing Procedure:
[0126] 1. Mix items 1, 2, 3 and 4 and granulate with purified
water. 2. Dry the granules at 50.degree. C. 3. Pass the granules
through suitable milling equipment. 4. Add item 5 and mix for three
minutes; compress on a suitable press.
TABLE-US-00004 b) Capsule Formulation: Item Ingredients mg/tablet
1. Compound of formula (I) 5 25 100 500 2. Hydrous Lactose 159 123
148 -- 3. Corn Starch 25 35 40 70 4. Talc 10 15 10 25 5. Magnesium
Stearate 1 2 2 5 Total 200 200 200 600
Manufacturing Procedure:
[0127] 1. Mix items 1, 2 and 3 in a suitable mixer for 30 minutes.
2. Add items 4 and 5 and mix for 3 minutes. 3. Fill into a suitable
capsule.
[0128] In one further embodiment of the invention the
pharmaceutical compositions according to the invention are two
separate formulations for said type II anti-CD20 antibody and said
Bcl-2 inhibitor.
[0129] The active ingredients may also be entrapped in
microcapsules prepared. for example, by coacervation techniques or
by interracial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition. Osol, A. Ed. (1980).
[0130] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0131] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0132] The invention relates in part to a method for the treatment
of a patient suffering from cancer, particularly a CD20-expressing
cancer, comprising co-administering, to a patient in need of such
treatment, a type II anti-CD20 antibody and a selective Bcl-2
inhibitor. Said type II anti-CD20 antibody and anti-Bcl-2 active
agent are administered in effective amounts.
[0133] In certain embodiments, a dosing cycle is for 28 days.
[0134] In certain embodiments of a method of treating cancer in a
patient as provided herein, the method comprises administering the
type II anti-CD20 antibody and the selective Bcl-2 for one or more
dosing cycles to the patient. In one embodiment, the one or more
dosing cycles each last for at least one week. In another
embodiment, the one or more dosing cycles are each for at least two
weeks. three weeks, four weeks, five weeks, six weeks, seven weeks,
eight weeks, nine weeks, or for more than nine weeks. In one
embodiment, each dosing cycle is four weeks.
[0135] In one embodiment, the therapeutic agents are administered
to the patient for one dosing cycle.
[0136] In another embodiment, therapeutic agents are administered
to the patient for more than one dosing cycle, for instance, for
two, three, four, five, six, seven or more than seven dosing
cycles. As an example, where a dosing cycle is four weeks, and the
patient is administered with one or both therapeutic agents over
six dosing cycles, the treatment regimen will be for 24 weeks, such
as illustrated in the dosing schemes shown in FIG. 3.
[0137] In certain embodiments, methods are provided for the
treatment of a cancer in a patient in need thereof comprising
administering to said human a GA101 antibody and/or GDC-0199 in
multiple dosing cycles.
[0138] In certain embodiments, the GA101 antibody and GDC-199 are
both administered to the patient in one or more dosing cycles of
the multiple dosing cycles, and one of the GA101 antibody and
GDC-0199 are administered in one or more dosing cycles of the
multiple dosing cycles.
[0139] In certain embodiments of the methods of treatment provided
herein, the therapeutic agents are administered to the patent in a
dosing scheme comprising two or three treatment phases, where each
treatment phase comprises at least one dosing cycle that differs
from the dosing cycle from other treatment phases. For example, in
one embodiment where the dosing cycle comprises four weeks, the
type II anti-CD20 antibody can be administered to the patient once
a week for two or more weeks of the first dosing cycle (e.g., a
first treatment phase), and administered once per dosing cycle in
the dosing cycles that follow the first dosing cycle (e.g., a
second treatment phase).
[0140] In certain embodiments of the methods of treatment provided
herein, the type II anti-CD20 antibody is administered to the
patient once a week for at least one week of a dosing cycle. In
some embodiments, where the dosing cycle is for two or more weeks,
the type II anti-CD20 antibody is administered to the patient once
per dosing cycle.
[0141] In certain embodiments of the methods of treatment provided
herein, GDC-0199 is administered once per day of a dosing
cycle.
[0142] Both GDC-0199 and the type II anti-CD20 antibody can, for
example, be administered to the patient in a dosing cycle. In
certain dosing cycles, one therapeutic agent alone is administered
to the patient.
[0143] In certain embodiments of the methods provided wherein the
therapeutic agents are administered to the patent in a dosing
scheme comprising multiple dosing cycles, the multiple dosing
cycles comprise a first treatment phase having dosing cycles
wherein in each dosing cycle the type 11 anti-CD20 antibody is
administered once per dosing cycle and GDC-0199 is administered
each day of the dosing cycle. Such dosing cycles in the first
treatment phase can, for example, each be for four weeks. In some
embodiments, the multiple dosing cycles can further comprise a
second treatment phase wherein the type 11 anti-CD20 antibody alone
is administered to the patient or wherein GDC-0199 alone is
administered to the patient (e.g., a maintenance phase).
[0144] In some embodiments of the methods provided herein wherein
the type 11 anti-CD20 antibody and GDC-0199 are both administered
to the patient for one or more dosing cycles (e.g., in a treatment
phase), the patient can then be administered with GDC-0199 alone
(e.g., in a maintenance phase).
[0145] In certain embodiments where GDC-0199 alone is administered
to the patient following combination therapy, GDC-0199 can, for
example, be administered once a day, once every other day, once
every three days, four, five or six days, or once a week, to the
patient.
[0146] In certain embodiments wherein the type II anti-CD20
antibody alone is administered to the patient following combination
therapy, the type II anti-CD20 antibody can, for example, be
administered once a week, once every two weeks or once per month,
to the patient.
[0147] In certain embodiments of the methods of treatment provided
herein, the amounts of GDC-0199 per dose administered to the
patient are increased during a first dosing cycle. See, e.g., FIG.
2 for an exemplary dosing scheme where amounts of GDC-0199
administered to patients in the first dosing cycle escalate from 50
mg doses the first week, to 100 mg doses the second week, to 300 mg
doses the third week).
[0148] In certain embodiments, escalating doses of GDC-0199 are
administered to the patient prior to administration of the type II
anti-CD20 antibody. In other embodiments, escalating doses of
GDC-0199 are administered to the patient after the type II
anti-CD20 antibody has been administered to the patient.
[0149] In some embodiments of the method of treatment provided
herein, the amount of GDC-0199 administered to the patient per dose
is increased during the first dosing cycle from initial amounts of
between 10 mg to 80 mg to final amounts of between 190 mg to 400
mg. In certain embodiments, the amount of GDC-0199 per dose
administered to the patients begins with 50 mg or 100 mg. and is
increased to 300 mg per dose. In some embodiments, the amount of
GDC-0199 in the initial doses administered to the patient can, for
example, be between 20 mg to 60 mg (e.g., 20 mg, 25 mg, 30 mg, 35
mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg doses). followed by dose
amounts of 100 mg, 200 mg, 300 mg or more of GDC-0199.
[0150] In certain embodiments of the methods provided herein, doses
of GDC-0199 are administered to the patient in increasing amounts
prior to the first administration of the type 11 anti-CD20
antibody. In some embodiments, doses of GDC-0199 are administered
to the patient in increasing amounts after the first administration
of the type II anti-CD20 antibody.
[0151] As used herein, the term "patient" typically refers to a
human in need of treatment with type II anti-CD20 antibody (e.g. a
patient suffering from CD20 expressing cancer) for any purpose,
and, in one embodiment, a human in need of such a treatment to
treat cancer, or a precancerous condition or lesion. However, the
term "patient" can also refer to non-human animals, preferably
mammals such as dogs, cats, horses, cows, pigs, sheep and non-human
primates, among others.
[0152] The invention further comprises a type II anti-CD20 antibody
for the treatment of CD20 expressing cancer in combination with a
selective Bcl-2 inhibitor.
[0153] The invention further comprises a type II anti-CD20 antibody
for the treatment of a patient suffering from a CD20 expressing
cancer in combination a selective Bcl-2 inhibitor.
[0154] The invention further comprises a type II anti-CD20 antibody
and a selective Bel-2 inhibitor for use in the treatment of CD20
expressing cancer.
[0155] The invention further comprises a type II anti-CD20 antibody
and a selective Bcl-2 inhibitor for use in the treatment of a
patient suffering from a CD20 expressing cancer.
[0156] In one embodiment, said selective Bcl-2 inhibitor is
ABT-199.
[0157] In one embodiment, said type II anti-CD20 antibody has a
ratio of the binding capacities to CD20 on Raji cells (ATCC-No.
CCL-86) of said type II anti-CD20 antibody compared to rituximab of
0.3 to 0.6, and in one embodiment, 0.35 to 0.55, and in another
embodiment, 0.4 to 0.5.
[0158] In one embodiment, said type II anti-CD20 antibody is a
GA101 antibody.
[0159] In one embodiment, said type II anti-CD20 antibody has
increased antibody dependent cellular cytotoxicity (ADCC).
[0160] In certain embodiments of the methods of treatment of a
cancer in a patient provided herein, the cancer is a non-solid
tumor. In one embodiment, the non-solid tumor is a CD20 expressing
non-solid tumor. Exemplary non-solid tumors that can be treated in
the methods provided herein, include, for instance, a leukemia or a
lymphoma. In one embodiment, the non-solid tumor is a B cell
lymphoma.
[0161] In one embodiment, the CD20 expressing cancer is a B-Cell
Non-Hodgkin's lymphoma (NHL).
[0162] In one embodiment, said type II anti-CD20 antibody is a
monoclonal antibody.
[0163] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
EXAMPLES
Example 1. Treating Lymphoma with a Combination of GDC-0199 and
Obinutuzumab
[0164] Nonclinical data supports the hypothesis that the
combination of GDC-0199 and GA101 (in this case, obinutuzumab) will
show more anti-tumor activity than when each drug is administered
alone. The study used a non-Hodgkin's lymphoma (NHL) xenograft
model of aggressive lymphoma, the diffuse large B-cell lymphoma
(DLBCL) derived cell line SU.DHL-4. Obinutuzumab was administered
at a dose of 1 mg/kg IV, once a week for 3 weeks and achieved tumor
stasis followed by growth delay. GDC-0199 was administered at 100
mg/kg QD for 21 days and also demonstrated stasis followed by tumor
growth delay. However, the combination of GDC-0199 and obinutuzumab
induced a greater than additive effect resulting in tumor
regressions (5 of 8 partial regressions (PRs); see FIG. 1). Three
weeks of combination therapy resulted in enhanced tumor growth
inhibition (TGI) (118% TGI), compared with 76% (GA101) and 80% (GDC
0199) TGI observed with single agent administration (see FIG. 1).
Increased tumor regressions (5 PRs) were also observed when GA101
was combined with GDC 0199, compared with single agent
administration. Additionally. TGI was sustained in the combination
treatment group after treatment ended on day 21 since 116% TGI was
observed at day 31 (10 days after dosing ended), versus 30% TGI
with GA101 and 25% TGI with GDC 0199 as single agents. In summary,
GA101 in combination with GDC 0199 resulted in increased TGI and
tumor regressions compared to each agent administered separately in
a NHL xenograft model.
[0165] In this study the vehicles for GA-101 and GDC-0199 were
saline and 60% phosal, respectively. In FIG. 1, the dose of
GDC-0199 is expressed as free base equivalents in mg/kg of body
weight. Results are expressed as the fitted tumor volume as
determined by linear mixed effects modeling for each treatment
group vs. time in days, where Day 0 is the first day of
treatment.
Example 2. A Phase Ib Multicenter Study of GDC-0199 and
Obinutuzumab in Patients with Relapsed or Refractory or Previously
Untreated Chronic Lymphocytic Leukemia
[0166] Two schedules will be evaluated: Schedule A (FIG. 2),
consisting of GDC-0199 administered in escalating doses for 3 weeks
prior to the first obinutuzumab infusion, and Schedule B (FIG. 3).
consisting of obinutuzumab administered first followed by
escalating dose levels of GDC-0199. Schedule A and Cohort 1 of
Schedule B will be enrolled in parallel. In addition, the
dose-finding stage will evaluate whether the administration of
GDC-0199 prior to the first obinutuzumab infusion (Schedule A) will
result in a lower incidence of infusion reactions, thereby reducing
the need for split doses of obinutuzumab and corticosteroid
premedication.
[0167] The expansion stage will include two expansion cohorts of 20
patients each (relapsed/refractory and previously untreated CLL)
and will evaluate the safety and preliminary efficacy of the
selected combination dose and schedule.
[0168] In Schedule A, the combination treatment of GDC-0199 and
obinutuzumab will be administered for a total of 7 cycles of 28
days each, including a total of 8 infusions of obinutuzumab and
GDC-0199 QD.
[0169] In Schedule B. the combination treatment of GDC-0199 and
obinutuzumab will be administered for a total of 6 cycles of 28
days each, including a total of 9 infusions of obinutuzumab (8
doses; first dose will be split into two infusions) and GDC-0199
QD.
[0170] GDC-0199 monotherapy may be continued in patients beyond 6-7
cycles of combination treatment described above (e.g., if they have
acceptable toxicity and have not yet achieved maximal clinical
response (i.e., are having continued improvement/reduction in tumor
burden that has not yet stabilized for at least 2 months)). Such
patients may continue GDC-0199 monotherapy until they have achieved
maximal response or up to 1 year after the last patient is
enrolled, whichever occurs first.
Example 3. Antitumor Activity of Combined Treatment of GDC-0199 and
Type II Anti-CD20 Antibody (Obinutuzumab) as Compared to Combined
Treatment of GDC-0199 and a Type I Anti-CD20 Antibody
(Rituximab)
[0171] Test agents. The type II anti-CD20 antibody was the
GA101antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed
in WO 2005/044859 (termed B-HH6-B-KV1 GE therein, also known as
obinutuzumab or RO5072759), which was provided as stock solution
(conc. 9.4 mg/ml) from Roche GlycArt, Schlieren, Switzerland.
Antibody buffer included histidine, trehalose and polysorbate 20.
Antibody solution was diluted appropriately in PBS from stock for
prior injections. GDC-0199 was obtained from Genentech Inc.,
Calif., USA.
[0172] Cell line and culture conditions. The human Z138 mantle cell
lymphoma cell line is routinely cultured in DMEM supplemented with
10% fetal bovine serum (PAA Laboratories, Austria) and 2 mM
L-glutamine at 37.degree. C. in a water-saturated atmosphere at 5%
CO.sub.2. Cells were co-injected with MATRIGEL.
[0173] Animals. Female SCID beige mice; age 5-6 weeks at arrival
(purchased from Charles River. Sulzfeld, Germany) were maintained
under specific-pathogen-free condition with daily cycles of 12 h
light/12 h darkness according to committed guidelines (GV-Solas;
Felasa; TierschG). Experimental study protocol was reviewed and
approved by local government (Regierung von Oberbayern;
registration no. 55.2-1-54-2531.2-26-09). After arrival animals
were maintained in the quarantine part of the animal facility for
one week to get accustomed to new environment and for observation.
Continuous health monitoring was carried out on regular basis. Diet
food (Altromin Spezialfutter GmbH & Co. KG) and water
(filtered) were provided ad libitum.
[0174] Monitoring. Animals were controlled daily for clinical
symptoms and detection of adverse effects. For monitoring
throughout the experiment body weight of animals was documented two
times weekly and tumor volume was measured by caliper after
staging.
[0175] Treatment of animals. Animal treatment was started at the
day of randomisation 18 days after tumor cell inoculation.
RO5072759 or rituximab were administered as single agent, i.p.,
once weekly (day 18, 25, 32) for 3 weeks at dosages of 1 mg/kg. The
corresponding vehicle was administered on the same days. GCD-0199
was given p.o. once daily (from day 18 to day 34), over 17 days at
a dosage of 100 mg/kg. In the combination therapy groups, the
antibodies and GDC-0199 were administered at the same dosages and
on the same days.
[0176] Tumor growth inhibition study in vivo. Results of therapy on
tumor volume development are shown in FIG. 4. On day 35 after tumor
cell inoculation, there was a tumor growth inhibition of 32%, 59%,
73%, 96% or 106% (regression) in the animals given rituximab,
GDC-0199, RO5072759, combination of GDC-0199 plus rituximab or
combination of GDC-0199 plus RO5072759, respectively, compared to
the control group.
Example 4. Administration of GDC-0199 as a Single Agent Following
Combination with Obinutuzumab Results in Significant Delay in Tumor
Regrowth
[0177] This example describes results using the DLBCL SU-DH L-4
xenograft model, which discussed in Example 1 above. Initially,
GDC-0199 was dosed orally for 21 continuous days in combination
with GA101 (in this example, obinutuzumab) at 1 mg/kg for 3 weeks.
The latter resulted in enhanced TGI (91%). compared with 54%
(GA101) and 24% (GDC-0199) TGI observed with each agent alone (FIG.
5). At day 22 tumor bearing mice in the combination cohort
continued to be dosed with GDC-0199 alone at 100 mg/kg for an
additional 24 days. The latter resulted in a significant delay in
tumor regrowth when compared to mice treated with the combination
of GA101 and GDC-0199 over a 21 day period (time to tumor
progression of the combination cohort=38 days vs. continued
treatment with GDC-0199=45 days (FIG. 5). Thus, single-agent
treatment with GDC-0199 following combination with GA101 sustains
efficacy in vivo. These results support a benefit for maintenance
therapy with GDC-0199.
[0178] In FIG. 5, the control is saline vehicle for GA101 plus 60%
phosal vehicle for GDC-0199. GA101 was dosed intravenously once a
week (QW) for 3 weeks and GDC-0199 was dosed orally and daily (QD)
for 21 days (QD.times.21) as single agents or in combination. As
explained above, a cohort of tumor bearing mice was also dosed with
GDC-0199 alone for an additional 24 days after combination
treatment ended on day 21 (QD.times.45). Under the x-axis,
treatment periods are denoted by solid black lines (-) for the
combination cohorts while the continued single agent GDC-0199
treatment is denoted by dashed black line (---).
[0179] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
1016PRTArtificialSequence of HVR-H1 of GA101 Antibody 1Gly Tyr Ala
Phe Ser Tyr1 528PRTArtificialSequence of HVR-H2 of GA101 Antibody
2Phe Pro Gly Asp Gly Asp Thr Asp1 5310PRTArtificialSequence of
HVR-H3 of GA101 Antibody 3Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr1
5 10416PRTArtificialSequence of HVR-L1 of GA101 Antibody 4Arg Ser
Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr1 5 10
1557PRTArtificialSequence of HVR-L2 of GA101 Antibody 5Gln Met Ser
Asn Leu Val Ser1 569PRTArtificialSequence of HVR-L3 of GA101
Antibody 6Ala Gln Asn Leu Glu Leu Pro Tyr Thr1
57119PRTArtificialSequence of VH of GA101 Antibody 7Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55
60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1158115PRTArtificialSequence of VL of GA101 Antibody 8Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn
Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Ala Gln Asn 85 90 95Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 110Arg Thr Val 1159448PRTArtificialSequence
of Heavy Chain Full Sequence of GA101 Antibody 9Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60Lys
Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44510219PRTArtificialSequence of Light Chain Full Sequence of GA101
Antibody 10Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu
Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu
Val Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150
155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 215
* * * * *