U.S. patent application number 14/863125 was filed with the patent office on 2016-03-24 for methods of using anti-cd79b immunoconjugates.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Yu-Waye Chu, Andrew Polson, Michael Wenger, Shang-Fan Yu.
Application Number | 20160082120 14/863125 |
Document ID | / |
Family ID | 54261107 |
Filed Date | 2016-03-24 |
United States Patent
Application |
20160082120 |
Kind Code |
A1 |
Polson; Andrew ; et
al. |
March 24, 2016 |
METHODS OF USING ANTI-CD79b IMMUNOCONJUGATES
Abstract
Provided herein are methods of treating B-cell proliferative
disorders in particular Follicular Lymphoma and/or Diffuse Large
B-Cell Lymphoma using immunoconjugates comprising anti-CD79b
antibodies in combination with additional therapeutic agents.
Inventors: |
Polson; Andrew; (San
Francisco, CA) ; Yu; Shang-Fan; (Millbrae, CA)
; Chu; Yu-Waye; (Burlingame, CA) ; Wenger;
Michael; (Los Altos Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
54261107 |
Appl. No.: |
14/863125 |
Filed: |
September 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62054257 |
Sep 23, 2014 |
|
|
|
62076823 |
Nov 7, 2014 |
|
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62136324 |
Mar 20, 2015 |
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Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 16/3061 20130101; A61K 31/4184 20130101; A61K 47/6889
20170801; A61K 2300/00 20130101; A61K 47/6849 20170801; A61K
47/6803 20170801; C07K 16/2887 20130101; A61K 45/06 20130101; A61K
31/553 20130101; A61K 2039/54 20130101; A61K 2039/545 20130101;
A61K 2039/507 20130101; A61K 31/635 20130101; C07K 2317/24
20130101; A61K 39/39558 20130101; A61K 47/6867 20170801; A61K
31/454 20130101; C07K 16/2803 20130101; A61P 35/00 20180101; A61K
39/39558 20130101; A61K 2300/00 20130101; A61K 31/4184 20130101;
A61K 2300/00 20130101; A61K 31/635 20130101; A61K 2300/00 20130101;
A61K 31/454 20130101; A61K 2300/00 20130101; A61K 31/553 20130101;
A61K 2300/00 20130101; A61K 39/3955 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/4184 20060101 A61K031/4184; A61K 39/395
20060101 A61K039/395 |
Claims
1. A method for treating a B-cell proliferative disorder in an
individual comprising administering to the individual an effective
amount of (a) an immunoconjugate comprising an anti-CD79b antibody
linked to a cytotoxic agent, (b) an anti-CD20 antibody, and (c) an
alkylating agent.
2. The method of claim 1, wherein the anti-CD20 antibody is
rituximab.
3. The method of claim 1, wherein the anti-CD20 antibody is a
humanized B-Lyl antibody.
4. The method of claim 3, wherein the humanized B-Lyl antibody is
obinituzumab.
5. The method of claim 1, wherein the anti-CD20 antibody is
ofatumumab, ublituximab, and/or ibritumomab tiuxetan.
6. The method of claim 1, wherein the alkylating agent is
4-[5-[Bis(2-chloroethy)amino]-1-methylbenzimidazol-2-yl]butanoic
acid and salts thereof.
7. The method of claim 6, wherein the alkylating agent is
bendamustine.
8. The method of claim 7, wherein the cytotoxic agent is an
antimitotic agent.
9. The method of claim 8, wherein the antimitotic agent is an
inhibitor of the polymerization of tubulin.
10. The method of claim 7, wherein the immunoconjugate has the
formula Ab-(L-D)p, wherein: (a) Ab is the antibody which binds
CD79b; (b) L is a linker; (c) D is the cytotoxic agent and the
cytotoxic agent is selected from a maytansinoid or an auristatin;
and (d) p ranges from 1-8.
11. The method of claim 10, wherein D is an auristatin.
12. The method of claim 10, wherein D has formula D.sub.E
##STR00020## and wherein R.sup.2 and R.sup.6 are each methyl,
R.sup.3 and R.sup.4 are each isopropyl, R.sup.5 is H, R.sup.7 is
sec-butyl, each R.sup.8 is independently selected from CH.sub.3,
O--CH.sub.3, OH, and H; R.sup.9 is H; and R.sup.18 is
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl.
13. The method of claim 10, wherein D is MMAE.
14. The method of claim 13, wherein the linker is cleavable by a
protease.
15. The method of claim 14, wherein the linker comprises a val-cit
dipeptide or a Phe-homoLys dipeptide.
16. The method of claim 13, wherein the linker is acid-labile.
17. The method of claim 16, wherein the linker comprises
hydrazone.
18. The method of claim 10 having the formula: ##STR00021## wherein
S is a sulfur atom.
19. The method of claim 18, wherein p ranges from 2-5.
20. The method of claim 19, wherein the antibody is a monoclonal
antibody.
21. The method of claim 20, wherein the antibody is a human,
humanized, or chimeric antibody.
22. The method of claim 21, wherein the antibody comprises (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO:21; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO:22; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO:23; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:24; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:25; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.
23. The method of claim 22, wherein the antibody comprises (a) a VH
comprising the amino acid sequence of SEQ ID NO:19 and (b) a VL
sequence comprises the amino acid sequence of SEQ ID NO:20.
24. The method of claim 23, wherein the antibody comprises (a) a
heavy chain comprising the amino acid sequence of SEQ ID NO:36 and
(b) a light chain comprising the amino acid sequence of SEQ ID
NO:35.
25. The method of claim 23, wherein the antibody comprises (a) a
heavy chain comprising the amino acid sequence of SEQ ID NO:37 and
(b) a light chain comprising the amino acid sequence of SEQ ID
NO:35.
26. The method of claim 23, wherein the antibody comprises (a) a
heavy chain comprising the amino acid sequence of SEQ ID NO:36 and
(b) a light chain comprising the amino acid sequence of SEQ ID
NO:38.
27. The method of claim 23, wherein the B-cell proliferative
disorder is cancer.
28. The method of claim 27, wherein the B-cell proliferative
disorder is lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,
relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,
refractory indolent NHL, chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute
lymphocytic leukemia (ALL), or mantle cell lymphoma.
29. The method of claim 27, wherein the B-cell proliferative
disorder is NHL, such as indolent NHL and/or aggressive NHL.
30. The method of claim 27, wherein the B-cell proliferative
disorder is indolent follicular lymphoma or diffuse large B-cell
lymphoma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims priority to U.S. Provisional
Application Ser. No. 62/054,257, filed on Sep. 23, 2014, and U.S.
Provisional Application Ser. No. 62/076,823, filed on Nov. 7, 2014,
and U.S. Provisional Application Ser. No. 62/136,324, filed on Mar.
20, 2015 the contents of which are incorporated by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing
submitted via EFS-Web and hereby incorporated by reference in its
entirety. Said ASCII copy, created on Sep. 22, 2015, is named
P32333-US-3_SL.txt, and is 64,601 bytes in size.
FIELD OF THE INVENTION
[0003] Provided herein are methods of treating B-cell proliferative
disorders in particular Follicular Lymphoma and/or Diffuse Large
B-Cell Lymphoma using immunoconjugates comprising anti-CD79b
antibodies in combination with additional therapeutic agents.
BACKGROUND OF THE INVENTION
[0004] CD79b is the signaling component of the B-cell receptor
which acts as a covalent heterodimer containing CD79a (i.e.,
Ig.alpha. or mb-1) and CD79b (i.e., Ig.beta. or B29). CD79b
contains an extracellular immunoglobulin (Ig) domain, a
transmembrane domain, and an intracellular signaling domain, an
immunoreceptor tyrosine-based activation motif (ITAM) domain. CD79
is expressed on B-cells and, for example, in Non-Hodgkin's Lymphoma
cells (NHLs) (Cabezudo et al., Haematologica 84:413-418 (1999);
D'Arena et al., Am. J. Hematol. 64: 275-281 (2000); Olejniczak et
al., Immunol. Invest. 35: 93-114 (2006)). CD79a and CD79b and sIg
are all required for surface expression of the CD79 (Matsuuchi et
al., Curr. Opin. Immunol. 13(3): 270-7)).
[0005] B-cell proliferative disorders are generally treated with
some combination of surgery, radiation therapy and/or drug
treatment. Accumulated empirical clinical experience, supported by
animal models, supports the hypothesis that cytotoxic drugs may be
more effective when given in combination to achieve additive or
synergistic effects. However, a caveat to the hypothesis is that
success requires the ability to combine drugs at their respective
effective doses without unacceptable side-effects and avoiding
possible pharmacokinetic interactions. Further, although it may
seem reasonable to combine a targeted agent with the standard of
care, clinical experience indicates that differences in
administration regimens and the dosages of each agents has an
effect on efficacy of the treatment. These factors have led to the
clinical failure of many combinations. See, e.g., Al-Lazikani et
al., Nature Biotechnology 30:679-692 (2012). There is a need in the
art for new treatment regimens for treating B-cell proliferative
disorders including treatments comprising agents that target CD79b
(e.g., anti-CD79b immunoconjugates).
[0006] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
SUMMARY
[0007] Provided herein are methods of treating a B-cell
proliferative disorder in an individual comprising (a) an
immunoconjugate comprising an antibody which binds CD79b linked to
a cytotoxic agent and (b) an additional therapeutic agent.
[0008] In particular, provided herein are methods for treating a
B-cell proliferative disorder in an individual comprising
administering to the individual an effective amount of (a) an
immunoconjugate comprising an anti-CD79b antibody linked to a
cytotoxic agent and (b) an alkylating agent. In some embodiments,
provided herein are methods for treating a B-cell proliferative
disorder in an individual comprising administering to the
individual an effective amount of (a) an immunoconjugate comprising
an anti-CD79b antibody linked to a cytotoxic agent, (b) an
anti-CD20 antibody, and (c) an alkylating agent.
[0009] In some embodiments of any of the methods, the anti-CD20
antibody is rituximab. In some embodiments, rituximab is
administered at about 375 mg/m.sup.2. In some embodiments of any of
the methods, the anti-CD20 antibody is a humanized B-Lyl antibody.
In some embodiments, the humanized B-Lyl antibody is obinituzumab.
In some embodiments, obinituzumab is administered at about 1000
mg/m.sup.2. In some embodiments of any of the methods, the
anti-CD20 antibody is ofatumumab, ublituximab, and/or ibritumomab
tiuxetan.
[0010] In some embodiments of any of the methods, the alkylating
agent is
4-[5-[Bis(2-chloroethy)amino]-1-methylbenzimidazol-2-yl]butanoic
acid and salts thereof. In some embodiments of any of the methods,
the alkylating agent is bendamustine. In some embodiments,
bendamustine is administered at about 25-120 mg/m.sup.2. In some
embodiments, bendamustine is administered at about 90
mg/m.sup.2.
[0011] In some embodiments of any of the methods, the cytotoxic
agent is an antimitotic agent. In some embodiments, the antimitotic
agent is an inhibitor of the polymerization of tubulin.
[0012] In some embodiments of any of the methods, the
immunoconjugate has the formula Ab-(L-D)p, wherein: (a) Ab is the
antibody which binds CD79b; (b) L is a linker; (c) D is the
cytotoxic agent and the cytotoxic agent is selected from a
maytansinoid or an auristatin; and (d) p ranges from 1-8.
[0013] In some embodiments of any of the methods, D is an
auristatin. In some embodiments of any of the methods, D has
formula D.sub.E
##STR00001##
and wherein R.sup.2 and R.sup.6 are each methyl, R.sup.3 and
R.sup.4 are each isopropyl, R.sup.5 is H, R.sup.7 is sec-butyl,
each R.sup.8 is independently selected from CH.sub.3, O--CH.sub.3,
OH, and H; R.sup.9 is H; and R.sup.18 is
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl. In some embodiments of
any of the methods, D is MMAE.
[0014] In some embodiments of any of the methods, the linker is
cleavable by a protease. In some embodiments, the linker comprises
a val-cit dipeptide or a Phe-homoLys dipeptide.
[0015] In some embodiments of any of the methods, the linker is
acid-labile. In some embodiments, the linker comprises
hydrazone.
[0016] In some embodiments of any of the methods, the formula
is:
##STR00002##
wherein S is a sulfur atom.
[0017] In some embodiments of any of the methods, p ranges from
2-5.
[0018] In some embodiments of any of the methods, the antibody is a
monoclonal antibody. In some embodiments, the antibody is a human,
humanized, or chimeric antibody.
[0019] In some embodiments of any of the methods, the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:26.
[0020] In some embodiments of any of the methods, the antibody
comprises (a) a VH comprising the amino acid sequence of SEQ ID
NO:19 and (b) a VL sequence comprises the amino acid sequence of
SEQ ID NO:20. In some embodiments of any of the methods, the
antibody comprises (a) a heavy chain comprising the amino acid
sequence of SEQ ID NO:36 and (b) a light chain comprising the amino
acid sequence of SEQ ID NO:35.
[0021] In some embodiments of any of the methods, the antibody is a
cysteine engineered antibody. In some embodiments, the antibody
comprises an engineered cysteine at position 118 according to EU
numbering convention of the heavy chain (A118C). In some
embodiments, the antibody comprises an engineered cysteine at
position 205 according to Kabat numbering convention of the light
chain (V205C). In some embodiments of any of the methods, the
cytotoxic agent is linked to the anti-CD79b antibody through the
engineered cysteine (e.g., at position 118 according to EU
numbering convention of the heavy chain and/or at position 205
according to Kabat numbering convention of the light chain). In
some embodiments of any of the methods, the antibody comprises (a)
a heavy chain comprising the amino acid sequence of SEQ ID NO:37
and (b) a light chain comprising the amino acid sequence of SEQ ID
NO:35. In some embodiments of any of the methods, the antibody
comprises (a) a heavy chain comprising the amino acid sequence of
SEQ ID NO:36 and (b) a light chain comprising the amino acid
sequence of SEQ ID NO:38.
[0022] In some embodiments of any of the methods, the B-cell
proliferative disorder is cancer. In some embodiments, the B-cell
proliferative disorder is lymphoma, non-Hodgkins lymphoma (NHL),
aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,
refractory NHL, refractory indolent NHL, chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell
leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell
lymphoma. In some embodiments, the B-cell proliferative disorder is
NHL, such as indolent NHL and/or aggressive NHL. In some
embodiments, the B-cell proliferative disorder is indolent
follicular lymphoma or diffuse large B-cell lymphoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows change in tumor volume (mm.sup.3) upon
treatment of WSU-CLCL2 (Diffuse Large B-cell Lymphoma with (a)
huMA79bv28-MC-vc-PAB-MMAE, (b) rituximab+bendamustine, and (c)
huMA79bv28-MC-vc-PAB-MMAE+rituximab+bendamustine.
huMA79bv28-MC-vc-PAB-MMAE: 2 mg/kg, iv, once on day 0; anti-CD20
(rituximab): 30 mg/kg, ip, once on day 0, and bendamustine: 30
mg/kg, iv, once on day 0.
[0024] FIG. 2 shows change in tumor volume (mm.sup.3) upon
treatment of tumor xenografts model of Granta-519 human mantle-cell
lymphoma with (a) vehicle, (b) huMA79bv28-MC-vc-PAB-MMAE
(DCDS4501A), (c) ABT-199, and (d) huMA79bv28-MC-vc-PAB-MMAE
(DCDS4501A)+ABT-199. huMA79bv28-MC-vc-PAB-MMAE (DCDS4501A): 1
mg/kg, iv, once on day 0 and ABT-199: 100 mg/kg, po, qd21.
[0025] FIG. 3A-B shows change in tumor volume (mm.sup.3) upon
treatment of tumor xenografts model of WSU-DLCL2 (DLBCL) and TMD8
(ABC-DLBCL) with various combination therapy regimens including
huMA79bv28-MC-vc-PAB-MMAE.
[0026] FIG. 4 shows change in tumor volume (mm.sup.3) upon
treatment of tumor xenografts model of WSU-DLCL2 (DLBCL) with
various combination therapy regimens huMA79bv28-MC-vc-PAB-MMAE.
DETAILED DESCRIPTION
[0027] Provided herein are methods of treating B-cell proliferative
disorders such as indolent and aggressive NHL using combinations of
immunoconjugates comprising an antibody which binds CD79b linked to
a cytotoxic agent (i.e., anti-CD79b immunoconjugate) and additional
therapeutic agents, in particular, in some embodiments, the
immunoconjugates comprise an antimitotic agent such as an inhibitor
of the polymerization of tubulin.
I. GENERAL TECHNIQUES
[0028] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature, such
as, "Molecular Cloning: A Laboratory Manual", second edition
(Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait,
ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987);
"Methods in Enzymology" (Academic Press, Inc.); "Current Protocols
in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and
periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et
al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal
Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et
al., 2001).
II. DEFINITIONS
[0029] The term "CD79b", as used herein, refers to any native CD79b
from any vertebrate source, including mammals such as primates
(e.g., humans, cynomologus monkey (cyno)) and rodents (e.g., mice
and rats), unless otherwise indicated. Human CD79b is also referred
herein to as "Ig.beta.," "B29," "DNA225786" or "PRO36249." An
exemplary CD79b sequence including the signal sequence is shown in
SEQ ID NO:1. An exemplary CD79b sequence without the signal
sequence is shown in SEQ ID NO:2. The term "CD79b" encompasses
"full-length," unprocessed CD79b as well as any form of CD79b that
results from processing in the cell. The term also encompasses
naturally occurring variants of CD79b, e.g., splice variants,
allelic variants and isoforms. The CD79b polypeptides described
herein may be isolated from a variety of sources, such as from
human tissue types or from another source, or prepared by
recombinant or synthetic methods. A "native sequence CD79b
polypeptide" comprises a polypeptide having the same amino acid
sequence as the corresponding CD79b polypeptide derived from
nature. Such native sequence CD79b polypeptides can be isolated
from nature or can be produced by recombinant or synthetic means.
The term "native sequence CD79b polypeptide" specifically
encompasses naturally-occurring truncated or secreted forms of the
specific CD79b polypeptide (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the
polypeptide.
[0030] "CD20" as used herein refers to the human B-lymphocyte
antigen CD20 (also known as CD20, B-lymphocyte surface antigen B1,
Leu-16, Bp35, BM5, and LF5; the sequence is characterized by the
SwissProt database entry P11836) is a hydrophobic transmembrane
protein with a molecular weight of approximately 35 kD located on
pre-B and mature B lymphocytes. (Valentine, M. A., et al., J. Biol.
Chem. 264(19) (1989 11282-11287; Tedder, T. F., et al, Proc. Natl.
Acad. Sci. U.S.A. 85 (1988) 208-12; Stamenkovic, I., et al., J.
Exp. Med. 167 (1988) 1975-80; Einfeld, D. A. et al., EMBO J. 7
(1988) 711-7; Tedder, T. F., et al., J. Immunol. 142 (1989)
2560-8). The corresponding human gene is Membrane-spanning
4-domains, subfamily A, member 1, also known as MS4A1. This gene
encodes a member of the membrane-spanning 4A gene family. Members
of this nascent protein family are characterized by common
structural features and similar intron/exon splice boundaries and
display unique expression patterns among hematopoietic cells and
nonlymphoid tissues. This gene encodes the B-lymphocyte surface
molecule which plays a role in the development and differentiation
of B-cells into plasma cells. This family member is localized to
11q12, among a cluster of family members. Alternative splicing of
this gene results in two transcript variants which encode the same
protein.
[0031] The terms "CD20" and "CD20 antigen" are used interchangeably
herein, and include any variants, isoforms and species homologs of
human CD20 which are naturally expressed by cells or are expressed
on cells transfected with the CD20 gene. Binding of an antibody of
the invention to the CD20 antigen mediate the killing of cells
expressing CD20 (e.g., a tumor cell) by inactivating CD20. The
killing of the cells expressing CD20 may occur by one or more of
the following mechanisms: Cell death/apoptosis induction, ADCC and
CDC. Synonyms of CD20, as recognized in the art, include
B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16,
Bp35, BM5, and LF5.
[0032] The term "expression of the CD20" antigen is intended to
indicate a significant level of expression of the CD20 antigen in a
cell, e.g., a T- or B-Cell. In one embodiment, patients to be
treated according to the methods of this invention express
significant levels of CD20 on a B-cell 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 (IHC) detection, FACS or via PCR-based
detection of the corresponding mRNA.
[0033] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0034] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0035] 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.
[0036] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g., scFv); and multispecific antibodies
formed from antibody fragments.
[0037] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0038] The term "epitope" refers to the particular site on an
antigen molecule to which an antibody binds.
[0039] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0040] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0041] The term "anti-CD79b antibody" or "an antibody that binds to
CD79b" refers to an antibody that is capable of binding CD79b with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting CD79b. Preferably,
the extent of binding of an anti-CD79b antibody to an unrelated,
non-CD79b protein is less than about 10% of the binding of the
antibody to CD79b as measured, e.g., by a radioimmunoassay (RIA).
In certain embodiments, an antibody that binds to CD79b has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In certain
embodiments, anti-CD79b antibody binds to an epitope of CD79b that
is conserved among CD79b from different species.
[0042] The term "anti-CD20 antibody" according to the invention
refers to an antibody that is capable of binding CD20 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting CD20. Preferably,
the extent of binding of an anti-CD20 antibody to an unrelated,
non-CD20 protein is less than about 10% of the binding of the
antibody to CD20 as measured, e.g., by a radioimmunoassay (RIA). In
certain embodiments, an antibody that binds to CD20 has a
dissociation constant (Kd) of .ltoreq.1 04, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In certain
embodiments, anti-CD20 antibody binds to an epitope of CD20 that is
conserved among CD20 from different species.
[0043] The term "PD-1 axis binding antagonist" refers to a molecule
that inhibits the interaction of a PD-1 axis binding partner with
either one or more of its binding partner, so as to remove T-cell
dysfunction resulting from signaling on the PD-1 signaling
axis--with a result being to restore or enhance T-cell function
(e.g., proliferation, cytokine production, target cell killing). As
used herein, a PD-1 axis binding antagonist includes a PD-1 binding
antagonist, a PD-L1 binding antagonist and a PD-L2 binding
antagonist.
[0044] The term "PD-1 binding antagonist" refers to a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-1 with one or
more of its binding partners, such as PD-L1, PD-L2. In some
embodiments, the PD-1 binding antagonist is a molecule that
inhibits the binding of PD-1 to one or more of its binding
partners. In a specific aspect, the PD-1 binding antagonist
inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example,
PD-1 binding antagonists include anti-PD-1 antibodies, antigen
binding fragments thereof, immunoadhesins, fusion proteins,
oligopeptides and other molecules that decrease, block, inhibit,
abrogate or interfere with signal transduction resulting from the
interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a
PD-1 binding antagonist reduces the negative co-stimulatory signal
mediated by or through cell surface proteins expressed on T
lymphocytes mediated signaling through PD-1 so as render a
dysfunctional T-cell less dysfunctional (e.g., enhancing effector
responses to antigen recognition). In some embodiments, the PD-1
binding antagonist is an anti-PD-1 antibody. In a specific aspect,
a PD-1 binding antagonist is MDX-1106 (nivolumab) described herein.
In another specific aspect, a PD-1 binding antagonist is MK-3475
(lambrolizumab) described herein. In another specific aspect, a
PD-1 binding antagonist is CT-011 (pidilizumab) described herein.
In another specific aspect, a PD-1 binding antagonist is AMP-224
described herein.
[0045] The term "PD-L1 binding antagonist" refers to a molecule
that decreases, blocks, inhibits, abrogates or interferes with
signal transduction resulting from the interaction of PD-L1 with
either one or more of its binding partners, such as PD-1, B7-1. In
some embodiments, a PD-L1 binding antagonist is a molecule that
inhibits the binding of PD-L1 to its binding partners. In a
specific aspect, the PD-L1 binding antagonist inhibits binding of
PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding
antagonists include anti-PD-L1 antibodies, antigen binding
fragments thereof, immunoadhesins, fusion proteins, oligopeptides
and other molecules that decrease, block, inhibit, abrogate or
interfere with signal transduction resulting from the interaction
of PD-L1 with one or more of its binding partners, such as PD-1,
B7-1. In one embodiment, a PD-L1 binding antagonist reduces the
negative co-stimulatory signal mediated by or through cell surface
proteins expressed on T lymphocytes mediated signaling through
PD-L1 so as to render a dysfunctional T-cell less dysfunctional
(e.g., enhancing effector responses to antigen recognition). In
some embodiments, a PD-L1 binding antagonist is an anti-PD-L1
antibody. In a specific aspect, an anti-PD-L1 antibody is
YW243.55.S70 described herein. In another specific aspect, an
anti-PD-L1 antibody is MDX-1105 described herein. In still another
specific aspect, an anti-PD-L1 antibody is MPDL3280A described
herein. In still another specific aspect, an anti-PD-L1 antibody is
MEDI4736 described herein.
[0046] The term "PD-L2 binding antagonist" refers to a molecule
that decreases, blocks, inhibits, abrogates or interferes with
signal transduction resulting from the interaction of PD-L2 with
either one or more of its binding partners, such as PD-1. In some
embodiments, a PD-L2 binding antagonist is a molecule that inhibits
the binding of PD-L2 to one or more of its binding partners. In a
specific aspect, the PD-L2 binding antagonist inhibits binding of
PD-L2 to PD-1. In some embodiments, the PD-L2 antagonists include
anti-PD-L2 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-L2 with either
one or more of its binding partners, such as PD-1. In one
embodiment, a PD-L2 binding antagonist reduces the negative
co-stimulatory signal mediated by or through cell surface proteins
expressed on T lymphocytes mediated signaling through PD-L2 so as
render a dysfunctional T-cell less dysfunctional (e.g., enhancing
effector responses to antigen recognition). In some embodiments, a
PD-L2 binding antagonist is an immunoadhesin.
[0047] The term "dysfunction" in the context of immune dysfunction,
refers to a state of reduced immune responsiveness to antigenic
stimulation. The term includes the common elements of both
exhaustion and/or anergy in which antigen recognition may occur,
but the ensuing immune response is ineffective to control infection
or tumor growth.
[0048] The term "dysfunctional", as used herein, also includes
refractory or unresponsive to antigen recognition, specifically,
impaired capacity to translate antigen recognition into down-stream
T-cell effector functions, such as proliferation, cytokine
production (e.g., IL-2) and/or target cell killing.
[0049] The term "anergy" refers to the state of unresponsiveness to
antigen stimulation resulting from incomplete or insufficient
signals delivered through the T-cell receptor (e.g., increase in
intracellular Ca.sup.+2 in the absence of ras-activation). T cell
anergy can also result upon stimulation with antigen in the absence
of co-stimulation, resulting in the cell becoming refractory to
subsequent activation by the antigen even in the context of
costimulation. The unresponsive state can often be overriden by the
presence of Interleukin-2. Anergic T-cells do not undergo clonal
expansion and/or acquire effector functions.
[0050] The term "exhaustion" refers to T cell exhaustion as a state
of T cell dysfunction that arises from sustained TCR signaling that
occurs during many chronic infections and cancer. It is
distinguished from anergy in that it arises not through incomplete
or deficient signaling, but from sustained signaling. It is defined
by poor effector function, sustained expression of inhibitory
receptors and a transcriptional state distinct from that of
functional effector or memory T cells. Exhaustion prevents optimal
control of infection and tumors. Exhaustion can result from both
extrinsic negative regulatory pathways (e.g., immunoregulatory
cytokines) as well as cell intrinsic negative regulatory
(costimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).
[0051] "Enhancing T-cell function" means to induce, cause or
stimulate a T-cell to have a sustained or amplified biological
function, or renew or reactivate exhausted or inactive T-cells.
Examples of enhancing T-cell function include: increased secretion
of .gamma.-interferon from CD8.sup.+ T-cells, increased
proliferation, increased antigen responsiveness (e.g., viral,
pathogen, or tumor clearance) relative to such levels before the
intervention. In one embodiment, the level of enhancement is as
least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%,
and/or 200%. The manner of measuring this enhancement is known to
one of ordinary skill in the art.
[0052] A "T cell dysfunctional disorder" is a disorder or condition
of T-cells characterized by decreased responsiveness to antigenic
stimulation. In a particular embodiment, a T-cell dysfunctional
disorder is a disorder that is specifically associated with
inappropriate increased signaling through PD-1. In another
embodiment, a T-cell dysfunctional disorder is one in which T-cells
are anergic or have decreased ability to secrete cytokines,
proliferate, or execute cytolytic activity. In a specific aspect,
the decreased responsiveness results in ineffective control of a
pathogen or tumor expressing an immunogen. Examples of T cell
dysfunctional disorders characterized by T-cell dysfunction include
unresolved acute infection, chronic infection and tumor
immunity.
[0053] "Tumor immunity" refers to the process in which tumors evade
immune recognition and clearance. Thus, as a therapeutic concept,
tumor immunity is "treated" when such evasion is attenuated, and
the tumors are recognized and attacked by the immune system.
Examples of tumor recognition include tumor binding, tumor
shrinkage and tumor clearance.
[0054] "Immunogenecity" refers to the ability of a particular
substance to provoke an immune response. Tumors are immunogenic and
enhancing tumor immunogenicity aids in the clearance of the tumor
cells by the immune response. Examples of enhancing tumor
immunogenicity include treatment with a PD-1 axis binding
antagonist and an anti-CD79b immunoconjugate (e.g.,
anti-CD79b-MC-vc-PAB-MMAE).
[0055] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0056] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007). The "variable
region" or "variable domain" of an antibody refers to the
amino-terminal domains of the heavy or light chain of the antibody.
The variable domain of the heavy chain may be referred to as "VH."
The variable domain of the light chain may be referred to as "VL."
These domains are generally the most variable parts of an antibody
and contain the antigen-binding sites.
[0057] "Isolated nucleic acid encoding an anti-CD79b antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0058] 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.
[0059] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0060] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0061] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0062] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0063] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0064] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0065] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0066] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0067] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0068] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0069] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur 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).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0070] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0071] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g., B-cell receptor); and B-cell activation.
[0072] "CD79b polypeptide variant" means a CD79b polypeptide,
preferably an active CD79b polypeptide, as defined herein having at
least about 80% amino acid sequence identity with a full-length
native sequence CD79b polypeptide sequence as disclosed herein, a
CD79b polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a CD79b polypeptide, with or
without the signal peptide, as disclosed herein or any other
fragment of a full-length CD79b polypeptide sequence as disclosed
herein (such as those encoded by a nucleic acid that represents
only a portion of the complete coding sequence for a full-length
CD79b polypeptide). Such CD79b polypeptide variants include, for
instance, CD79b polypeptides wherein one or more amino acid
residues are added, or deleted, at the N- or C-terminus of the
full-length native amino acid sequence. Ordinarily, a CD79b
polypeptide variant will have at least about 80% amino acid
sequence identity, alternatively at least about 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% amino acid sequence identity, to a full-length native
sequence CD79b polypeptide sequence as disclosed herein, a CD79b
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a CD79b polypeptide, with or
without the signal peptide, as disclosed herein or any other
specifically defined fragment of a full-length CD79b polypeptide
sequence as disclosed herein. Ordinarily, CD79b variant
polypeptides are at least about 10 amino acids in length,
alternatively at least about 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 amino acids
in length, or more. Optionally, CD79b variant polypeptides will
have no more than one conservative amino acid substitution as
compared to the native CD79b polypeptide sequence, alternatively no
more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid
substitution as compared to the native CD79b polypeptide
sequence.
[0073] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0074] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
[0075] 100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0076] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0077] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0078] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0079] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome. More specific examples include,
but are not limited to, relapsed or refractory NHL, front line low
grade NHL, Stage III/IV NHL, chemotherapy resistant NHL, precursor
B lymphoblastic leukemia and/or lymphoma, small lymphocytic
lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic
leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic
lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma,
lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic
marginal zone lymphoma, extranodal marginal zone--MALT lymphoma,
nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma
and/or plasma cell myeloma, low grade/follicular lymphoma,
intermediate grade/follicular NHL, mantle cell lymphoma, follicle
center lymphoma (follicular), intermediate grade diffuse NHL,
diffuse large B-cell lymphoma, aggressive NHL (including aggressive
front-line NHL and aggressive relapsed NHL), NHL relapsing after or
refractory to autologous stem cell transplantation, primary
mediastinal large B-cell lymphoma, primary effusion lymphoma, high
grade immunoblastic NHL, high grade lymphoblastic NHL, high grade
small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma,
precursor (peripheral) large granular lymphocytic leukemia, mycosis
fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas,
anaplastic large cell lymphoma, angiocentric lymphoma.
[0080] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0081] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0082] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0083] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0084] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, reduction of
free light chain, preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, decreasing the rate of
disease progression, amelioration or palliation of the disease
state, and remission or improved prognosis. In some embodiments,
the antibodies described herein are used to delay development of a
disease or to slow the progression of a disease.
[0085] The term "CD79b-positive cancer" refers to a cancer
comprising cells that express CD79b on their surface. In some
embodiments, expression of CD79b on the cell surface is determined,
for example, using antibodies to CD79b in a method such as
immunohistochemistry, FACS, etc. Alternatively, CD79b mRNA
expression is considered to correlate to CD79b expression on the
cell surface and can be determined by a method selected from in
situ hybridization and RT-PCR (including quantitative RT-PCR).
[0086] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during, or after
administration of the other treatment modality to the
individual.
[0087] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include erlotinib (TARCEVA.RTM., Genentech/OSI Pharm.), bortezomib
(VELCADE.RTM., Millennium Pharm.), disulfiram, epigallocatechin
gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin),
radicicol, lactate dehydrogenase A (LDH-A), fulvestrant
(FASLODEX.RTM., AstraZeneca), sunitib (SUTENT.RTM., Pfizer/Sugen),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
Novartis), finasunate (VATALANIB.RTM., Novartis), oxaliplatin
(ELOXATIN.RTM., Sanofi), 5-FU (5-fluorouracil), leucovorin,
Rapamycin (Sirolimus, RAPAMUNE.RTM., Wyeth), Lapatinib
(TYKERB.RTM., GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336),
sorafenib (NEXAVAR.RTM., Bayer Labs), gefitinib (IRESSA.RTM.,
AstraZeneca), AG1478, alkylating agents such as thiotepa and
CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including topotecan and
irinotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogs);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
adrenocorticosteroids (including prednisone and prednisolone);
cyproterone acetate; 5.alpha.-reductases including finasteride and
dutasteride); vorinostat, romidepsin, panobinostat, valproic acid,
mocetinostat dolastatin; aldesleukin, talc duocarmycin (including
the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin;
pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards
such as chlorambucil, chlomaphazine, chlorophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosoureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;
antibiotics such as the enediyne antibiotics (e.g., calicheamicin,
especially calicheamicin .gamma.1I and calicheamicin .omega.1I
(Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. (doxorubicin), morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, everolimus, sotrataurin,
idarubicin, marcellomycin, mitomycins such as mitomycin C,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogs such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfomithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology,
Princeton, N.J.), ABRAXANE.RTM. (Cremophor-free),
albumin-engineered nanoparticle formulations of paclitaxel
(American Pharmaceutical Partners, Schaumberg, Ill.), and
TAXOTERE.RTM. (docetaxel, doxetaxel; Sanofi-Aventis);
chloranmbucil; GEMZAR.RTM. (gemcitabine); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. (vinorelbine);
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
capecitabine (XELODA.RTM.); ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; and pharmaceutically acceptable salts, acids and
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone, and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and leucovovin.
Additional examples include of chemotherapeutic agents include
bendamustine (TREANDA.RTM.), ibrutinib, lenalidomide, and/or
idelalisib (GS-1101).
[0088] Additional examples of chemotherapeutic agents include
anti-hormonal agents that act to regulate, reduce, block, or
inhibit the effects of hormones that can promote the growth of
cancer, and are often in the form of systemic, or whole-body
treatment. They may be hormones themselves. Examples include
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene (EVISTA.RTM.), droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene (FARESTON.RTM.); anti-progesterones; estrogen
receptor down-regulators (ERDs); estrogen receptor antagonists such
as fulvestrant (FASLODEX.RTM.); agents that function to suppress or
shut down the ovaries, for example, leutinizing hormone-releasing
hormone (LHRH) agonists such as leuprolide acetate (LUPRON.RTM. and
ELIGARD.RTM.), goserelin acetate, buserelin acetate and
tripterelin; anti-androgens such as flutamide, nilutamide and
bicalutamide; and aromatase inhibitors that inhibit the enzyme
aromatase, which regulates estrogen production in the adrenal
glands, such as, for example, 4(5)-imidazoles, aminoglutethimide,
megestrol acetate (MEGASE.RTM.), exemestane (AROMASIN.RTM.),
formestanie, fadrozole, vorozole (RIVISOR.RTM.), letrozole
(FEMARA.RTM.), and anastrozole (ARIMIDEX.RTM.). In addition, such
definition of chemotherapeutic agents includes bisphosphonates such
as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.), etidronate
(DIDROCAL.RTM.), NE-58095, zoledronic acid/zoledronate
(ZOMETA.RTM.), alendronate (FOSAMAX.RTM.), pamidronate
(AREDIA.RTM.), tiludronate (SKELID.RTM.), or risedronate
(ACTONEL.RTM.); as well as troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); anti-sense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
[0089] In some embodiments, the chemotherapeutic agent includes
topoisomerase 1 inhibitor (e.g., LURTOTECAN.RTM.); an anti-estrogen
such as fulvestrant; a Kit inhibitor such as imatinib or EXEL-0862
(a tyrosine kinase inhibitor); EGFR inhibitor such as erlotinib or
cetuximab; an anti-VEGF inhibitor such as bevacizumab; arinotecan;
rmRH (e.g., ABARELIX.RTM.); lapatinib and lapatinib ditosylate (an
ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also
known as GW572016); 17AAG (geldanamycin derivative that is a heat
shock protein (Hsp) 90 poison), and pharmaceutically acceptable
salts, acids or derivatives of any of the above.
[0090] Chemotherapetuic agent also includes antibodies such as
alemtuzumab (Campath), bevacizumab (AVASTIN.RTM., Genentech);
cetuximab (ERBITUX.RTM., Imclone); panitumumab (VECTIBIX.RTM.,
Amgen), rituximab (RITUXAN.RTM., Genentech/Biogen Idec),
ublituximab, ofatumumab, ibritumomab tiuxetan, pertuzumab
(OMNITARG.RTM., 2C4, Genentech), trastuzumab (HERCEPTIN.RTM.,
Genentech), tositumomab (Bexxar, Corixia), and the antibody drug
conjugate, gemtuzumab ozogamicin (MYLOTARG.RTM., Wyeth). Additional
humanized monoclonal antibodies with therapeutic potential as
agents in combination with the compounds include: apolizumab,
aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine,
cantuzumab mertansine, cedelizumab, certolizumab pegol,
cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab,
epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab
ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab,
lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab,
natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab,
omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab,
pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab,
resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab,
sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab,
tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin,
tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab,
and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and
Abbott Laboratories) which is a recombinant exclusively
human-sequence, full-length IgG1 .lamda. antibody genetically
modified to recognize interleukin-12 p40 protein.
[0091] As used herein, the term "cytokine" refers generically to
proteins released by one cell population that act on another cell
as intercellular mediators or have an autocrine effect on the cells
producing the proteins. Examples of such cytokines include
lymphokines, monokines; interleukins ("ILs") such as IL-1,
IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10,
IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as
IL-23), IL-31, including PROLEUKIN.RTM. rIL-2; a tumor-necrosis
factor such as TNF-.alpha. or TNF-.beta., TGF-.beta.1-3; and other
polypeptide factors including leukemia inhibitory factor ("LIF"),
ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"),
cardiotrophin ("CT"), and kit ligand ("KL").
[0092] As used herein, the term "chemokine" refers to soluble
factors (e.g., cytokines) that have the ability to selectively
induce chemotaxis and activation of leukocytes. They also trigger
processes of angiogenesis, inflammation, wound healing, and
tumorigenesis. Example chemokines include IL-8, a human homolog of
murine keratinocyte chemoattractant (KC).
[0093] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0094] "Alkyl" is C.sub.1-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms. Examples are methyl
(Me, --CH.sub.3), ethyl (Et, --CH.sub.2CH.sub.3), 1-propyl (n-Pr,
n-propyl, --CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (i-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3.
[0095] The term "C.sub.1-C.sub.8 alkyl," as used herein refers to a
straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 8 carbon atoms. Representative "C.sub.1-C.sub.8
alkyl" groups include, but are not limited to, -methyl, -ethyl,
-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl,
-n-nonyl and -n-decyl; while branched C.sub.1-C.sub.8 alkyls
include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,
-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C.sub.1-C.sub.8
alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl,
-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,
1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butyryl,
-2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1 butynyl. A
C.sub.1-C.sub.8 alkyl group can be unsubstituted or substituted
with one or more groups including, but not limited to,
--C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl), -aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2--NHC(O)R', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN; where each R' is independently selected
from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0096] The term "C.sub.1-C.sub.12 alkyl," as used herein refers to
a straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 12 carbon atoms. A C.sub.1-C.sub.12 alkyl group
can be unsubstituted or substituted with one or more groups
including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--SO.sub.3R', --S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3,
--NH.sub.2, --NH(R'), --N(R').sub.2 and --CN; where each R' is
independently selected from H, --C.sub.1-C.sub.8 alkyl and
aryl.
[0097] The term "C.sub.1-C.sub.6 alkyl," as used herein refers to a
straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 6 carbon atoms. Representative "C.sub.1-C.sub.6
alkyl" groups include, but are not limited to, -methyl, -ethyl,
-n-propyl, -n-butyl, -n-pentyl, and n-hexyl; while branched
C.sub.1-C.sub.6 alkyls include, but are not limited to, -isopropyl,
-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;
unsaturated C.sub.1-C.sub.6 alkyls include, but are not limited to,
-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl,
-1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,
-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A
C.sub.1-C.sub.6 alkyl group can be unsubstituted or substituted
with one or more groups, as described above for C.sub.1-C.sub.8
alkyl group.
[0098] The term "C.sub.1-C.sub.4 alkyl," as used herein refers to a
straight chain or branched, saturated or unsaturated hydrocarbon
having from 1 to 4 carbon atoms. Representative "C.sub.1-C.sub.4
alkyl" groups include, but are not limited to, -methyl, -ethyl,
-n-propyl, -n-butyl; while branched C.sub.1-C.sub.4 alkyls include,
but are not limited to, -isopropyl, -sec-butyl, -isobutyl,
-tert-butyl; unsaturated C.sub.1-C.sub.4 alkyls include, but are
not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, and
-isobutylenyl. A C.sub.1-C.sub.4 alkyl group can be unsubstituted
or substituted with one or more groups, as described above for
C.sub.1-C.sub.8 alkyl group.
[0099] "Alkoxy" is an alkyl group singly bonded to an oxygen.
Exemplary alkoxy groups include, but are not limited to, methoxy
(--OCH.sub.3) and ethoxy (--OCH.sub.2CH.sub.3). A "C.sub.1-C.sub.5
alkoxy" is an alkoxy group with 1 to 5 carbon atoms. Alkoxy groups
may can be unsubstituted or substituted with one or more groups, as
described above for alkyl groups.
[0100] "Alkenyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp.sup.2 double bond.
Examples include, but are not limited to: ethylene or vinyl
(--CH.dbd.CH.sub.2), allyl (--CH.sub.2CH.dbd.CH.sub.2),
cyclopentenyl (--C.sub.5H.sub.7), and 5-hexenyl (--CH.sub.2
CH.sub.2CH.sub.2CH.sub.2CH.dbd.CH.sub.2). A "C.sub.2-C.sub.8
alkenyl" is a hydrocarbon containing 2 to 8 normal, secondary,
tertiary or cyclic carbon atoms with at least one site of
unsaturation, i.e. a carbon-carbon, sp.sup.2 double bond.
[0101] "Alkynyl" is C.sub.2-C.sub.18 hydrocarbon containing normal,
secondary, tertiary or cyclic carbon atoms with at least one site
of unsaturation, i.e. a carbon-carbon, sp triple bond. Examples
include, but are not limited to: acetylenic (--C.ident.CH) and
propargyl (--CH.sub.2C.ident.CH). A "C.sub.2-C.sub.8 alkynyl" is a
hydrocarbon containing 2 to 8 normal, secondary, tertiary or cyclic
carbon atoms with at least one site of unsaturation, i.e. a
carbon-carbon, sp triple bond.
[0102] "Alkylene" refers to a saturated, branched or straight chain
or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two
monovalent radical centers derived by the removal of two hydrogen
atoms from the same or two different carbon atoms of a parent
alkane. Typical alkylene radicals include, but are not limited to:
methylene (--CH.sub.2--) 1,2-ethyl (--CH.sub.2CH.sub.2--),
1,3-propyl (--CH.sub.2CH.sub.2CH.sub.2--), 1,4-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like.
[0103] A "C.sub.1-C.sub.10 alkylene" is a straight chain, saturated
hydrocarbon group of the formula --(CH.sub.2).sub.1-10--. Examples
of a C.sub.1-C.sub.10 alkylene include methylene, ethylene,
propylene, butylene, pentylene, hexylene, heptylene, ocytylene,
nonylene and decalene.
[0104] "Alkenylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkene. Typical alkenylene radicals include, but are not
limited to: 1,2-ethylene (--CH.dbd.CH--).
[0105] "Alkynylene" refers to an unsaturated, branched or straight
chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and
having two monovalent radical centers derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent alkyne. Typical alkynylene radicals include, but are not
limited to: acetylene (--C.ident.C--), propargyl
(--CH.sub.2C.ident.C--), and 4-pentynyl
(--CH.sub.2CH.sub.2CH.sub.2C.ident.C--).
[0106] "Aryl" refers to a carbocyclic aromatic group. Examples of
aryl groups include, but are not limited to, phenyl, naphthyl and
anthracenyl. A carbocyclic aromatic group or a heterocyclic
aromatic group can be unsubstituted or substituted with one or more
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0107] A "C.sub.5-C.sub.20 aryl" is an aryl group with 5 to 20
carbon atoms in the carbocyclic aromatic rings. Examples of
C.sub.5-C.sub.20 aryl groups include, but are not limited to,
phenyl, naphthyl and anthracenyl. A C.sub.5-C.sub.20 aryl group can
be substituted or unsubstituted as described above for aryl groups.
A "C.sub.5-C.sub.14 aryl" is an aryl group with 5 to 14 carbon
atoms in the carbocyclic aromatic rings. Examples of
C.sub.5-C.sub.14 aryl groups include, but are not limited to,
phenyl, naphthyl and anthracenyl. A C.sub.5-C.sub.14 aryl group can
be substituted or unsubstituted as described above for aryl
groups.
[0108] An "arylene" is an aryl group which has two covalent bonds
and can be in the ortho, meta, or para configurations as shown in
the following structures:
##STR00003##
in which the phenyl group can be unsubstituted or substituted with
up to four groups including, but not limited to, --C.sub.1-C.sub.8
alkyl, --O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0109] "Arylalkyl" refers to an acyclic alkyl radical in which one
of the hydrogen atoms bonded to a carbon atom, typically a terminal
or sp.sup.3 carbon atom, is replaced with an aryl radical. Typical
arylalkyl groups include, but are not limited to, benzyl,
2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,
2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,
2-naphthophenylethan-1-yl and the like. The arylalkyl group
comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, including
alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to
6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
[0110] "Heteroarylalkyl" refers to an acyclic alkyl radical in
which one of the hydrogen atoms bonded to a carbon atom, typically
a terminal or sp.sup.3 carbon atom, is replaced with a heteroaryl
radical. Typical heteroarylalkyl groups include, but are not
limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The
heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g., the
alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the
heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl
moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from
N, O, P, and S. The heteroaryl moiety of the heteroarylalkyl group
may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and S), for example: a
bicyclo[4,5], [5,5], [5,6], or [6,6] system.
[0111] "Substituted alkyl," "substituted aryl," and "substituted
arylalkyl" mean alkyl, aryl, and arylalkyl respectively, in which
one or more hydrogen atoms are each independently replaced with a
substituent. Typical substituents include, but are not limited to,
--X, --R, --O.sup.-, --OR, --SR, --S.sup.-, --NR.sub.2, --NR.sub.3,
.dbd.NR, --CX.sub.3, --CN, --OCN, --SCN, --N.dbd.C.dbd.O, --NCS,
--NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3, NC(.dbd.O)R,
--C(.dbd.O)R, --C(.dbd.O)NR.sub.2, --SO.sub.3.sup.-, --SO.sub.3H,
--S(.dbd.O).sub.2R, --OS(.dbd.O).sub.2OR, --S(.dbd.O).sub.2NR,
--S(.dbd.O)R, --OP(.dbd.O)(OR).sub.2, --P(.dbd.O)(OR).sub.2,
--PO.sup.-.sub.3, --PO.sub.3H.sub.2, --C(.dbd.O)R, --C(.dbd.O)X,
--C(.dbd.S)R, --CO.sub.2R, --CO.sub.2.sup.-, --C(.dbd.S)OR,
--C(.dbd.O)SR, --C(.dbd.S)SR, --C(.dbd.O)NR.sub.2,
--C(.dbd.S)NR.sub.2, --C(.dbd.NR)NR.sub.2, where each X is
independently a halogen: F, Cl, Br, or I; and each R is
independently --H, C.sub.2-C.sub.18 alkyl, C.sub.6-C.sub.20 aryl,
C.sub.3-C.sub.14 heterocycle, protecting group or prodrug moiety.
Alkylene, alkenylene, and alkynylene groups as described above may
also be similarly substituted.
[0112] "Heteroaryl" and "heterocycle" refer to a ring system in
which one or more ring atoms is a heteroatom, e.g., nitrogen,
oxygen, and sulfur. The heterocycle radical comprises 3 to 20
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. A
heterocycle may be a monocycle having 3 to 7 ring members (2 to 6
carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S)
or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1
to 3 heteroatoms selected from N, O, P, and S), for example: a
bicyclo[4,5], [5,5], [5,6], or [6,6] system.
[0113] Exemplary heterocycles are described, e.g., in Paquette, Leo
A., "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin,
New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The
Chemistry of Heterocyclic Compounds, A series of Monographs" (John
Wiley & Sons, New York, 1950 to present), in particular Volumes
13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
[0114] Examples of heterocycles include by way of example and not
limitation pyridyl, dihydroypyridyl, tetrahydropyridyl(piperidyl),
thiazolyl, tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,
pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,
indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl,
pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,
tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl.
[0115] By way of example and not limitation, carbon bonded
heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine,
position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,
or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or
thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4
of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more
typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,
4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,
5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,
5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or
5-thiazolyl.
[0116] By way of example and not limitation, nitrogen bonded
heterocycles are bonded at position 1 of an aziridine, azetidine,
pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,
imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,
2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole,
indoline, 1H-indazole, position 2 of a isoindole, or isoindoline,
position 4 of a morpholine, and position 9 of a carbazole, or
.beta.-carboline. Still more typically, nitrogen bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0117] A "C.sub.3-C.sub.8 heterocycle" refers to an aromatic or
non-aromatic C.sub.3-C.sub.8 carbocycle in which one to four of the
ring carbon atoms are independently replaced with a heteroatom from
the group consisting of O, S and N. Representative examples of a
C.sub.3-C.sub.8 heterocycle include, but are not limited to,
benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl,
isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,
imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl,
pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl,
isoxazolyl and tetrazolyl. A C.sub.3-C.sub.8 heterocycle can be
unsubstituted or substituted with up to seven groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; wherein each R' is independently selected from H,
--C.sub.1-C.sub.8 alkyl and aryl.
[0118] "C.sub.3-C.sub.8 heterocyclo" refers to a C.sub.3-C.sub.8
heterocycle group defined above wherein one of the heterocycle
group's hydrogen atoms is replaced with a bond. A C.sub.3-C.sub.8
heterocyclo can be unsubstituted or substituted with up to six
groups including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0119] A "C.sub.3-C.sub.20 heterocycle" refers to an aromatic or
non-aromatic C.sub.3-C.sub.8 carbocycle in which one to four of the
ring carbon atoms are independently replaced with a heteroatom from
the group consisting of O, S and N. A C.sub.3-C.sub.20 heterocycle
can be unsubstituted or substituted with up to seven groups
including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--O--(C.sub.1-C.sub.8 alkyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2--NHC(O)R',
--S(O).sub.2R', --S(O)R', --OH, -halogen, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN; wherein each R' is independently
selected from H, --C.sub.1-C.sub.8 alkyl and aryl.
[0120] "C.sub.3-C.sub.20 heterocyclo" refers to a C.sub.3-C.sub.20
heterocycle group defined above wherein one of the heterocycle
group's hydrogen atoms is replaced with a bond.
[0121] "Carbocycle" means a saturated or unsaturated ring having 3
to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a
bicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still more
typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring
atoms, e.g., arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6]
system, or 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6]
system. Examples of monocyclic carbocycles include cyclopropyl,
cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and
cyclooctyl.
[0122] A "C.sub.3-C.sub.8 carbocycle" is a 3-, 4-, 5-, 6-, 7- or
8-membered saturated or unsaturated non-aromatic carbocyclic ring.
Representative C.sub.3-C.sub.8 carbocycles include, but are not
limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl,
-cyclopentadienyl, -cyclohexyl, -cyclohexenyl,
-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,
-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and
-cyclooctadienyl. A C.sub.3-C.sub.8 carbocycle group can be
unsubstituted or substituted with one or more groups including, but
not limited to, --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8
alkyl), -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2--NHC(O)R', --S(O).sub.2R', --S(O)R',
--OH, -halogen, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN; where each R' is independently selected from H,
--C.sub.1-C.sub.8 alkyl and aryl.
[0123] A "C.sub.3-C.sub.8 carbocyclo" refers to a C.sub.3-C.sub.8
carbocycle group defined above wherein one of the carbocycle
groups' hydrogen atoms is replaced with a bond.
[0124] "Linker" refers to a chemical moiety comprising a covalent
bond or a chain of atoms that covalently attaches an antibody to a
drug moiety. In various embodiments, linkers include a divalent
radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl,
moieties such as: --(CR.sub.2).sub.nO(CR.sub.2).sub.n--, repeating
units of alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and
alkylamino (e.g., polyethyleneamino, Jeffamine.TM.); and diacid
ester and amides including succinate, succinamide, diglycolate,
malonate, and caproamide. In various embodiments, linkers can
comprise one or more amino acid residues, such as valine,
phenylalanine, lysine, and homolysine.
[0125] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0126] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0127] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[0128] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0129] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds
(1994) John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and L, or R and S, are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and 1 or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or 1 meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these stereoisomers are identical except that
they are mirror images of one another. A specific stereoisomer may
also be referred to as an enantiomer, and a mixture of such isomers
is often called an enantiomeric mixture. A 50:50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species, devoid of optical activity.
[0130] "Leaving group" refers to a functional group that can be
substituted by another functional group. Certain leaving groups are
well known in the art, and examples include, but are not limited
to, a halide (e.g., chloride, bromide, iodide), methanesulfonyl
(mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl
(triflate), and trifluoromethylsulfonate.
[0131] The term "protecting group" refers to a substituent that is
commonly employed to block or protect a particular functionality
while reacting other functional groups on the compound. For
example, an "amino-protecting group" is a substituent attached to
an amino group that blocks or protects the amino functionality in
the compound. Suitable amino-protecting groups include, but are not
limited to, acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC),
benzyloxycarbonyl (CBZ) and 9-fluorenylmethylenoxycarbonyl (Fmoc).
For a general description of protecting groups and their use, see
T. W. Greene, Protective Groups in Organic Synthesis, John Wiley
& Sons, New York, 1991, or a later edition.
[0132] As used herein and in the appended claims, the singular
forms "a," "or," and "the" include plural referents unless the
context clearly dictates otherwise.
[0133] Reference to "about" a value or parameter herein includes
(and describes) variations that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X".
[0134] It is understood that aspects and variations of the
invention described herein include "consisting of" and/or
"consisting essentially of" aspects and variations.
[0135] A. Methods of Use
[0136] Provided herein are methods of treating a B-cell
proliferative disorder in an individual comprising (a) an
immunoconjugate comprising an antibody which binds CD79b linked to
a cytotoxic agent and (b) an additional therapeutic agent. In some
embodiments, the additional therapeutic agent is a chemotherapeutic
agent. In some embodiments, the additional therapeutic agent is
cytotoxic agent.
[0137] Provided herein are methods for treating a B-cell
proliferative disorder in an individual comprising administering to
the individual an effective amount of (a) an immunoconjugate
comprising an anti-CD79b antibody linked to a cytotoxic agent
(i.e., anti-CD79b immunoconjugate and (b) an alkylating agent. In
particular, provided herein are methods for treating a B-cell
proliferative disorder in an individual comprising administering to
the individual an effective amount of (a) an immunoconjugate
comprising an anti-CD79b antibody linked to a cytotoxic agent
(i.e., anti-CD79b immunoconjugate), (b) an anti-CD20 antibody, and
(c) an alkylating agent. In some embodiments, the anti-CD20
antibody is rituximab. In some embodiments, the anti-CD20 antibody
is a humanized B-Lyl antibody. In some embodiments, the humanized
B-Lyl antibody is obinituzumab. In some embodiments, the anti-CD20
antibody is ofatumumab, ublituximab, and/or ibritumomab tiuxetan.
In some embodiments, the alkylating agent is
4-[5-[Bis(2-chloroethy)amino]-1-methylbenzimidazol-2-yl]butanoic
acid and salts thereof. In some embodiments, the alkylating agent
is bendamustine. In some embodiments, the anti-CD79b
immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE.
[0138] In addition, provided herein are methods for treating a
B-cell proliferative disorder in an individual comprising
administering to the individual an effective amount of (a) an
immunoconjugate comprising an anti-CD79b antibody linked to a
cytotoxic agent (i.e., anti-CD79b immunoconjugate) and (b) a BCL-2
inhibitor. In particular, provided herein are methods for treating
a B-cell proliferative disorder in an individual comprising
administering to the individual an effective amount of (a) an
immunoconjugate comprising an anti-CD79b antibody linked to a
cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) an
anti-CD20 antibody, and (c) a BCL-2 inhibitor. In some embodiments,
the anti-CD20 antibody is rituximab. In some embodiments, the
anti-CD20 antibody is a humanized B-Lyl antibody. In some
embodiments, the humanized B-Lyl antibody is obinituzumab. In some
embodiments, the anti-CD20 antibody is ofatumumab, ublituximab,
and/or ibritumomab tiuxetan. In some embodiments, the BCL-2
inhibitor is
4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-
-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfony-
l)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and salts thereof.
In some embodiments, the BCL-2 inhibitor is venetoclax (CAS#:
1257044-40-8). In some embodiments, the anti-CD79b immunoconjugate
is huMA79bv28-MC-vc-PAB-MMAE.
[0139] Also provided herein are methods for treating a B-cell
proliferative disorder in an individual comprising administering to
the individual an effective amount of (a) an immunoconjugate
comprising an anti-CD79b antibody linked to a cytotoxic agent
(i.e., anti-CD79b immunoconjugate) and (b) a phosphoinositide
3-kinase (PI3K) inhibitor. For example, provided herein are methods
for treating a B-cell proliferative disorder in an individual
comprising administering to the individual an effective amount of
(a) an immunoconjugate comprising an anti-CD79b antibody linked to
a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) an
anti-CD20 antibody, and (c) a phosphoinositide 3-kinase (PI3K)
inhibitor. In some embodiments, the anti-CD20 antibody is
rituximab. In some embodiments, the anti-CD20 antibody is a
humanized B-Lyl antibody. In some embodiments, the humanized B-Lyl
antibody is obinituzumab. In some embodiments, the anti-CD20
antibody is ofatumumab, ublituximab, and/or ibritumomab tiuxetan.
In some embodiments, the PI3K inhibitor inhibits delta isoform PI3K
(i.e., P110.delta.). In some embodiments, the PI3K inhibitor is
5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolino-
ne and salts thereof. In some embodiments, the PI3K inhibitor is
idelalisib (CAS#: 870281-82-6). In some embodiments, the PI3K
inhibitor inhibits alpha and delta isoforms of PI3K. In some
embodiments, the PI3K inhibitor is
2-{3-[2-(1-Isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]i-
midazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide
and salts thereof. In some embodiments, the anti-CD79b
immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE.
[0140] Also provided herein are methods for treating a B-cell
proliferative disorder in an individual comprising administering to
the individual an effective amount of (a) an immunoconjugate
comprising an anti-CD79b antibody linked to a cytotoxic agent
(i.e., anti-CD79b immunoconjugate) and (b) a Bruton's tyrosine
kinase (BTK) inhibitor. In some embodiments, provided herein are
methods for treating a B-cell proliferative disorder in an
individual comprising administering to the individual an effective
amount of (a) an immunoconjugate comprising an anti-CD79b antibody
linked to a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b)
an anti-CD20 antibody, and (c) a Bruton's tyrosine kinase (BTK)
inhibitor. In some embodiments, the anti-CD20 antibody is
rituximab. In some embodiments, the anti-CD20 antibody is a
humanized B-Lyl antibody. In some embodiments, the humanized B-Lyl
antibody is obinituzumab. In some embodiments, the anti-CD20
antibody is ofatumumab, ublituximab, and/or ibritumomab tiuxetan.
In some embodiments, the BTK inhibitor is
1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]p-
iperidin-1-yl]prop-2-en-1-one and salts thereof. In some
embodiments, the BTK inhibitor is ibrutinib (CAS#: 936563-96-1). In
some embodiments, the anti-CD79b immunoconjugate is
huMA79bv28-MC-vc-PAB-MMAE.
[0141] Provided herein are also methods for treating a B-cell
proliferative disorder in an individual comprising administering to
the individual an effective amount of (a) an immunoconjugate
comprising an anti-CD79b antibody linked to a cytotoxic agent
(i.e., anti-CD79b immunoconjugate) and (b) thalidomide or a
derivative thereof. For example, provided herein are methods for
treating a B-cell proliferative disorder in an individual
comprising administering to the individual an effective amount of
(a) an immunoconjugate comprising an anti-CD79b antibody linked to
a cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) an
anti-CD20 antibody, and (c) thalidomide or a derivative thereof. In
some embodiments, the anti-CD20 antibody is rituximab. In some
embodiments, the anti-CD20 antibody is a humanized B-Lyl antibody.
In some embodiments, the humanized B-Lyl antibody is obinituzumab.
In some embodiments, the anti-CD20 antibody is ofatumumab,
ublituximab, and/or ibritumomab tiuxetan. In some embodiments, the
thalidomide or a derivative thereof is (RS)-3-(4-Amino-1-oxo
1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione and salts
thereof. In some embodiments, the thalidomide or a derivative
thereof is lendalidomide (CAS#: 191732-72-6). In some embodiments,
the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE.
[0142] Provided herein are also methods for treating a B-cell
proliferative disorder in an individual comprising administering to
the individual an effective amount of (a) an immunoconjugate
comprising an anti-CD79b antibody linked to a cytotoxic agent
(i.e., anti-CD79b immunoconjugate) and (b) a PD-1 axis binding
antagonist. For example, provided herein are methods for treating a
B-cell proliferative disorder in an individual comprising
administering to the individual an effective amount of (a) an
immunoconjugate comprising an anti-CD79b antibody linked to a
cytotoxic agent (i.e., anti-CD79b immunoconjugate), (b) an
anti-CD20 antibody, and (c) a PD-1 axis binding agent. In some
embodiments, the anti-CD20 antibody is rituximab. In some
embodiments, the anti-CD20 antibody is a humanized B-Lyl antibody.
In some embodiments, the humanized B-Lyl antibody is obinituzumab.
In some embodiments, the anti-CD20 antibody is ofatumumab,
ublituximab, and/or ibritumomab tiuxetan. In some embodiments, the
anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some
embodiments, the PD-1 axis binding antagonist is selected from the
group consisting of a PD-1 binding antagonist, a PD-L1 binding
antagonist and a PD-L2 binding antagonist. PD-1 (programmed death
1) is also referred to in the art as "programmed cell death 1",
PDCD1, CD279 and SLEB2. PD-L1 (programmed death ligand 1) is also
referred to in the art as "programmed cell death 1 ligand 1",
PDCD1LG1, CD274, B7-H, and PDL1. PD-L1 (programmed death ligand 1),
also known as PDL1, B7-H1, B7-4, CD274, and B7-H, is a
transmembrane protein, and its interaction with PD-1 inhibits
T-cell activation and cytokine production. PD-L2 (programmed death
ligand 2) is also referred to in the art as "programmed cell death
1 ligand 2", PDCD1LG2, CD273, B7-DC, Btdc, and PDL2. In some
embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and
PD-L2. In some embodiments, the PD-1 axis binding antagonist is a
PD-1 binding antagonist. In some embodiments, the PD-1 binding
antagonist inhibits the binding of PD-1 to its ligand binding
partners. In some embodiments, the PD-1 binding antagonist inhibits
the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding
antagonist inhibits the binding of PD-1 to PD-L2. In some
embodiments, the PD-1 binding antagonist inhibits the binding of
PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding
antagonist is an antibody. In some embodiments, the PD-1 binding
antagonist is selected from the group consisting of MDX-1106
(nivolumab), MK-3475 (pembrolizumab, lambrolizumab), CT-011
(pidilizumab), and AMP-224. Nivolumab, also known as MDX-1106-04,
MDX-1106, ONO-4538, BMS-936558, and OPDIVO.RTM., is an anti-PD-1
antibody described in WO2006/121168. In some embodiments, the
anti-PD-1 antibody is Nivolumab (CAS Registry Number: 946414-94-4).
Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab,
KEYTRUDA.RTM., and SCH-900475, is an anti-PD-1 antibody described
in WO2009/114335. CT-011, also known as hBAT, hBAT-1 or
pidilizumab, is an anti-PD-1 antibody described in WO2009/101611.
AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble
receptor described in WO2010/027827 and WO2011/066342. In some
embodiments, the PD-1 axis binding antagonist is a PD-L1 binding
antagonist. In some embodiments, the PD-L1 binding antagonist
inhibits the binding of PD-L1 to PD-1. In some embodiments, the
PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In
some embodiments, the PD-L1 binding antagonist inhibits the binding
of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1
binding antagonist is an antibody. In some embodiments, the PD-L1
binding antagonist is selected from the group consisting of:
YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. Antibody
YW243.55.S70 is an anti-PD-L1 described in WO 2010/077634.
MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody
described in WO2007/005874. MEDI4736, is an anti-PD-L1 monoclonal
antibody described in WO2011/066389 and US2013/034559. Examples of
anti-PDL1 antibodies that can be used in the methods described
herein are described in PCT patent application WO 2010/077634 A1
and U.S. Pat. No. 8,217,149, which are incorporated herein by
reference. In some embodiments, the PD-1 axis binding antagonist is
an antibody. In some embodiments, the PD-1 axis binding antagonist
is a PD-L2 binding antagonist. In some embodiments, the PD-L2
binding antagonist is an antibody. In some embodiments, the PD-L2
binding antagonist is an immunoadhesin. In some embodiments, the
combination method enhances inhibition of tumor growth, increased
response rates and/or durable responses.
[0143] In some embodiments, an activating co-stimulatory molecule
may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or
CD127. In some embodiments, the agonist directed against an
activating co-stimulatory molecule is an agonist antibody that
binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or
CD127. In some embodiments, method further comprises
administeration in conjunction with an antagonist directed against
an inhibitory co-stimulatory molecule. In some embodiments, an
inhibitory co-stimulatory molecule may include CTLA-4 (also known
as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO,
TIGIT, MICA/B, or arginase. In some embodiments, the antagonist
directed against an inhibitory co-stimulatory molecule is an
antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA,
LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some
embodiments, method further comprises administeration in
conjunction with a treatment comprising adoptive transfer of a T
cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric
antigen receptor (CAR).
[0144] In some embodiments of any of the methods, the cytotoxic
agent is an antimitotic agent. Antimitotic agents are known in the
art as well as inhibitors of the polymerization of tubulin. See
e.g., Perez, Mol. Cancer Ther. 8:2086-2095 (2009), Doronina et al.,
Nat. Biotechnol. 21:778-784 (2003), and Doronina et al., Bioconjug
Chem. 17:114-124 (2006). In some embodiments, the antimitotic agent
includes, but is not limited to, a maytansinoid, a dolastatin, an
auristatin, and/or analogs and/or derivatives thereof. In some
embodiments, the antimitotic agent is an auristatin and/or analog
and/or derivative thereof. In some embodiments, the auristatin
and/or analog and/or derivative thereof is MMAE. In some
embodiments, the auristatin and/or analog and/or derivative thereof
is MMAF.
[0145] In a further aspect, the invention provides for the use of
an anti-CD79b immunoconjugate in the manufacture or preparation of
a medicament for use in combination with an additional therapeutic
agent. For example, provided herein is the use of an anti-CD79b
immunoconjugate in the manufacture or preparation of a medicament
for use in combination with an anti-CD20 antibody and an alkylating
agent (e.g., bendamustine). In one such embodiment, the method
further comprises administering to the individual an effective
amount of at least one additional therapeutic agent.
[0146] An "individual" according to any of the above embodiments
may be a human.
[0147] In one embodiment, B-cell proliferative disease includes,
but is not limited 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, and/or j) Hodgkin's disease.
[0148] In some embodiments of any of the methods, the B-cell
proliferative disorder is cancer. In some embodiments, the B-cell
proliferative disorder is lymphoma, non-Hodgkins lymphoma (NHL),
aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,
refractory NHL, refractory indolent NHL, chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell
leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell
lymphoma. In some embodiments, the B-cell proliferative disorder is
NHL, such as indolent NHL and/or aggressive NHL. In some
embodiments, the B-cell proliferative disorder is indolent
follicular lymphoma or diffuse large B-cell lymphoma (DLBCL). In
some embodiments, the DLBCL is activated B cell DLBCL (ABC DLBCL).
In some embodiments, the DLBCL is germinal center B-cell like DLBCL
(GCB DLBCL). In some embodiment, the DLBCL is BCL2 positive (e.g.,
positive for BCL2 gene rearrangement, t(14;18)(q32;q21)). In some
embodiments, the DLBCL is BCL2 negative (e.g., negative for BCL2
gene rearrangement, t(14;18)(q32;q21)).
[0149] In some embodiments of any of the methods, the B-cell
proliferative disorder is a histologically confirmed FL (Grade 1,
2, or 3a) or DLBCL. In some embodiments, the individual has
received at least one prior therapy for FL or DLBCL. In some
embodiments, the patient has received prior bendamustine and the
duration must have been >1 year (for patients who have relapse
disease after a prior regimen). In some embodiments, at least one
bi-dimensionally measurable lesion on imaging scan defined as
>1.5 cm in its longest dimension; confirmed availability of
archival or freshly collected tumor tissue meeting protocol-defined
specifications prior to study enrollment; Life expectancy of at
least 24 weeks; Eastern Cooperative Oncology Group (ECOG)
Performance Status of 0, 1, or 2; adequate hematological function;
and/or, for women of childbearing potential, a negative serum
pregnancy test result within 7 days prior to commencement of
dosing.
[0150] In some embodiments, the individual does not have a history
of severe allergic or anaphylactic reactions to humanized or murine
monoclonal antibodies (MAbs, or recombinant antibody-related fusion
proteins) or known sensitivity or allergy to murine products,
contraindication to bendamustine, rituximab, or obinutuzumab. In
some embodiments, the individual does not have a history of
sensitivity to mannitol, prior use of any MAb,
radioimmunoconjugate, or antibody-drug conjugate (ADC) within 4
weeks before Cycle 1 Day 1, treatment with radiotherapy,
chemotherapy, immunotherapy, immunosuppressive therapy, and/or any
investigational agent for the purposes of treating cancer within 2
weeks prior to Cycle 1 Day 1, ongoing corticosteroid use >30
mg/day prednisone or equivalent, for purposes other than lymphoma
symptom control, completion of autologous SCT within 100 days prior
to Cycle 1 Day 1, prior allogeneic SCT, eligibility for autologous
SCT (patients with relapsed/refractory DLBCL), Grade 3b FL, history
of transformation of indolent disease to DLBCL, primary CNS
lymphoma, current Grade>1 peripheral neuropathy, evidence of
significant, uncontrolled concomitant diseases that could affect
compliance with the protocol or interpretation of results,
including significant cardiovascular disease (such as New York
Heart Association Class III or IV cardiac disease, myocardial
infarction within the last 6 months, unstable arrhythmias, or
unstable angina) or significant pulmonary disease (including
obstructive pulmonary disease and history of bronchospasm), known
active bacterial, viral, fungal, mycobacterial, parasitic, or other
infection (excluding fungal infections of nail beds) at study
enrollment or any major episode of infection requiring treatment
with intravenous (IV) antibiotics or hospitalization within 4 weeks
prior to Cycle 1 Day 1, patients with suspected or latent
tuberculosis, positive test results for chronic hepatitis B virus
(HBV) infection or for hepatitis C virus (HCV) antibody, known
infection with HIV or human T-cell leukemia virus 1 (HTLV-1) virus,
women who are pregnant or lactating or who intend to become
pregnant within a year of the last dose of rituximab or
obinutuzumab, and/or evidence of laboratory abnormalities in
standard renal, hepatic or coagulation function tests.
[0151] In some embodiments of any of the methods, the B-cell
proliferative disorder is a relapsed or refractory B-cell
proliferative disorder. In some embodiments, relapsed or refractory
B-cell proliferative disorder as used herein includes patients who
have received at least 1 prior chemotherapy containing treatment
regimen. In some embodiments, relapsed patients generally have
developed progressive disease following a response to the prior
chemotherapy-containing treatment regimen. In some embodiments,
refractory patients have generally failed to respond or relapsed
within 6 months to the last prior chemotherapy-containing regimen.
In some embodiments, relapsed/refractory follicular lymphoma (FL)
patients who have relapsed to prior regimen(s) after having a
documented history of response (complete response [CR], CR
unconfirmed [CRu], or partial response [PR]) of >/=6 months in
duration from completion of regimen(s); refractory to any prior
regimen, defined as no response to the prior therapy, or
progression within 6 months of completion of the last dose of
therapy. In some embodiments, relapsed/refractory DLBCL patients
are patients who are ineligible for second-line stem cell
transplant (SCT), with progressive disease or no response (stable
disease [SD])<6 months from start of initial therapy; patients
who are ineligible for second-line SCT, with disease relapse after
initial response of >/=6 months from start of initial therapy;
patients who are ineligible for third-line (or beyond) SCT, with
progressive disease or no response (SD)<6 months from start of
prior therapy; patients who are ineligible for third-line (or
beyond) SCT with disease relapse after initial response of >/=6
months from start of prior therapy.
[0152] In some embodiments, the individual having a B-cell
proliferative disorder is previously untreated. In some
embodiments, previously untreated as used herein includes patients
diagnosed with a B-cell proliferative disease, 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.
[0153] An immunoconjugate provided herein (and any additional
therapeutic agent) for use in any of the therapeutic methods
described herein can be administered by any suitable means,
including parenteral, intrapulmonary, and intranasal, and, if
desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g., by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0154] In some embodiments of any of the methods, if the
administration is intravenous the initial infusion time for the
anti-CD79b immunoconjugate or the additional therapeutic agent 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).
[0155] The terms "co-administration" or "co-administering" refer to
the administration of the anti-CD79b immunoconjugate and the
additional therapeutic agent as two separate formulations (or as
one single formulation). The co-administration can be simultaneous
or sequential in either order, wherein preferably there is a time
period while both (or all) active agents simultaneously exert their
biological activities. The anti-CD79b immunoconjugate and the
additional therapeutic agent are co-administered either
simultaneously or sequentially. In some embodiments, when both
therapeutic agents are co-administered sequentially the dose is
administered either on the same day in two separate
administrations, or one of the agents is administered on day 1 and
the second is co-administered on day 2 to day 7, preferably on day
2 to 4. Thus in one embodiment the term "sequentially" means within
7 days after the dose of the first component, preferably within 4
days after the dose of the first component; and the term
"simultaneously" means at the same time. The term
"co-administration" with respect to the maintenance doses of said
the anti-CD79b immunoconjugate and the additional therapeutic agent
means that the maintenance doses can be either co-administered
simultaneously, if the treatment cycle is appropriate for both
drugs, e.g., every week. Or anti-CD79b immunoconjugate is e.g.,
administered e.g., every first to third day and the additional
therapeutic is administered every week. Or the maintenance doses
are co-administered sequentially, either within one or within
several days.
[0156] Anti-CD79b immunoconjugates and additional therapeutic
agents provided herein for use in any of the therapeutic methods
described herein would be formulated, dosed, and administered in a
fashion consistent with good medical practice. Factors for
consideration in this context include the particular disorder being
treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the
site of delivery of the agent, the method of administration, the
scheduling of administration, and other factors known to medical
practitioners. The immunoconjugate need not be, but is optionally
formulated with one or more agents currently used to prevent or
treat the disorder in question.
[0157] The amount of co-administration of the anti-CD79b
immunoconjugate and the additional therapeutic agent 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. The anti-CD79b
immunoconjugate and the additional therapeutic agent are suitably
co-administered to the patient at one time or over a series of
treatments e.g., on the same day or on the day after.
[0158] In some embodiments of any of the methods, the dosage of
anti-CD79b immunoconjugate (such as huMA79bv28-MC-vc-PAB-MMAE) is
between about any of 1.4-5 mg/kg, 1.8-4 mg/kg, 1.8-3.2 mg/kg,
and/or 1.8-2.4 mg/kg. In some embodiments of any of the methods,
the dosage of anti-CD79 immunoconjugate is about any of 1.4, 1.8,
2.0, 2.2, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, and/or 4.8 mg/kg. In some
embodiments, the dosage of anti-CD79b immunoconjugate is about 1.8
mg/kg. In some embodiments, the dosage of anti-CD79b
immunoconjugate is about 2.4 mg/kg. In some embodiments, the dosage
of anti-CD79b immunoconjugate is about 3.2 mg/kg. In some
embodiments, the dosage of anti-CD79b immunoconjugate is about 3.6
mg/kg. In some embodiments of any of the methods, the anti-CD79b
immunoconjugate is administered q3wk. In some embodiments, the
anti-CD79b immunoconjugate is administered via intravenous
infusion. The dosage administered via infusion is in the range of
about 1 .mu.g/m2 to about 10,000 .mu.g/m2 per dose, generally one
dose per week for a total of one, two, three or four doses.
Alternatively, the dosage range is of about 1 .mu.g/m2 to about
1000 .mu.g/m2, about 1 .mu.g/m2 to about 800 .mu.g/m2, about 1
.mu.g/m2 to about 600 .mu.g/m2, about 1 .mu.g/m2 to about 400
.mu.g/m2, about 10 .mu.g/m2 to about 500 .mu.g/m2, about 10
.mu.g/m2 to about 300 .mu.g/m2, about 10 .mu.g/m2 to about 200
.mu.g/m2, and about 1 .mu.g/m2 to about 200 .mu.g/m2. The dose may
be administered once per day, once per week, multiple times per
week, but less than once per day, multiple times per month but less
than once per day, multiple times per month but less than once per
week, once per month or intermittently to relieve or alleviate
symptoms of the disease. Administration may continue at any of the
disclosed intervals until remission of the tumor or symptoms of the
lymphoma, leukemia being treated. Administration may continue after
remission or relief of symptoms is achieved where such remission or
relief is prolonged by such continued administration.
[0159] In some embodiments of any of the methods, the dosage of the
anti-CD20 antibody is between about 300-1600 mg/m.sup.2 and/or
300-2000 mg In some embodiments of any of the methods, the dosage
of the anti-CD20 antibody is about any of 300, 375, 600, 1000, or
1250 mg/m.sup.2 and/or 300, 1000, or 2000 mg. In some embodiments,
the anti-CD20 antibody is rituximab and the dosage administered is
375 mg/m.sup.2. In some embodiments, the anti-CD20 antibody is
obinutuzumab and the dosage administered is 1000 mg/m.sup.2. In
some embodiments, the anti-CD20 antibody is administered q3wk. In
some embodiments, the dosage of said afucosylated anti-CD20
antibody (preferably the afocusylated humanized B-Lyl antibody) may
be 800 to 1600 mg (in one embodiment 800 to 1200 mg) on day 1, 8,
15 of a 3- to 6-weeks-dosage-cycle and then in a dosage of 400 to
1200 (in one embodiment 800 to 1200 mg on day 1 of up to nine 3- to
4-weeks-dosage-cycles. In some embodiments, the dose is a flat dose
1000 mg in a three-weeks-dosage schedule, with the possibility of
an additional cycle of a flat dose of 1000 mg in the second
week.
[0160] Exemplary dosing regimens for the combination therapy of
anti-CD79b immunoconjugates (such as huMA79bv28-MC-vc-PAB-MMAE) and
other agents include, but are not limited to, anti-CD79
immunoconjugate (such as huMA79bv28-MC-vc-PAB-MMAE) administered at
about 1.4-5 mg/kg q3wk, plus 375 mg/m.sup.2 q3wk rituximab, and
25-120 mg/m.sup.2 bendamustine (e.g., bendamustine hydrochloride)
dl and 2 of q3wk daily. In some embodiments, the anti-CD79
immunoconjugate is administered at about any of 1.8 mg/kg, 2.0
mg/kg, 2.2 mg/kg, 2.4 mg/kg, 3.2 mg/kg, or 4.0 mg/kg. In some
embodiments, the anti-CD79b immunoconjugate is administered at
about 1.8 mg/kg. In some embodiments, the anti-CD79b
immunoconjugate is administered at about 2.4 mg/kg. In some
embodiments, bendamustine is administered at about 90
mg/m.sup.2.
[0161] Another exemplary dosage regime for the combination therapy
of anti-CD79b immunoconjugates (such as huMA79bv28-MC-vc-PAB-MMAE)
and other agents include, but are not limited to, anti-CD79
immunoconjugate (such as huMA79bv28-MC-vc-PAB-MMAE) administered
1.4-5 mg/kg q3wk, plus 1000 m g/m.sup.2 q3wk obinutuzumab, and
25-120 mg/m.sup.2 bendamustine dl and 2 of q3wk daily. In some
embodiments, the anti-CD79 immunoconjugate is administered at about
any of 1.8 mg/kg, 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 3.2 mg/kg, or
4.0 mg/kg. In some embodiments, the anti-CD79b immunoconjugate is
administered at about 1.8 mg/kg. In some embodiments, the
anti-CD79b immunoconjugate is administered at about 2.4 mg/kg. In
some embodiments, bendamustine is administered at about 90
mg/m.sup.2.
[0162] B. Agents for Use in the Methods Described Herein
[0163] Provided herein are anti-CD79b immunoconjugates and
additional therapeutic agents for use in the methods described
herein. In some embodiments, the additional therapeutic agent is an
antibody. In some embodiments, the additional therapeutic agent is
a small molecule. In some embodiments, the additional therapeutic
agent is an anti-CD20 antibody and bendamustine.
[0164] 1. Anti-CD79b Immunoconjugates Comprising Anti-CD79b
Antibodies and Other Embodiments
[0165] Provided herein are anti-CD79b antibodies for the anti-CD79b
immunoconjugates for use in the methods described herein comprising
anti-CD79b immunoconjugates and an additional therapeutic
agent.
[0166] In some embodiments, the methods herein provide an
immunoconjugate comprising an anti-CD79b antibody comprising at
least one, two, three, four, five, or six HVRs selected from (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d)
HVR-L1 comprising an amino acid sequence of SEQ ID NO: 24; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some
such embodiments, the immunoconjugate comprises at least one of:
(i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23,
and/or (ii) HVR-L1 comprising an amino acid sequence of SEQ ID NO:
24.
[0167] In some embodiments, provided herein for use in the methods
are immunoconjugates comprising an anti-CD79b antibody comprising
at least one, two, three, four, five, or six HVRs selected from (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d)
HVR-L1 comprising an amino acid sequence of SEQ ID NO: 24; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some
such embodiments, the immunoconjugate comprises at least one of:
(i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23,
and/or (ii) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:24. In one aspect, provided herein are immunoconjugates
comprising an anti-CD79b immunoconjugate comprising at least one,
at least two, or all three VH HVR sequences selected from (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b)
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; and (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23. In some
embodiments, the immunoconjugate comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO: 23. In another embodiment, the
immunoconjugate comprises HVR-H3 comprising the amino acid sequence
of SEQ ID NO: 23 and HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 26. In a further embodiment, the immunoconjugate
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:
23, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22. In a
further embodiment, the immunoconjugate comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 22; and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO: 23.
[0168] In another aspect, the immunoconjugate comprises an
anti-CD79b antibody comprising at least one, at least two, or all
three VL HVR sequences selected from (a) HVR-L1 comprising an amino
acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino
acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 26. In another aspect, provided herein
are immunoconjugates comprising at least one, at least two, or all
three VL HVR sequences selected from (a) HVR-L1 comprising the
amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the
amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 26. In one embodiment, the
immunoconjugate comprises (a) HVR-L1 comprising an amino acid
sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate
comprises an HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 24. In some embodiments, the immunoconjugate comprises an
HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. In some
embodiments, the immunoconjugate comprises (a) HVR-L1 comprising
the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the
amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 26.
[0169] In another aspect, the immunoconjugate comprises an
anti-CD79b antibody comprising (a) a VH domain comprising at least
one, at least two, or all three VH HVR sequences selected from (i)
HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (ii)
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and
(iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID
NO:23; and (b) a VL domain comprising at least one, at least two,
or all three VL HVR sequences selected from (i) HVR-L1 comprising
an amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2 comprising the
amino acid sequence of SEQ ID NO: 25, and (iii) HVR-L3 comprising
the amino acid sequence of SEQ ID NO: 26. In some such embodiments,
the immunoconjugate comprises at least one of: (i) HVR-H3
comprising the amino acid sequence of SEQ ID NO: 23, and/or (ii)
HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24.
[0170] In another aspect, the provided herein are immunoconjugates
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:
22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23;
(d) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 24; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some
such embodiments, the immunoconjugate comprises at least one of:
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23 and/or
HVR-L1 comprising an amino acid sequence of SEQ ID NO: 24. In
another aspect, provided are immunoconjugates comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO: 26.
[0171] In any of the above embodiments, the anti-CD79b
immunoconjugates comprises a humanized anti-CD79b antibody. In one
embodiment, an anti-CD79b antibody comprises HVRs as in any of the
above embodiments, and further comprises a human acceptor
framework, e.g., a human immunoglobulin framework or a human
consensus framework. In certain embodiments, the human acceptor
framework is the human VL kappa 1 (VL.sub.KI) framework and/or the
VH framework VH.sub.III. In some embodiments, a humanized
anti-CD79b antibody comprises (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO: 23; (d) HVR-L1 comprising an amino acid
sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 26. In some embodiments, a humanized
anti-CD79b antibody comprises (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 26.
[0172] In another aspect, an anti-CD79b immunoconjugate comprises a
heavy chain variable domain (VH) sequence having at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to the amino acid sequence of SEQ ID NO: 19. In certain
embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of
SEQ ID NO: 19 contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-CD79b immunoconjugate comprising that
sequence retains the ability to bind to CD79b. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO: 19. In certain embodiments, a
total of 1 to 5 amino acids have been substituted, inserted and/or
deleted in SEQ ID NO: 19. In certain embodiments, substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs).
[0173] Optionally, the anti-CD79b immunoconjugate comprises the VH
sequence of any one of SEQ ID NO: 19, including post-translational
modifications of that sequence. In some embodiments, the anti-CD79b
immunoconjugate comprises the VH sequence of SEQ ID NO: 19,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three HVRs
selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 21, (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO: 22, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO: 17 or SEQ ID NO: 23.
[0174] In some embodiments, an anti-CD79b immunoconjugate comprises
a light chain variable domain (VL) having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO: 20. In certain embodiments, a VL
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to the amino acid sequence of SEQ ID NO: 20
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-CD79b immunoconjugate comprising that sequence retains the
ability to bind to CD79b. In certain embodiments, a total of 1 to
10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 20. In certain embodiments, a total of 1 to 5 amino
acids have been substituted, inserted and/or deleted in SEQ ID NO:
20. In certain embodiments, the substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-CD79b immunoconjugate comprises the VL
sequence of any one of SEQ ID NO: 20, including post-translational
modifications of that sequence. In some embodiments, the anti-CD79b
immunoconjugate comprises the VL sequence of SEQ ID NO: 20,
including post-translational modifications of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs
selected from (a) HVR-L1 comprising an amino acid sequence of SEQ
ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO: 26. In some embodiments, the VL comprises one, two or three
HVRs selected from (a) HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ
ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ
ID NO: 26.
[0175] In another aspect, an anti-CD79b immunoconjugate comprises a
VH as in any of the embodiments provided above, and a VL as in any
of the embodiments provided above. In some embodiments, the
antibody comprises the VH and VL sequences in SEQ ID NO: 19 and SEQ
ID NO: 20, respectively, including post-translational modifications
of those sequences.
[0176] In a further aspect, provided herein are anti-CD79b
immunoconjugates that binds to the same epitope as an anti-CD79b
antibody provided herein. For example, in certain embodiments,
immunoconjugate is provided that binds to the same epitope as an
anti-CD79b antibody comprising a VH sequence of SEQ ID NO: 19 and a
VL sequence of SEQ ID NO: 20.
[0177] In a further aspect of the invention, an anti-CD79b
immunoconjugate according to any of the above embodiments comprises
a monoclonal antibody, including a chimeric, humanized or human
antibody. In one embodiment, an anti-CD79b immunoconjugate
comprises an antibody fragment, e.g., a Fv, Fab, Fab', scFv,
diabody, or F(ab').sub.2 fragment. In another embodiment, the
immunoconjugate comprises a substantially full length antibody,
e.g., an IgG1 antibody or other antibody class or isotype as
defined herein.
[0178] 2. Anti-CD20 Antibodies and Other Embodiments
[0179] Provided herein are anti-CD20 antibodies for use in the
methods described herein comprising anti-CD79b immunoconjugates and
an additional therapeutic agent.
[0180] Depending on binding properties and biological activities of
anti-CD20 antibodies to the CD20 antigen, two types of anti-CD20
antibodies (type I and type II anti-CD20 antibodies) can be
distinguished according to Cragg, M. S., et al., Blood 103 (2004)
2738-2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052,
see Table 1.
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 IgGl
isotype) ADCC activity (if IgG1 isotype) Full binding capacity
Reduced binding capacity Homotypic aggregation Stronger homotypic
aggregation Apoptosis induction upon cross- Strong cell death
induction without linking cross-linking
[0181] Examples of type I anti-CD20 antibodies include e.g.,
rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in
WO 2005/103081), 2F2 IgG1 (as disclosed and WO 2004/035607 and WO
2005/103081) and 2H7 IgG1 (as disclosed in WO 2004/056312). In some
embodiments, the anti-CD20 antibody is rituximab antibody. In some
embodiments, the rituximab antibody (reference antibody; example of
a type I anti-CD20 antibody) is a genetically engineered chimeric
human gamma 1 murine constant domain containing monoclonal antibody
directed against the human CD20 antigen. However this antibody is
not glycoengineered and not afocusylates and thus has an amount of
fucose of at least 85%. This chimeric antibody contains human gamma
1 constant domains and is identified by the name "C2B8" in U.S.
Pat. No. 5,736,137 (Andersen, et. al.) issued on Apr. 17, 1998,
assigned to IDEC Pharmaceuticals Corporation. Rituximab is approved
for the treatment of patients with relapsed or refracting low-grade
or follicular, CD20 positive, B-cell non-Hodgkin's lymphoma. In
vitro mechanism of action studies have shown that rituximab
exhibits human complement-dependent cytotoxicity (CDC) (Reff, M.
E., et. al, Blood 83(2) (1994) 435-445). Additionally, it exhibits
activity in assays that measure antibody-dependent cellular
cytotoxicity (ADCC).
[0182] In some embodiments, the anti-CD20 antibodies are an
afucosylated anti-CD20 antibody.
[0183] Examples of type II anti-CD20 antibodies include e.g.,
humanized B-Lyl antibody IgG1 (a chimeric humanized IgG1 antibody
as disclosed in WO 2005/044859), 11B8 IgG1 (as disclosed in WO
2004/035607), and AT80 IgG1. Typically type II anti-CD20 antibodies
of the IgG1 isotype show characteristic CDC properties. Type II
anti-CD20 antibodies have a decreased CDC (if IgG1 isotype)
compared to type I antibodies of the IgG1 isotype. In one
embodiment said type II anti-CD20 antibody, e.g., a GA101 antibody,
has increased antibody dependent cellular cytotoxicity (ADCC). In
some embodiments, the type II anti-CD20 antibodies, more preferably
an afucosylated humanized B-Lyl antibody as described in WO
2005/044859 and WO 2007/031875.
[0184] In some embodiments, the anti-CD20 antibody is GA101
antibody. In some embodiments, the 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:5, an HVR-H2 comprising the amino acid
sequence of SEQ ID NO:6, an HVR-H3 comprising the amino acid
sequence of SEQ ID NO:7, an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:8, an HVR-L2 comprising the amino acid
sequence of SEQ ID NO:9, and an HVR-L3 comprising the amino acid
sequence of SEQ ID NO:10; (2) an antibody comprising a VH domain
comprising the amino acid sequence of SEQ ID NO:11 and a VL domain
comprising the amino acid sequence of SEQ ID NO:12, (3) an antibody
comprising an amino acid sequence of SEQ ID NO:13 and an amino acid
sequence of SEQ ID NO: 14; (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:13 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: 14. In one
embodiment, the GA101 antibody is an IgG1 isotype antibody.
[0185] In some embodiments, the anti-CD20 antibody is a humanized
B-Lyl antibody. In some embodiments, the humanized B-Lyl antibody
refers to humanized B-Lyl antibody as disclosed in WO 2005/044859
and WO 2007/031875, which were obtained from the murine monoclonal
anti-CD20 antibody B-Lyl (variable region of the murine heavy chain
(VH): SEQ ID NO: 3; variable region of the murine light chain (VL):
SEQ ID NO: 4--see Poppema, S. and Visser, L., Biotest Bulletin 3
(1987) 131-139) by chimerization with a human constant domain from
IgG1 and following humanization (see WO 2005/044859 and WO
2007/031875). The humanized B-Lyl antibodies are disclosed in
detail in WO 2005/044859 and WO 2007/031875.
[0186] In one embodiment, the humanized B-Lyl antibody has variable
region of the heavy chain (VH) selected from group of SEQ ID
NO:15-16 and 40-55 (corresponding to B-HH2 to B-HH9 and B-HL8 to
B-HL17 of WO 2005/044859 and WO 2007/031875). In one specific
embodiment, such variable domain is selected from the group
consisting of SEQ ID NO: 15, 16, 42, 44, 46, 48 and 50
(corresponding to B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 and
B-HL13 of WO 2005/044859 and WO 2007/031875). In one specific
embodiment, the humanized B-Lyl antibody has variable region of the
light chain (VL) of SEQ ID NO:55 (corresponding to B-KV1 of WO
2005/044859 and WO 2007/031875). In one specific embodiment, the
humanized B-Lyl antibody has a variable region of the heavy chain
(VH) of SEQ ID NO:42 (corresponding to B-HH6 of WO 2005/044859 and
WO 2007/031875) and a variable region of the light chain (VL) of
SEQ ID NO:55 (corresponding to B-KV1 of WO 2005/044859 and WO
2007/031875). Furthermore in one embodiment, the humanized B-Lyl
antibody is an IgG1 antibody. According to the invention such
afocusylated humanized B-Lyl antibodies are glycoengineered (GE) in
the Fc region according to the procedures described in WO
2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al.,
Nature Biotechnol. 17 (1999) 176-180 and WO 99/154342. In one
embodiment, the afucosylated glyco-engineered humanized B-Lyl is
B-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody is
obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No.
4, 2012, p. 453). As used herein, obinutuzumab is synonymous for
GA101 or RO5072759. This replaces all previous versions (e.g., Vol.
25, No. 1, 2011, p. 75-76), and is formerly known as afutuzumab
(recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009, p.
176; Vol. 22, No. 2, 2008, p. 124). In some embodiments, the
humanized B-Lyl antibody is an antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO:17 and a light
chain comprising the amino acid sequence of SEQ ID NO:18 or an
antigen-binding fragment thereof. In some embodiments, the
humanized B-Lyl antibody comprises a heavy chain variable region
comprising the three heavy chain CDRs of SEQ ID NO:17 and a light
chain variable region comprising the three light chain CDRs of SEQ
ID NO:18.
[0187] In some embodiments, the humanized B-Lyl antibody is an
afucosylated glyco-engineered humanized B-Lyl. Such glycoengineered
humanized B-Lyl antibodies have an altered pattern of glycosylation
in the Fc region, preferably having a reduced level of fucose
residues. Preferably the amount of fucose is about 60% or less of
the total amount of oligosaccharides at Asn297 (in one embodiment
the amount of fucose is between about 40% and about 60%, in another
embodiment the amount of fucose is about 50% or less, and in still
another embodiment the amount of fucose is about 30% or less).
Furthermore the oligosaccharides of the Fc region are preferably
bisected. These glycoengineered humanized B-Lyl antibodies have an
increased ADCC.
[0188] The "ratio of the binding capacities to CD20 on Raji cells
(ATCC-No. CCL-86) of an anti-CD20 antibodies compared to rituximab"
is determined by direct immunofluorescence measurement (the mean
fluorescence intensities (MFI) is measured) using said anti-CD20
antibody conjugated with Cy5 and rituximab conjugated with Cy5 in a
FACSArray (Becton Dickinson) with Raji cells (ATCC-No. CCL-86), as
described in Example No. 2, and calculated as follows:
Ratio of the binding capacities to CD 20 on Raji cells ( ATCC - No
. CCL - 86 ) = MFI ( Cy 5 - anti - CD 20 antibody ) MFI ( Cy 5 -
rituximab ) .times. Cy 5 - labeling ratio ( Cy 5 - rituximab ) Cy 5
- labeling ratio ( Cy 5 - anti - CD 20 antibody ) ##EQU00001##
[0189] MFI is the mean fluorescent intensity. The "Cy5-labeling
ratio" as used herein means the number of Cy5-label molecules per
molecule antibody.
[0190] 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.
[0191] 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.
[0192] One accepted in vitro ADCC assay is as follows: [0193] 1)
the assay uses target cells that are known to express the target
antigen recognized by the antigen-binding region of the antibody;
[0194] 2) the assay uses human peripheral blood mononuclear cells
(PBMCs), isolated from blood of a randomly chosen healthy donor, as
effector cells; [0195] 3) the assay is carried out according to
following protocol: [0196] 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; [0197] 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; [0198] iii) 100 microliters of the final target cell
suspension above are transferred to each well of a 96-well
microtiter plate; [0199] 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; [0200] 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); [0201] 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); [0202]
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.; [0203] 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; [0204] ix) the cell-free supernatant
from each well is harvested and the experimentally released
radioactivity (ER) is quantified using a gamma counter; [0205] 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);
[0206] 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).
[0207] In some embodiments, the "increased ADCC" can be obtained
by, for example, mutating and/or glycoengineering of said
antibodies. In one embodiment, the antibody is glycoengineered to
have a biantennary oligosaccharide attached to the Fc region of the
antibody that is bisected by GlcNAc. 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). 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)).
[0208] In some embodiments, 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.
[0209] In a further aspect of the invention, an anti-CD20 antibody
according to any of the above embodiments is a monoclonal antibody,
including a human antibody. In one embodiment, an anti-CD20
antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv,
diabody, or F(ab').sub.2 fragment. In another embodiment, the
antibody is a substantially full length antibody, e.g., an IgG1
antibody, IgG2a antibody or other antibody class or isotype as
defined herein.
[0210] In a further aspect, an antibody according to any of the
above embodiments may incorporate any of the features, singly or in
combination, as described in below.
[0211] 1. Antibody Affinity
[0212] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.50 nM, .ltoreq.10 nM, .ltoreq.5 nM, .ltoreq.1 nM,
.ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM, and
optionally is .gtoreq.10.sup.-13 M. (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).
[0213] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA) performed with the Fab version of an antibody
of interest and its antigen as described by the following assay.
Solution binding affinity of Fabs for antigen is measured by
equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen antigen
are mixed with serial dilutions of a Fab of interest (e.g.,
consistent with assessment of the anti-VEGF antibody, Fab-12, in
Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of
interest is then incubated overnight; however, the incubation may
continue for a longer period (e.g., about 65 hours) to ensure that
equilibrium is reached. Thereafter, the mixtures are transferred to
the capture plate for incubation at room temperature (e.g., for one
hour). The solution is then removed and the plate washed eight
times with 0.1% polysorbate 20 (TWEEN-20.RTM.) in PBS. When the
plates have dried, 150 .mu.l/well of scintillant
(MICROSCINT-20.TM.; Packard) is added, and the plates are counted
on a TOPCOUNT.TM. gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding
assays.
[0214] According to another embodiment, Kd is measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds
10.sup.6M.sup.-1s.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0215] 2. Antibody Fragments
[0216] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthiin, in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0217] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0218] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0219] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g., E.
coli or phage), as described herein.
[0220] 3. Chimeric and Humanized Antibodies
[0221] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0222] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0223] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0224] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0225] 4. Human Antibodies
[0226] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0227] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0228] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0229] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0230] 5. Library-Derived Antibodies
[0231] Antibodies for use in the methods described herein may be
isolated by screening combinatorial libraries for antibodies with
the desired activity or activities. For example, a variety of
methods are known in the art for generating phage display libraries
and screening such libraries for antibodies possessing the desired
binding characteristics. Such methods are reviewed, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N. J., 2001) and further
described, e.g., in the McCafferty et al., Nature 348:552-554;
Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol.
Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in
Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N. J.,
2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132(2004).
[0232] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0233] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0234] 6. Multispecific Antibodies
[0235] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g., a bispecific antibody is useful in a
method described herein. Multispecific antibodies are monoclonal
antibodies that have binding specificities for at least two
different sites. In certain embodiments, one of the binding
specificities is for one antigen (e.g., CD79b) and the other is for
any other antigen. In certain embodiments, one of the binding
specificities is for one antigen (e.g., CD79b) and the other is for
CD3. See, e.g., U.S. Pat. No. 5,821,337. In certain embodiments,
bispecific antibodies may bind to two different epitopes of an
antigen (e.g., CD79b). Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express the antigen (e.g.,
CD79b). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments.
[0236] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bispecific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0237] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g., US 2006/0025576A1).
[0238] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to
CD79b as well as another, different antigen (see, US 2008/0069820,
for example).
[0239] 7. Antibody Variants
[0240] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0241] a. Substitution, Insertion, and Deletion Variants
[0242] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of "preferred
substitutions." More substantial changes are provided in Table 1
under the heading of "exemplary substitutions," and as further
described below in reference to amino acid side chain classes.
Amino acid substitutions may be introduced into an antibody of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0243] Amino acids may be grouped according to common side-chain
properties:
[0244] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0245] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0246] (3) acidic: Asp, Glu;
[0247] (4) basic: His, Lys, Arg;
[0248] (5) residues that influence chain orientation: Gly, Pro;
[0249] (6) aromatic: Trp, Tyr, Phe.
[0250] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0251] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g., a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g., binding
affinity).
[0252] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0253] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0254] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex is used to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0255] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g., for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0256] b. Glycosylation Variants
[0257] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0258] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH.sub.2 domain of the Fc region. See, e.g., Wright
et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include
various carbohydrates, e.g., mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached
to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some embodiments, modifications of the
oligosaccharide in an antibody of the invention may be made in
order to create antibody variants with certain improved
properties.
[0259] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about +3 amino acids upstream
or downstream of position 297, i.e., between positions 294 and 300,
due to minor sequence variations in antibodies. Such fucosylation
variants may have improved ADCC function. See, e.g., US Patent
Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to
"defucosylated" or "fucose-deficient" antibody variants include: US
2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki
et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of
producing defucosylated antibodies include Lec13 CHO cells
deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1,
Presta, L; and WO 2004/056312 A1, Adams et al., especially at
Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0260] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0261] c. Fc Region Variants
[0262] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g., a substitution) at one or more amino acid
positions.
[0263] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc(RIII only, whereas monocytes express Fc(RI,
Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Nonlimiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc.
Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,
Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No.
5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361
(1987)). Alternatively, non-radioactive assays methods may be
employed (see, for example, ACTI.TM. non-radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View,
Calif.; and CytoTox 96.RTM. non-radioactive cytotoxicity assay
(Promega, Madison, Wis.). Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al. Proc. Nat'l Acad.
Sci. USA 95:652-656 (1998). C1q binding assays may also be carried
out to confirm that the antibody is unable to bind C1q and hence
lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO
2006/029879 and WO 2005/100402. To assess complement activation, a
CDC assay may be performed (see, for example, Gazzano-Santoro et
al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0264] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0265] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0266] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0267] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0268] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0269] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0270] d. Cysteine Engineered Antibody Variants
[0271] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an anti-CD79b antibody are substituted with
cysteine residues. In particular embodiments, the substituted
residues occur at accessible sites of the antibody. By substituting
those residues with cysteine, reactive thiol groups are thereby
positioned at accessible sites of the antibody and may be used to
conjugate the antibody to other moieties, such as drug moieties or
linker-drug moieties, to create an immunoconjugate, as described
further herein. In certain embodiments, any one or more of the
following residues may be substituted with cysteine: V205 (Kabat
numbering) of the light chain; A118 (EU numbering) of the heavy
chain; and S400 (EU numbering) of the heavy chain Fc region.
Cysteine engineered antibodies may be generated as described, e.g.,
in U.S. Pat. No. 7,521,541.
[0272] e. Antibody Derivatives
[0273] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Nonlimiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0274] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0275] C. Recombinant Methods and Compositions
[0276] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an antibody described
herein is provided. Such nucleic acid may encode an amino acid
sequence comprising the VL and/or an amino acid sequence comprising
the VH of the antibody (e.g., the light and/or heavy chains of the
antibody). In a further embodiment, one or more vectors (e.g.,
expression vectors) comprising such nucleic acid are provided. In a
further embodiment, a host cell comprising such nucleic acid is
provided. In one such embodiment, a host cell comprises (e.g., has
been transformed with): (1) a vector comprising a nucleic acid that
encodes an amino acid sequence comprising the VL of the antibody
and an amino acid sequence comprising the VH of the antibody, or
(2) a first vector comprising a nucleic acid that encodes an amino
acid sequence comprising the VL of the antibody and a second vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VH of the antibody. In one embodiment, the host cell
is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid
cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of
making an antibody is provided, wherein the method comprises
culturing a host cell comprising a nucleic acid encoding the
antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0277] For recombinant production of an antibody, nucleic acid
encoding an antibody, e.g., as described above, is isolated and
inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0278] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N. J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0279] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0280] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0281] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0282] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0283] D. Assays
[0284] Antibodies provided herein may be identified, screened for,
or characterized for their physical/chemical properties and/or
biological activities by various assays known in the art.
[0285] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
BIACore.RTM., FACS, or Western blot.
[0286] In another aspect, competition assays may be used to
identify an antibody that competes with any of the antibodies
described herein for binding to the target antigen. In certain
embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by an
antibody described herein. Detailed exemplary methods for mapping
an epitope to which an antibody binds are provided in Morris (1996)
"Epitope Mapping Protocols," in Methods in Molecular Biology vol.
66 (Humana Press, Totowa, N.J.).
[0287] In an exemplary competition assay, immobilized antigen is
incubated in a solution comprising a first labeled antibody that
binds to antigen (e.g., any of the antibodies described herein) and
a second unlabeled antibody that is being tested for its ability to
compete with the first antibody for binding to antigen. The second
antibody may be present in a hybridoma supernatant. As a control,
immobilized antigen is incubated in a solution comprising the first
labeled antibody but not the second unlabeled antibody. After
incubation under conditions permissive for binding of the first
antibody to antigen, excess unbound antibody is removed, and the
amount of label associated with immobilized antigen is measured. If
the amount of label associated with immobilized antigen is
substantially reduced in the test sample relative to the control
sample, then that indicates that the second antibody is competing
with the first antibody for binding to antigen. See Harlow and Lane
(1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0288] E. Immunoconjugates
[0289] Provided herein are also immunoconjugates comprising an
anti-CD79b antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes (i.e., a radioconjugate) for use
in the methods described herein.
[0290] Immunoconjugates allow for the targeted delivery of a drug
moiety to a tumor, and, in some embodiments intracellular
accumulation therein, where systemic administration of unconjugated
drugs may result in unacceptable levels of toxicity to normal cells
(Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).
[0291] Antibody-drug conjugates (ADC) are targeted chemotherapeutic
molecules which combine properties of both antibodies and cytotoxic
drugs by targeting potent cytotoxic drugs to antigen-expressing
tumor cells (Teicher, B. A. (2009) Current Cancer Drug Targets
9:982-1004), thereby enhancing the therapeutic index by maximizing
efficacy and minimizing off-target toxicity (Carter, P. J. and
Senter P. D. (2008) The Cancer Jour. 14(3):154-169; Chari, R. V.
(2008) Acc. Chem. Res. 41:98-107.
[0292] The ADC compounds of the invention include those with
anticancer activity. In some embodiments, the ADC compounds include
an antibody conjugated, i.e. covalently attached, to the drug
moiety. In some embodiments, the antibody is covalently attached to
the drug moiety through a linker. The antibody-drug conjugates
(ADC) of the invention selectively deliver an effective dose of a
drug to tumor tissue whereby greater selectivity, i.e. a lower
efficacious dose, may be achieved while increasing the therapeutic
index ("therapeutic window").
[0293] The drug moiety (D) of the antibody-drug conjugates (ADC)
may include any compound, moiety or group that has a cytotoxic or
cytostatic effect. Drug moieties may impart their cytotoxic and
cytostatic effects by mechanisms including but not limited to
tubulin binding, DNA binding or intercalation, and inhibition of
RNA polymerase, protein synthesis, and/or topoisomerase. Exemplary
drug moieties include, but are not limited to, a maytansinoid,
dolastatin, auristatin, calicheamicin, anthracycline, duocarmycin,
vinca alkaloid, taxane, trichothecene, CC1065, camptothecin,
elinafide, and stereoisomers, isosteres, analogs, and derivatives
thereof that have cytotoxic activity. Nonlimiting examples of such
immunoconjugates are discussed in further detail below.
[0294] 1. Exemplary Antibody-Drug Conjugates
[0295] An exemplary embodiment of an antibody-drug conjugate (ADC)
compound comprises an antibody (Ab) which targets a tumor cell, a
drug moiety (D), and a linker moiety (L) that attaches Ab to D. In
some embodiments, the antibody is attached to the linker moiety (L)
through one or more amino acid residues, such as lysine and/or
cysteine. In some embodiments of any of the methods, the
immunoconjugate has the formula Ab-(L-D)p, wherein: (a) Ab is the
antibody which binds a MM cell surface protein; (b) L is a linker;
(c) D is a cytotoxic agent; and (d) p ranges from 1-8.
[0296] An exemplary ADC has Formula I:
Ab-(L-D).sub.p I
where p is 1 to about 20. In some embodiments, the number of drug
moieties that can be conjugated to an antibody is limited by the
number of free cysteine residues. In some embodiments, free
cysteine residues are introduced into the antibody amino acid
sequence by the methods described herein. Exemplary ADC of Formula
I include, but are not limited to, antibodies that have 1, 2, 3, or
4 engineered cysteine amino acids (Lyon, R. et al (2012) Methods in
Enzym. 502:123-138). In some embodiments, one or more free cysteine
residues are already present in an antibody, without the use of
engineering, in which case the existing free cysteine residues may
be used to conjugate the antibody to a drug. In some embodiments,
an antibody is exposed to reducing conditions prior to conjugation
of the antibody in order to generate one or more free cysteine
residues.
[0297] a) Exemplary Linkers
[0298] A "Linker" (L) is a bifunctional or multifunctional moiety
that can be used to link one or more drug moieties (D) to an
antibody (Ab) to form an antibody-drug conjugate (ADC) of Formula
I. In some embodiments, antibody-drug conjugates (ADC) can be
prepared using a Linker having reactive functionalities for
covalently attaching to the drug and to the antibody. For example,
in some embodiments, a cysteine thiol of an antibody (Ab) can form
a bond with a reactive functional group of a linker or a
drug-linker intermediate to make an ADC.
[0299] In one aspect, a linker has a functionality that is capable
of reacting with a free cysteine present on an antibody to form a
covalent bond. Nonlimiting exemplary such reactive functionalities
include maleimide, haloacetamides, .alpha.-haloacetyl, activated
esters such as succinimide esters, 4-nitrophenyl esters,
pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides,
acid chlorides, sulfonyl chlorides, isocyanates, and
isothiocyanates. See, e.g., the conjugation method at page 766 of
Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, and
the Examples herein.
[0300] In some embodiments, a linker has a functionality that is
capable of reacting with an electrophilic group present on an
antibody. Exemplary such electrophilic groups include, but are not
limited to, aldehyde and ketone carbonyl groups. In some
embodiments, a heteroatom of the reactive functionality of the
linker can react with an electrophilic group on an antibody and
form a covalent bond to an antibody unit. Nonlimiting exemplary
such reactive functionalities include, but are not limited to,
hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine
carboxylate, and arylhydrazide.
[0301] A linker may comprise one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl (a
"PAB"), N-Succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and
4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("MCC"). Various
linker components are known in the art, some of which are described
below.
[0302] A linker may be a "cleavable linker," facilitating release
of a drug. Nonlimiting exemplary cleavable linkers include
acid-labile linkers (e.g., comprising hydrazone),
protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile
linkers, or disulfide-containing linkers (Chari et al., Cancer
Research 52:127-131 (1992); U.S. Pat. No. 5,208,020).
[0303] In certain embodiments, a linker has the following Formula
II:
-A.sub.a-W.sub.w--Y.sub.y-- II
wherein A is a "stretcher unit", and a is an integer from 0 to 1; W
is an "amino acid unit", and w is an integer from 0 to 12; Y is a
"spacer unit", and y is 0, 1, or 2; and Ab, D, and p are defined as
above for Formula I. Exemplary embodiments of such linkers are
described in U.S. Pat. No. 7,498,298, which is expressly
incorporated herein by reference.
[0304] In some embodiments, a linker component comprises a
"stretcher unit" that links an antibody to another linker component
or to a drug moiety. Nonlimiting exemplary stretcher units are
shown below (wherein the wavy line indicates sites of covalent
attachment to an antibody, drug, or additional linker
components):
##STR00004##
[0305] In some embodiments, a linker component comprises an "amino
acid unit". In some such embodiments, the amino acid unit allows
for cleavage of the linker by a protease, thereby facilitating
release of the drug from the immunoconjugate upon exposure to
intracellular proteases, such as lysosomal enzymes (Doronina et al.
(2003) Nat. Biotechnol. 21:778-784). Exemplary amino acid units
include, but are not limited to, dipeptides, tripeptides,
tetrapeptides, and pentapeptides. Exemplary dipeptides include, but
are not limited to, valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or
phe-lys); phenylalanine-homolysine (phe-homolys); and
N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides
include, but are not limited to, glycine-valine-citrulline
(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). An amino
acid unit may comprise amino acid residues that occur naturally
and/or minor amino acids and/or non-naturally occurring amino acid
analogs, such as citrulline. Amino acid units can be designed and
optimized for enzymatic cleavage by a particular enzyme, for
example, a tumor-associated protease, cathepsin B, C and D, or a
plasmin protease.
[0306] In some embodiments, a linker component comprises a "spacer"
unit that links the antibody to a drug moiety, either directly or
through a stretcher unit and/or an amino acid unit. A spacer unit
may be "self-immolative" or a "non-self-immolative." A
"non-self-immolative" spacer unit is one in which part or all of
the spacer unit remains bound to the drug moiety upon cleavage of
the ADC. Examples of non-self-immolative spacer units include, but
are not limited to, a glycine spacer unit and a glycine-glycine
spacer unit. In some embodiments, enzymatic cleavage of an ADC
containing a glycine-glycine spacer unit by a tumor-cell associated
protease results in release of a glycine-glycine-drug moiety from
the remainder of the ADC. In some such embodiments, the
glycine-glycine-drug moiety is subjected to a hydrolysis step in
the tumor cell, thus cleaving the glycine-glycine spacer unit from
the drug moiety.
[0307] A "self-immolative" spacer unit allows for release of the
drug moiety. In certain embodiments, a spacer unit of a linker
comprises a p-aminobenzyl unit. In some such embodiments, a
p-aminobenzyl alcohol is attached to an amino acid unit via an
amide bond, and a carbamate, methylcarbamate, or carbonate is made
between the benzyl alcohol and the drug (Hamann et al. (2005)
Expert Opin. Ther. Patents (2005) 15:1087-1103). In some
embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In
some embodiments, an ADC comprising a self-immolative linker has
the structure:
##STR00005##
wherein Q is --C.sub.1-C.sub.8 alkyl, --O--(C.sub.1-C.sub.8 alkyl),
-halogen, -nitro, or -cyno; m is an integer ranging from 0 to 4;
and p ranges from 1 to about 20. In some embodiments, p ranges from
1 to 10, 1 to 7, 1 to 5, or 1 to 4.
[0308] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically similar
to the PAB group, such as 2-aminoimidazol-5-methanol derivatives
(U.S. Pat. No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem.
Lett. 9:2237) and ortho- or para-aminobenzylacetals. In some
embodiments, spacers can be used that undergo cyclization upon
amide bond hydrolysis, such as substituted and unsubstituted
4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry
Biology 2:223), appropriately substituted bicyclo[2.2.1] and
bicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.
94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al
(1990) J. Org. Chem. 55:5867). Linkage of a drug to the
.alpha.-carbon of a glycine residue is another example of a
self-immolative spacer that may be useful in ADC (Kingsbury et al
(1984) J. Med. Chem. 27:1447).
[0309] In some embodiments, linker L may be a dendritic type linker
for covalent attachment of more than one drug moiety to an antibody
through a branching, multifunctional linker moiety (Sun et al
(2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215;
Sun et al (2003) Bioorganic & Medicinal Chemistry
11:1761-1768). Dendritic linkers can increase the molar ratio of
drug to antibody, i.e. loading, which is related to the potency of
the ADC. Thus, where an antibody bears only one reactive cysteine
thiol group, a multitude of drug moieties may be attached through a
dendritic linker.
[0310] Nonlimiting exemplary linkers are shown below in the context
of an ADC of Formula I:
##STR00006##
[0311] Further nonlimiting exemplary ADCs include the
structures:
##STR00007##
[0312] where X is:
##STR00008##
[0313] Y is:
##STR00009##
[0314] each R is independently H or C.sub.1-C.sub.6 alkyl; and n is
1 to 12.
[0315] Typically, peptide-type linkers can be prepared by forming a
peptide bond between two or more amino acids and/or peptide
fragments. Such peptide bonds can be prepared, for example,
according to a liquid phase synthesis method (e.g., E. Schroder and
K. Lubke (1965) "The Peptides", volume 1, pp 76-136, Academic
Press).
[0316] In some embodiments, a linker is substituted with groups
that modulate solubility and/or reactivity. As a nonlimiting
example, a charged substituent such as sulfonate (--SO.sub.3.sup.-)
or ammonium may increase water solubility of the linker reagent and
facilitate the coupling reaction of the linker reagent with the
antibody and/or the drug moiety, or facilitate the coupling
reaction of Ab-L (antibody-linker intermediate) with D, or D-L
(drug-linker intermediate) with Ab, depending on the synthetic
route employed to prepare the ADC. In some embodiments, a portion
of the linker is coupled to the antibody and a portion of the
linker is coupled to the drug, and then the Ab-(linker
portion).sup.a is coupled to drug-(linker portion).sup.b to form
the ADC of Formula I. In some such embodiments, the antibody
comprises more than one (linker portion).sup.a substituents, such
that more than one drug is coupled to the antibody in the ADC of
Formula I.
[0317] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with the following linker
reagents: bis-maleimido-trioxyethylene glycol (BMPEO),
N-(.beta.-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),
N-(.epsilon.-maleimidocaproyloxy) succinimide ester (EMCS),
N-[.gamma.-maleimidobutyryloxy]succinimide ester (GMBS),
1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)
(LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl
3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate
(SIA), succinimidyl(4-iodoacetyl)aminobenzoate (STAB),
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl
6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane
(IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and
succinimidyl-(4-vinylsulfone)benzoate (SVSB), and including
bis-maleimide reagents: dithiobismaleimidoethane (DTME),
1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane
(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE),
BM(PEG).sub.2 (shown below), and BM(PEG).sub.3 (shown below);
bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCl), active esters (such as disuccinimidyl suberate),
aldehydes (such as glutaraldehyde), bis-azido compounds (such as
bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such
as toluene 2,6-diisocyanate), and bis-active fluorine compounds
(such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments,
bis-maleimide reagents allow the attachment of the thiol group of a
cysteine in the antibody to a thiol-containing drug moiety, linker,
or linker-drug intermediate. Other functional groups that are
reactive with thiol groups include, but are not limited to,
iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl
disulfide, isocyanate, and isothiocyanate.
##STR00010##
[0318] Certain useful linker reagents can be obtained from various
commercial sources, such as Pierce Biotechnology, Inc. (Rockford,
Ill.), Molecular Biosciences Inc. (Boulder, Colo.), or synthesized
in accordance with procedures described in the art; for example, in
Toki et al (2002) J. Org. Chem. 67:1866-1872; Dubowchik, et al.
(1997) Tetrahedron Letters, 38:5257-60; Walker, M. A. (1995) J.
Org. Chem. 60:5352-5355; Frisch et al (1996) Bioconjugate Chem.
7:180-186; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189;
US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.
[0319] Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody. See, e.g.,
WO94/11026.
[0320] b) Exemplary Drug Moieties
[0321] (1) Maytansine and Maytansinoids
[0322] In some embodiments, an immunoconjugate comprises an
antibody conjugated to one or more maytansinoid molecules.
Maytansinoids are derivatives of maytansine, and are mitototic
inhibitors which act by inhibiting tubulin polymerization.
Maytansine was first isolated from the east African shrub Maytenus
serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered
that certain microbes also produce maytansinoids, such as
maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).
Synthetic maytansinoids are disclosed, for example, in U.S. Pat.
Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;
4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946;
4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866;
4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0323] Maytansinoid drug moieties are attractive drug moieties in
antibody-drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification or
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0324] Certain maytansinoids suitable for use as maytansinoid drug
moieties are known in the art and can be isolated from natural
sources according to known methods or produced using genetic
engineering techniques (see, e.g., Yu et al (2002) PNAS
99:7968-7973). Maytansinoids may also be prepared synthetically
according to known methods.
[0325] Exemplary maytansinoid drug moieties include, but are not
limited to, those having a modified aromatic ring, such as:
C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, for example, by
lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy
(or C-20-demethyl)+/-C-19-dechloro (U.S. Pat. Nos. 4,361,650 and
4,307,016) (prepared, for example, by demethylation using
Streptomyces or Actinomyces or dechlorination using LAH); and
C-20-demethoxy, C-20-acyloxy (--OCOR), +/-dechloro (U.S. Pat. No.
4,294,757) (prepared, for example, by acylation using acyl
chlorides), and those having modifications at other positions of
the aromatic ring.
[0326] Exemplary maytansinoid drug moieties also include those
having modifications such as: C-9-SH (U.S. Pat. No. 4,424,219)
(prepared, for example, by the reaction of maytansinol with
H.sub.2S or P.sub.2S.sub.5); C-14-alkoxymethyl(demethoxy/CH.sub.2
OR)(U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl
(CH.sub.2OH or CH.sub.2OAc) (U.S. Pat. No. 4,450,254) (prepared,
for example, from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.
4,364,866) (prepared, for example, by the conversion of maytansinol
by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and
4,315,929) (for example, isolated from Trewia nudlflora);
C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared,
for example, by the demethylation of maytansinol by Streptomyces);
and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by
the titanium trichloride/LAH reduction of maytansinol).
[0327] Many positions on maytansinoid compounds are useful as the
linkage position. For example, an ester linkage may be formed by
reaction with a hydroxyl group using conventional coupling
techniques. In some embodiments, the reaction may occur at the C-3
position having a hydroxyl group, the C-14 position modified with
hydroxymethyl, the C-15 position modified with a hydroxyl group,
and the C-20 position having a hydroxyl group. In some embodiments,
the linkage is formed at the C-3 position of maytansinol or a
maytansinol analogue.
[0328] Maytansinoid drug moieties include those having the
structure:
##STR00011## [0329] where the wavy line indicates the covalent
attachment of the sulfur atom of the maytansinoid drug moiety to a
linker of an ADC. Each R may independently be H or a
C.sub.1-C.sub.6 alkyl. The alkylene chain attaching the amide group
to the sulfur atom may be methanyl, ethanyl, or propyl, i.e., m is
1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020; Chari
et al (1992) Cancer Res. 52:127-131; Liu et al (1996) Proc. Natl.
Acad. Sci USA 93:8618-8623).
[0330] All stereoisomers of the maytansinoid drug moiety are
contemplated for the ADC of the invention, i.e. any combination of
R and S configurations at the chiral carbons (U.S. Pat. No.
7,276,497; U.S. Pat. No. 6,913,748; U.S. Pat. No. 6,441,163; U.S.
Pat. No. 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al
(2006) J. Med. Chem. 49:4392-4408, which are incorporated by
reference in their entirety). In some embodiments, the maytansinoid
drug moiety has the following stereochemistry:
##STR00012##
[0331] Exemplary embodiments of maytansinoid drug moieties include,
but are not limited to, DM1; DM3; and DM4, having the
structures:
##STR00013## [0332] wherein the wavy line indicates the covalent
attachment of the sulfur atom of the drug to a linker (L) of an
antibody-drug conjugate.
[0333] Other exemplary maytansinoid antibody-drug conjugates have
the following structures and abbreviations (wherein Ab is antibody
and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1
to 7, p is 1 to 5, or p is 1 to 4):
##STR00014##
[0334] Exemplary antibody-drug conjugates where DM1 is linked
through a BMPEO linker to a thiol group of the antibody have the
structure and abbreviation:
##STR00015## [0335] where Ab is antibody; n is 0, 1, or 2; and p is
1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is
1 to 5, or p is 1 to 4.
[0336] Immunoconjugates containing maytansinoids, methods of making
the same, and their therapeutic use are disclosed, for example, in
U.S. Pat. Nos. 5,208,020 and 5,416,064; US 2005/0276812 A1; and
European Patent EP 0 425 235 B1, the disclosures of which are
hereby expressly incorporated by reference. See also Liu et al.
Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996); and Chari et al.
Cancer Research 52:127-131 (1992).
[0337] In some embodiments, antibody-maytansinoid conjugates may be
prepared by chemically linking an antibody to a maytansinoid
molecule without significantly diminishing the biological activity
of either the antibody or the maytansinoid molecule. See, e.g.,
U.S. Pat. No. 5,208,020 (the disclosure of which is hereby
expressly incorporated by reference). In some embodiments, ADC with
an average of 3-4 maytansinoid molecules conjugated per antibody
molecule has shown efficacy in enhancing cytotoxicity of target
cells without negatively affecting the function or solubility of
the antibody. In some instances, even one molecule of
toxin/antibody is expected to enhance cytotoxicity over the use of
naked antibody.
[0338] Exemplary linking groups for making antibody-maytansinoid
conjugates include, for example, those described herein and those
disclosed in U.S. Pat. No. 5,208,020; EP Patent 0 425 235 B1; Chari
et al. Cancer Research 52:127-131 (1992); US 2005/0276812 A1; and
US 2005/016993 A1, the disclosures of which are hereby expressly
incorporated by reference.
[0339] (2) Auristatins and Dolastatins
[0340] Drug moieties include dolastatins, auristatins, and analogs
and derivatives thereof (U.S. Pat. No. 5,635,483; U.S. Pat. No.
5,780,588; U.S. Pat. No. 5,767,237; U.S. Pat. No. 6,124,431).
Auristatins are derivatives of the marine mollusk compound
dolastatin-10. While not intending to be bound by any particular
theory, dolastatins and auristatins have been shown to interfere
with microtubule dynamics, GTP hydrolysis, and nuclear and cellular
division (Woyke et al (2001) Antimicrob. Agents and Chemother.
45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and
antifungal activity (Pettit et al (1998) Antimicrob. Agents
Chemother. 42:2961-2965). The dolastatin/auristatin drug moiety may
be attached to the antibody through the N (amino) terminus or the C
(carboxyl) terminus of the peptidic drug moiety (WO 02/088172;
Doronina et al (2003) Nature Biotechnology 21(7):778-784; Francisco
et al (2003) Blood 102(4):1458-1465).
[0341] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties D.sub.E and D.sub.F,
disclosed in U.S. Pat. No. 7,498,298 and U.S. Pat. No. 7,659,241,
the disclosures of which are expressly incorporated by reference in
their entirety:
##STR00016## [0342] wherein the wavy line of D.sub.E and D.sub.F
indicates the covalent attachment site to an antibody or
antibody-linker component, and independently at each location:
[0343] R.sup.2 is selected from H and C.sub.1-C.sub.5 alkyl; [0344]
R.sup.3 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); [0345] R.sup.4
is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); [0346] R.sup.5
is selected from H and methyl; [0347] or R.sup.4 and R.sup.5
jointly form a carbocyclic ring and have the formula
--(CR.sup.aR.sup.b).sub.n-- wherein R.sup.a and R.sup.b are
independently selected from H, C.sub.1-C.sub.8 alkyl and
C.sub.3-C.sub.8 carbocycle and n is selected from 2, 3, 4, 5 and 6;
[0348] R.sup.6 is selected from H and C.sub.1-C.sub.8 alkyl; [0349]
R.sup.7 is selected from H, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8
carbocycle, aryl, C.sub.1-C.sub.8 alkyl-aryl, C.sub.1-C.sub.8
alkyl-(C.sub.3-C.sub.8 carbocycle), C.sub.3-C.sub.8 heterocycle and
C.sub.1-C.sub.8 alkyl-(C.sub.3-C.sub.8 heterocycle); [0350] each
R.sup.8 is independently selected from H, OH, C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 carbocycle and O--(C.sub.1-C.sub.8 alkyl);
[0351] R.sup.9 is selected from H and C.sub.1-C.sub.8 alkyl; [0352]
R.sup.10 is selected from aryl or C.sub.3-C.sub.8 heterocycle;
[0353] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.8 alkyl; [0354] R.sup.11 is selected from H,
C.sub.1-C.sub.20 alkyl, aryl, C.sub.3-C.sub.8 heterocycle,
--(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2; [0355] m is an integer
ranging from 1-1000; [0356] R.sup.13 is C.sub.2-C.sub.8 alkyl;
[0357] R.sup.14 is H or C.sub.1-C.sub.8 alkyl; [0358] each
occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.8 alkyl; [0359] each
occurrence of R.sup.16 is independently H, C.sub.1-C.sub.8 alkyl,
or --(CH.sub.2).sub.n--COOH; [0360] R.sup.18 is selected from
--C(R.sup.8).sub.2--C(R.sup.8).sub.2-aryl,
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8
heterocycle), and
--C(R.sup.8).sub.2--C(R.sup.8).sub.2--(C.sub.3-C.sub.8 carbocycle);
and [0361] n is an integer ranging from 0 to 6.
[0362] In one embodiment, R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H or methyl.
In an exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is --H, and R.sup.7 is sec-butyl.
[0363] In yet another embodiment, R.sup.2 and R.sup.6 are each
methyl, and R.sup.9 is --H.
[0364] In still another embodiment, each occurrence of R.sup.8 is
--OCH.sub.3.
[0365] In an exemplary embodiment, R.sup.3 and R.sup.4 are each
isopropyl, R.sup.2 and R.sup.6 are each methyl, R.sup.5 is --H,
R.sup.7 is sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3,
and R.sup.9 is --H.
[0366] In one embodiment, Z is --O-- or --NH--.
[0367] In one embodiment, R.sup.10 is aryl.
[0368] In an exemplary embodiment, R.sup.10 is -phenyl.
[0369] In an exemplary embodiment, when Z is --O--, R.sup.11 is
--H, methyl or t-butyl.
[0370] In one embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH).sub.n--N(R.sup.16).sub.2, and R.sup.16 is --C.sub.1-C.sub.8
alkyl or --(CH.sub.2).sub.n--COOH.
[0371] In another embodiment, when Z is --NH, R.sup.11 is
--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H.
[0372] An exemplary auristatin embodiment of formula D.sub.E is
MMAE, wherein the wavy line indicates the covalent attachment to a
linker (L) of an antibody-drug conjugate:
##STR00017##
[0373] An exemplary auristatin embodiment of formula D.sub.F is
MMAF, wherein the wavy line indicates the covalent attachment to a
linker (L) of an antibody-drug conjugate:
##STR00018##
[0374] Other exemplary embodiments include monomethylvaline
compounds having phenylalanine carboxy modifications at the
C-terminus of the pentapeptide auristatin drug moiety (WO
2007/008848) and monomethylvaline compounds having phenylalanine
sidechain modifications at the C-terminus of the pentapeptide
auristatin drug moiety (WO 2007/008603).
[0375] Nonlimiting exemplary embodiments of ADC of Formula I
comprising MMAE or MMAF and various linker components have the
following structures and abbreviations (wherein "Ab" is an
antibody; p is 1 to about 8, "Val-Cit" is a valine-citrulline
dipeptide; and "S" is a sulfur atom:
##STR00019##
[0376] Nonlimiting exemplary embodiments of ADCs of Formula I
comprising MMAF and various linker components further include
Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates comprising MMAF
attached to an antibody by a linker that is not proteolytically
cleavable have been shown to possess activity comparable to
immunoconjugates comprising MMAF attached to an antibody by a
proteolytically cleavable linker (Doronina et al. (2006)
Bioconjugate Chem. 17:114-124). In some such embodiments, drug
release is believed to be effected by antibody degradation in the
cell.
[0377] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to a liquid phase synthesis method (see, e.g., E.
Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965,
Academic Press). Auristatin/dolastatin drug moieties may, in some
embodiments, be prepared according to the methods of: U.S. Pat. No.
7,498,298; U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit
et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998)
Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin
Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol.
21(7):778-784.
[0378] In some embodiments, auristatin/dolastatin drug moieties of
formulas D.sub.E such as MMAE, and D.sub.E, such as MMAF, and
drug-linker intermediates and derivatives thereof, such as MC-MMAF,
MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using
methods described in U.S. Pat. No. 7,498,298; Doronina et al.
(2006) Bioconjugate Chem. 17:114-124; and Doronina et al. (2003)
Nat. Biotech. 21:778-784 and then conjugated to an antibody of
interest.
[0379] (3) Calicheamicin
[0380] In some embodiments, the immunoconjugate comprises an
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics, and analogues thereof, are
capable of producing double-stranded DNA breaks at sub-picomolar
concentrations (Hinman et al., (1993) Cancer Research 53:3336-3342;
Lode et al., (1998) Cancer Research 58:2925-2928). Calicheamicin
has intracellular sites of action but, in certain instances, does
not readily cross the plasma membrane. Therefore, cellular uptake
of these agents through antibody-mediated internalization may, in
some embodiments, greatly enhances their cytotoxic effects.
Nonlimiting exemplary methods of preparing antibody-drug conjugates
with a calicheamicin drug moiety are described, for example, in
U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S. Pat. No.
5,739,116; and U.S. Pat. No. 5,767,285.
[0381] (4) Other Drug Moieties
[0382] Drug moieties also include geldanamycin (Mandler et al
(2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)
Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al
(2002) Bioconjugate Chem. 13:786-791); and enzymatically active
toxins and fragments thereof, including, but not limited to,
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin and the tricothecenes. See,
e.g., WO 93/21232.
[0383] Drug moieties also include compounds with nucleolytic
activity (e.g., a ribonuclease or a DNA endonuclease).
[0384] In certain embodiments, an immunoconjugate may comprise a
highly radioactive atom. A variety of radioactive isotopes are
available for the production of radioconjugated antibodies.
Examples include At.sup.211, I.sup.131, I.sup.125, Y.sup.90,
Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. In some embodiments,
when an immunoconjugate is used for detection, it may comprise a
radioactive atom for scintigraphic studies, for example Tc.sup.99
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, MRI), such as
zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19,
carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
Zirconium-89 may be complexed to various metal chelating agents and
conjugated to antibodies, e.g., for PET imaging (WO
2011/056983).
[0385] The radio- or other labels may be incorporated in the
immunoconjugate in known ways. For example, a peptide may be
biosynthesized or chemically synthesized using suitable amino acid
precursors comprising, for example, one or more fluorine-19 atoms
in place of one or more hydrogens. In some embodiments, labels such
as Tc.sup.99, I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the antibody. In some
embodiments, yttrium-90 can be attached via a lysine residue of the
antibody. In some embodiments, the IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain
other methods.
[0386] In certain embodiments, an immunoconjugate may comprise an
antibody conjugated to a prodrug-activating enzyme. In some such
embodiments, a prodrug-activating enzyme converts a prodrug (e.g.,
a peptidyl chemotherapeutic agent, see WO 81/01145) to an active
drug, such as an anti-cancer drug. Such immunoconjugates are
useful, in some embodiments, in antibody-dependent enzyme-mediated
prodrug therapy ("ADEPT"). Enzymes that may be conjugated to an
antibody include, but are not limited to, alkaline phosphatases,
which are useful for converting phosphate-containing prodrugs into
free drugs; arylsulfatases, which are useful for converting
sulfate-containing prodrugs into free drugs; cytosine deaminase,
which is useful for converting non-toxic 5-fluorocytosine into the
anti-cancer drug, 5-fluorouracil; proteases, such as serratia
protease, thermolysin, subtilisin, carboxypeptidases and cathepsins
(such as cathepsins B and L), which are useful for converting
peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, which are useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase, which are
useful for converting glycosylated prodrugs into free drugs;
.beta.-lactamase, which is useful for converting drugs derivatized
with .beta.-lactams into free drugs; and penicillin amidases, such
as penicillin V amidase and penicillin G amidase, which are useful
for converting drugs derivatized at their amine nitrogens with
phenoxyacetyl or phenylacetyl groups, respectively, into free
drugs. In some embodiments, enzymes may be covalently bound to
antibodies by recombinant DNA techniques well known in the art.
See, e.g., Neuberger et al., Nature 312:604-608 (1984).
[0387] c) Drug Loading
[0388] Drug loading is represented by p, the average number of drug
moieties per antibody in a molecule of Formula I. Drug loading may
range from 1 to 20 drug moieties (D) per antibody. ADCs of Formula
I include collections of antibodies conjugated with a range of drug
moieties, from 1 to 20. The average number of drug moieties per
antibody in preparations of ADC from conjugation reactions may be
characterized by conventional means such as mass spectroscopy,
ELISA assay, and HPLC. The quantitative distribution of ADC in
terms of p may also be determined. In some instances, separation,
purification, and characterization of homogeneous ADC where p is a
certain value from ADC with other drug loadings may be achieved by
means such as reverse phase HPLC or electrophoresis.
[0389] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in certain exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached. In certain
embodiments, higher drug loading, e.g., p>5, may cause
aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates. In certain
embodiments, the average drug loading for an ADC ranges from 1 to
about 8; from about 2 to about 6; or from about 3 to about 5.
Indeed, it has been shown that for certain ADCs, the optimal ratio
of drug moieties per antibody may be less than 8, and may be about
2 to about 5 (U.S. Pat. No. 7,498,298).
[0390] In certain embodiments, fewer than the theoretical maximum
of drug moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, lysine residues
that do not react with the drug-linker intermediate or linker
reagent, as discussed below. Generally, antibodies do not contain
many free and reactive cysteine thiol groups which may be linked to
a drug moiety; indeed most cysteine thiol residues in antibodies
exist as disulfide bridges. In certain embodiments, an antibody may
be reduced with a reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or total reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, an antibody is subjected to denaturing conditions to
reveal reactive nucleophilic groups such as lysine or cysteine.
[0391] The loading (drug/antibody ratio) of an ADC may be
controlled in different ways, and for example, by: (i) limiting the
molar excess of drug-linker intermediate or linker reagent relative
to antibody, (ii) limiting the conjugation reaction time or
temperature, and (iii) partial or limiting reductive conditions for
cysteine thiol modification.
[0392] It is to be understood that where more than one nucleophilic
group reacts with a drug-linker intermediate or linker reagent,
then the resulting product is a mixture of ADC compounds with a
distribution of one or more drug moieties attached to an antibody.
The average number of drugs per antibody may be calculated from the
mixture by a dual ELISA antibody assay, which is specific for
antibody and specific for the drug. Individual ADC molecules may be
identified in the mixture by mass spectroscopy and separated by
HPLC, e.g., hydrophobic interaction chromatography (see, e.g.,
McDonagh et al (2006) Prot. Engr. Design & Selection
19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.
10:7063-7070; Hamblett, K. J., et al. "Effect of drug loading on
the pharmacology, pharmacokinetics, and toxicity of an anti-CD30
antibody-drug conjugate," Abstract No. 624, American Association
for Cancer Research, 2004 Annual Meeting, Mar. 27-31, 2004,
Proceedings of the AACR, Volume 45, March 2004; Alley, S. C., et
al. "Controlling the location of drug attachment in antibody-drug
conjugates," Abstract No. 627, American Association for Cancer
Research, 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the
AACR, Volume 45, March 2004). In certain embodiments, a homogeneous
ADC with a single loading value may be isolated from the
conjugation mixture by electrophoresis or chromatography.
[0393] d) Certain Methods of Preparing Immunoconjugates
[0394] An ADC of Formula I may be prepared by several routes
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
nucleophilic group of an antibody with a bivalent linker reagent to
form Ab-L via a covalent bond, followed by reaction with a drug
moiety D; and (2) reaction of a nucleophilic group of a drug moiety
with a bivalent linker reagent, to form D-L, via a covalent bond,
followed by reaction with a nucleophilic group of an antibody.
Exemplary methods for preparing an ADC of Formula I via the latter
route are described in U.S. Pat. No. 7,498,298, which is expressly
incorporated herein by reference.
[0395] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g., lysine, (iii) side chain thiol groups, e.g.,
cysteine, and (iv) sugar hydroxyl or amino groups where the
antibody is glycosylated. Amine, thiol, and hydroxyl groups are
nucleophilic and capable of reacting to form covalent bonds with
electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; and (iii) aldehydes, ketones, carboxyl, and
maleimide groups. Certain antibodies have reducible interchain
disulfides, i.e. cysteine bridges. Antibodies may be made reactive
for conjugation with linker reagents by treatment with a reducing
agent such as DTT (dithiothreitol) or tricarbonylethylphosphine
(TCEP), such that the antibody is fully or partially reduced. Each
cysteine bridge will thus form, theoretically, two reactive thiol
nucleophiles. Additional nucleophilic groups can be introduced into
antibodies through modification of lysine residues, e.g., by
reacting lysine residues with 2-iminothiolane (Traut's reagent),
resulting in conversion of an amine into a thiol. Reactive thiol
groups may also be introduced into an antibody by introducing one,
two, three, four, or more cysteine residues (e.g., by preparing
variant antibodies comprising one or more non-native cysteine amino
acid residues).
[0396] Antibody-drug conjugates of the invention may also be
produced by reaction between an electrophilic group on an antibody,
such as an aldehyde or ketone carbonyl group, with a nucleophilic
group on a linker reagent or drug. Useful nucleophilic groups on a
linker reagent include, but are not limited to, hydrazide, oxime,
amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide. In one embodiment, an antibody is modified to
introduce electrophilic moieties that are capable of reacting with
nucleophilic substituents on the linker reagent or drug. In another
embodiment, the sugars of glycosylated antibodies may be oxidized,
e.g., with periodate oxidizing reagents, to form aldehyde or ketone
groups which may react with the amine group of linker reagents or
drug moieties. The resulting imine Schiff base groups may form a
stable linkage, or may be reduced, e.g., by borohydride reagents to
form stable amine linkages. In one embodiment, reaction of the
carbohydrate portion of a glycosylated antibody with either
galactose oxidase or sodium meta-periodate may yield carbonyl
(aldehyde and ketone) groups in the antibody that can react with
appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, antibodies containing
N-terminal serine or threonine residues can react with sodium
meta-periodate, resulting in production of an aldehyde in place of
the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate
Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such an aldehyde can be
reacted with a drug moiety or linker nucleophile.
[0397] Exemplary nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0398] Nonlimiting exemplary cross-linker reagents that may be used
to prepare ADC are described herein in the section titled
"Exemplary Linkers." Methods of using such cross-linker reagents to
link two moieties, including a proteinaceous moiety and a chemical
moiety, are known in the art. In some embodiments, a fusion protein
comprising an antibody and a cytotoxic agent may be made, e.g., by
recombinant techniques or peptide synthesis. A recombinant DNA
molecule may comprise regions encoding the antibody and cytotoxic
portions of the conjugate either adjacent to one another or
separated by a region encoding a linker peptide which does not
destroy the desired properties of the conjugate.
[0399] In yet another embodiment, an antibody may be conjugated to
a "receptor" (such as streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a drug or radionucleotide).
[0400] F. Methods and Compositions for Diagnostics and
Detection
[0401] Provided herein are also methods and compositions for
diagnosis and/or detection of CD79b antibodies for use in the
methods described herein including detecting the presence of CD79b
in a biological sample for use in selecting patients for treating
using the methods described herein. The term "detecting" as used
herein encompasses quantitative or qualitative detection. A
"biological sample" comprises, e.g., a cell or tissue.
[0402] In one embodiment, an anti-CD79b antibody for use in a
method of diagnosis or detection is provided. In a further aspect,
a method of detecting the presence of CD79b in a biological sample
is provided. In certain embodiments, the method comprises
contacting the biological sample with an anti-CD79b antibody as
described herein under conditions permissive for binding of the
anti-CD79b antibody to CD79b, and detecting whether a complex is
formed between the anti-CD79b antibody and CD79b in the biological
sample. Such method may be an in vitro or in vivo method. In one
embodiment, an anti-CD79b antibody is used to select subjects
eligible for therapy with an anti-CD79b antibody, e.g., where CD79b
is a biomarker for selection of patients. In a further embodiment,
the biological sample is a cell and/or tissue (e.g., bone marrow
and/or blood).
[0403] In a further embodiment, an anti-CD79b antibody is used in
vivo to detect, e.g., by in vivo imaging, a CD79b-positive cancer
in a subject, e.g., for the purposes of diagnosing, prognosing, or
staging cancer, determining the appropriate course of therapy, or
monitoring response of a cancer to therapy. One method known in the
art for in vivo detection is immuno-positron emission tomography
(immuno-PET), as described, e.g., in van Dongen et al., The
Oncologist 12:1379-1389 (2007) and Verel et al., J. Nucl. Med.
44:1271-1281 (2003). In such embodiments, a method is provided for
detecting a CD79b-positive cancer in a subject, the method
comprising administering a labeled anti-CD79b antibody to a subject
having or suspected of having a CD79b-positive cancer, and
detecting the labeled anti-CD79b antibody in the subject, wherein
detection of the labeled anti-CD79b antibody indicates a
CD79b-positive cancer in the subject. In certain of such
embodiments, the labeled anti-CD79b antibody comprises an
anti-CD.sup.79b antibody conjugated to a positron emitter, such as
.sup.68Ga, .sup.18F, .sup.64Cu, .sup.86Y, .sup.76Br, .sup.89Zr, and
.sup.124I. In a particular embodiment, the positron emitter is
.sup.89Zr.
[0404] In further embodiments, a method of diagnosis or detection
comprises contacting a first anti-CD79b antibody immobilized to a
substrate with a biological sample to be tested for the presence of
CD79b, exposing the substrate to a second anti-CD79b antibody, and
detecting whether the second anti-CD79b is bound to a complex
between the first anti-CD79b antibody and CD79b in the biological
sample. A substrate may be any supportive medium, e.g., glass,
metal, ceramic, polymeric beads, slides, chips, and other
substrates. In certain embodiments, a biological sample comprises a
cell or tissue (e.g., blood and/or bone marrow). In certain
embodiments, the first or second anti-CD79b antibody is any of the
antibodies described herein.
[0405] Exemplary disorders that may be diagnosed or detected
according to any of the above embodiments include CD79b-positive
cancers, such as lymphoma, non-Hogkins lymphoma (NHL), aggressive
NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory
NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL),
small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),
acute lymphocytic leukemia (ALL), Burkitt's lymphoma, diffuse
B-cell lymphoma (DBCL), and mantle cell lymphoma, in particular,
NHL, follicular lymphoma, and/or DBCL. In some embodiments, a
CD79b-positive cancer is a cancer that expresses CD79b according to
a reverse-transcriptase PCR (RT-PCR) assay that detects CD79b mRNA.
In some embodiments, the RT-PCR is quantitative RT-PCR.
[0406] In certain embodiments, labeled anti-CD79b antibodies for
use in the methods described herein are provided. Labels include,
but are not limited to, labels or moieties that are detected
directly (such as fluorescent, chromophoric, electron-dense,
chemiluminescent, and radioactive labels), as well as moieties,
such as enzymes or ligands, that are detected indirectly, e.g.,
through an enzymatic reaction or molecular interaction. Exemplary
labels include, but are not limited to, the radioisotopes .sup.32P,
.sup.14C, .sup.125I, .sup.3H, and .sup.131I, fluorophores such as
rare earth chelates or fluorescein and its derivatives, rhodamine
and its derivatives, dansyl, umbelliferone, luceriferases, e.g.,
firefly luciferase and bacterial luciferase (U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish
peroxidase (HRP), alkaline phosphatase, .beta.-galactosidase,
glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase,
heterocyclic oxidases such as uricase and xanthine oxidase, coupled
with an enzyme that employs hydrogen peroxide to oxidize a dye
precursor such as HRP, lactoperoxidase, or microperoxidase,
biotin/avidin, spin labels, bacteriophage labels, stable free
radicals, and the like. In another embodiment, a label is a
positron emitter. Positron emitters include but are not limited to
.sup.68Ga, .sup.18F, .sup.64Cu, .sup.86Y, .sup.76Br, .sup.89Zr, and
.sup.124I. In a particular embodiment, a positron emitter is
.sup.89Zr.
[0407] G. Pharmaceutical Formulations
[0408] Pharmaceutical formulations of any of the agents described
herein (e.g., anti-CD79b immunoconjugates) for use in any of the
methods as described herein are prepared by mixing such antibody or
immunoconjugate having the desired degree of purity with one or
more optional pharmaceutically acceptable carriers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of lyophilized formulations or aqueous solutions.
Pharmaceutically acceptable carriers are generally nontoxic to
recipients at the dosages and concentrations employed, and include,
but are not limited to: buffers such as phosphate, citrate, and
other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG). Exemplary
pharmaceutically acceptable carriers herein further include
insterstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rHuPH20, are described in US Patent Publication
Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is
combined with one or more additional glycosaminoglycanases such as
chondroitinases.
[0409] Exemplary lyophilized antibody or immunoconjugate
formulations are described in U.S. Pat. No. 6,267,958. Aqueous
antibody or immunoconjugate formulations include those described in
U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations
including a histidine-acetate buffer.
[0410] The formulation herein may also contain more than one active
ingredient as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other.
[0411] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0412] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody or
immunoconjugate, which matrices are in the form of shaped articles,
e.g., films, or microcapsules.
[0413] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0414] H. Articles of Manufacture
[0415] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the disorder
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody or immunoconjugate of the
invention. The label or package insert indicates that the
composition is used for treating the condition of choice. Moreover,
the article of manufacture may comprise (a) a first container with
a composition contained therein, wherein the composition comprises
an antibody or immunoconjugate; and (b) a second container with a
composition contained therein, wherein the composition comprises a
further cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
or dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
EXAMPLES
[0416] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1
Anti-CD79b Immunoconjugate in Combination with Anti-CD20 Antibody
Plus Alkyating Agent (Bendamustine) in Lymphoma
[0417] The combination efficacy of anti-CD79b immunoconjugate
(anti-CD79b (huMA79b.v28)-MC-vc-PAB-MMAE ADC; polatuzumab vedotin;
Pola; DCDS4501A) with anti-CD20 antibody (rituximab) and
bendamustine was evaluated in a tumor xenograft model of WSU-DLCL2
human diffuse large B-cell lymphoma.
[0418] Female C.B-17 SCID mice (11-12 weeks old from Charles River
Laboratories; Hollister, Calif.) were each inoculated
subcutaneously in the flank with 20 million WSU-DLCL2 cells (DSMZ,
German Collection of Microorganisms an Cell Cultures; Braunschweig,
Germany). When the xenograft tumors reached desired volume, animals
were randomized into groups of 9 mice each and received a single
dose of treatments (referred to as Day 0). Anti-CD20 antibody
(rituximab) was given intraperitoneally at 30 mg/kg.
Anti-CD79b-MMAE ADC and bendamustine was given intravenously at 2
and 30 mg/kg, respectively.
[0419] Tumors were measured 1-2 times a week throughout the study
using UltraCal-IV calipers and tumor volume was calculated using
following formula: tumor volume (mm.sup.3)=0.5a.times.b.sup.2,
wherein a and b are the long and short diameters of the tumor,
respectively.
[0420] To appropriately analyze the repeated measurement of tumor
volumes from the same animals over time, a mixed modeling approach
was used (Pinheiro J, et al. nlme: linear and nonlinear mixed
effects models. 2009; R package, version 3.1-96). This approach
addressed both repeated measurements and modest dropout rates due
to non-treatment related removal of animals before the study end.
Cubic regression splines were used to fit a non-linear profile to
the time courses of log.sub.2 tumor volume at each dose level.
These non-linear profiles were then related to dose within the
mixed model. The results were plotted as fitted tumor volume of
each group over time.
[0421] In this study, as shown in FIG. 1, anti-CD79b-MMAE ADC
demonstrated clear inhibition of tumor growth, and the anti-tumor
activity was comparable with the combination of rituximab and
bendamustine at the doses tested. Additionally, the triple
combination of anti-CD79b-MMAE ADC with rituximab and bendamustine
resulted in significantly greater efficacy than the ADC or
rituximab/bendamustine doublet alone.
Example 2
A Study of CD79b-MC-Vc-PAB-MMAE in Combination with Anti-CD20
Antibody (Rituximab or Obinutuzumab) Plus Alkyating Agent
(Bendamustine) in Patients with Relapsed or Refractory Follicular
or Diffuse Large B-Cell Lymphoma
[0422] A multicenter, open-label study of polatuzumab vedotin
(anti-CD79b (huMA79b.v28)-MC-vc-PAB-MMAE; "Pola") administered by
intravenous (IV) infusion in combination with standard doses of
bendamustine (B) and rituximab (R) or obinutuzumab (G) in patients
with relapsed or refractory follicular lymphoma (FL) or diffuse
large B-cell lymphoma (DLBCL) is initiated. The study comprises
first stage dose-escalation stage, stage 2, and stage 3, and the
time on treatment is 18-24 weeks.
[0423] In the first stage, FL and DLBCL patients are enrolled into
separate cohorts for dose escalation of Pola in combination with R
and B or G and B. Pola is administered intravenously on Day 2 of
Cycle 1, then on Day 1 of each subsequent cycle. R is administered
at a dose of 375 mg/m.sup.2 intravenously on Day 1 of Cycle 1 and
on Day 1 of each subsequent cycle for up to six cycles. B is
administered intravenously (90 mg/m.sup.2) on Days 2 and 3 of Cycle
1, then on Days 1 and 2 of each subsequent cycle. G is administered
intravenously (1000 mg) on Days 1, 8, and 15 of Cycle 1 and on Day
1 of each subsequent cycle for up to six cycles. Complete response
(CR) rate is measured by positron emission tomography (PET) scan
and is determined by an Institutional Review Board.
[0424] In the second stage, randomized, separate FL and DLBCL
cohorts receive (a) Pola in combination with R and B or (b) R and B
alone. R is administered at a dose of 375 mg/m.sup.2 intravenously
on Day 1 of Cycle 1 and on Day 1 of each subsequent cycle for up to
six cycles. B is administered intravenously (90 mg/m.sup.2) on Days
2 and 3 of Cycle 1, then on Days 1 and 2 of each subsequent
cycle.
[0425] In the third stage, non-randomized, separate FL and DLBCL
cohorts receive Pola in combination with G and B. B is administered
intravenously (90 mg/m.sup.2) on Days 2 and 3 of Cycle 1, then on
Days 1 and 2 of each subsequent cycle. G is administered
intravenously (1000 mg) on Days 1, 8, and 15 of Cycle 1 and on Day
1 of each subsequent cycle for up to six cycles. Complete response
(CR) rate is measured by positron emission tomography (PET) scan
and is determined by an Institutional Review Board.
[0426] Inclusion criteria for patients on study includes: [0427]
Histologically confirmed FL (Grade 1, 2, or 3a) or DLBCL [0428]
Must have received at least one prior therapy for FL or DLBCL.
Patients must have either relapsed or have become refractory to a
prior regimen as defined below: [0429] (a) Relapsed/Refractory FL:
Patients who have relapsed to prior regimen(s) after having a
documented history of response (complete response [CR], CR
unconfirmed [CRu], or partial response [PR]) of >/=6 months in
duration from completion of regimen(s); refractory to any prior
regimen, defined as no response to the prior therapy, or
progression within 6 months of completion of the last dose of
therapy. [0430] (b) Relapsed/Refractory DLBCL: Patients who are
ineligible for second-line stem cell transplant (SCT), with
progressive disease or no response (stable disease [SD])<6
months from start of initial therapy; patients who are ineligible
for second-line SCT, with disease relapse after initial response of
>1=6 months from start of initial therapy; patients who are
ineligible for third-line (or beyond) SCT, with progressive disease
or no response (SD)<6 months from start of prior therapy;
patients who are ineligible for third-line (or beyond) SCT with
disease relapse after initial response of >1=6 months from start
of prior therapy. [0431] If the patient has received prior
bendamustine, response duration must have been >1 year (for
patients who have relapse disease after a prior regimen). [0432] At
least one bi-dimensionally measurable lesion on imaging scan
defined as >1.5 cm in its longest dimension; confirmed
availability of archival or freshly collected tumor tissue meeting
protocol-defined specifications prior to study enrollment; Life
expectancy of at least 24 weeks; Eastern Cooperative Oncology Group
(ECOG) Performance Status of 0, 1, or 2; adequate hematological
function; and, for women of childbearing potential, a negative
serum pregnancy test result within 7 days prior to commencement of
dosing.
[0433] Exclusion criteria for patients on study includes: history
of severe allergic or anaphylactic reactions to humanized or murine
monoclonal antibodies (MAbs, or recombinant antibody-related fusion
proteins) or known sensitivity or allergy to murine products,
contraindication to bendamustine, rituximab, or obinutuzumab,
history of sensitivity to mannitol, prior use of any MAb,
radioimmunoconjugate, or antibody-drug conjugate (ADC) within 4
weeks before Cycle 1 Day 1, treatment with radiotherapy,
chemotherapy, immunotherapy, immunosuppressive therapy, or any
investigational agent for the purposes of treating cancer within 2
weeks prior to Cycle 1 Day 1, ongoing corticosteroid use >30
mg/day prednisone or equivalent, for purposes other than lymphoma
symptom control, completion of autologous SCT within 100 days prior
to Cycle 1 Day 1, prior allogeneic SCT, eligibility for autologous
SCT (patients with relapsed/refractory DLBCL), Grade 3b FL, history
of transformation of indolent disease to DLBCL, primary CNS
lymphoma, current Grade>1 peripheral neuropathy, evidence of
significant, uncontrolled concomitant diseases that could affect
compliance with the protocol or interpretation of results,
including significant cardiovascular disease (such as New York
Heart Association Class III or IV cardiac disease, myocardial
infarction within the last 6 months, unstable arrhythmias, or
unstable angina) or significant pulmonary disease (including
obstructive pulmonary disease and history of bronchospasm), known
active bacterial, viral, fungal, mycobacterial, parasitic, or other
infection (excluding fungal infections of nail beds) at study
enrollment or any major episode of infection requiring treatment
with intravenous (IV) antibiotics or hospitalization within 4 weeks
prior to Cycle 1 Day 1, patients with suspected or latent
tuberculosis, positive test results for chronic hepatitis B virus
(HBV) infection or for hepatitis C virus (HCV) antibody, known
infection with HIV or human T-cell leukemia virus 1 (HTLV-1) virus,
women who are pregnant or lactating or who intend to become
pregnant within a year of the last dose of rituximab or
obinutuzumab, evidence of laboratory abnormalities in standard
renal, hepatic or coagulation function tests.
Example 3
Anti-CD79b Immunoconjugate in Combination with Bcl2 Inhibitor in
Lymphoma
[0434] The combination efficacy of anti-CD79b-MMAE ADC (DCDS4501A)
with a selective Bcl2 inhibitor (ABT-199 (i.e., venetoclax,
GDC-0199,
4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-
-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfony-
l)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, and/or
CAS#1257044-40-8) was evaluated in a tumor xenograft model of
Granta-519 human mantle-cell lymphoma.
[0435] Female C.B-17 SCID mice (8 weeks old from Charles River
Laboratories; Hollister, Calif.) were each inoculated in the flank
with 20 million Granta-519 cells. When the xenograft tumors reached
desired volume, animals were randomized into groups of 9 mice each
and received treatments (Day 0 of the study).
Anti-CD79b-MC-vc-PAB-MMAE ADC was dosed once intravenously at 1
mg/kg and ABT-199 were given orally once a day for 21 days at 100
mg/kg.
[0436] Tumors were measured 1-2 times a week throughout the study
using UltraCal-IV calipers and tumor volume was calculated using
following formula: tumor volume (mm.sup.3)=0.5a.times.b.sup.2,
wherein a and b are the long and short diameters of the tumor,
respectively.
[0437] To appropriately analyze the repeated measurement of tumor
volumes from the same animals over time, a mixed modeling approach
was used (Pinheiro J, et al. nlme: linear and nonlinear mixed
effects models. 2009; R package, version 3.1-96). This approach
addresses both repeated measurements and modest dropout rates due
to non-treatment related removal of animals before the study end.
Cubic regression splines were used to fit a non-linear profile to
the time courses of log.sub.2 tumor volume at each dose level.
These non-linear profiles were then related to dose within the
mixed model. The results were plotted as fitted tumor volume of
each group over time.
[0438] In this study, as shown in FIG. 2, treatment with
anti-CD79b-MC-vc-PAB-MMAE ADC alone caused modest tumor growth
delay while ABT-199 monotherapy did not result in anti-tumor
activity. However, the combination of anti-CD79b-MC-vc-PAB-MMAE ADC
and ABT-199 resulted in greater efficacy, causing tumor
regressions, than either agent alone. The combination of
anti-CD79b-MC-vc-PAB-MMAE ADC and ABT-199 was well-tolerated based
on minimal changes in animal body weights during the treatment
period.
Example 4
Anti-CD79b Immunoconjugate in Combination Therapy in Lymphoma
[0439] The combination efficacy of anti-CD79b-MC-vc-PAB-MMAE ADC
(DCDS4501A; huMA79bv28-MC-vc-PAB-MMAE) with various combination
therapies was evaluated in a tumor xenograft model of WSU-DLCL2
(DLBCL).
[0440] Female C.B-17 SCID mice (14 weeks old from Charles River
Laboratories; Hollister, Calif.) were each inoculated in the flank
with 20 million WSU-DLCL2 (DLBCL) cells. When the xenograft tumors
reached desired volume, animals were randomized and received
treatments (Day 0 of the study). There were six treatment groups
(1) vehicle, (2) anti-CD79b-vcMMAE, (3) G-CHP
(GA101-cyclophosphamide, doxorubicine, and prednisone), (4)
G-bendamustine (GA101-bendamustine), (5) G-CHP
(GA101-cyclophosphamide, doxorubicine, and
prednisone)+anti-CD79b-MC-vc-PAB-MMAE, and (6) G-bendamustine
(GA101-bendamustine)+anti-CD79b-MC-vc-PAB-MMAE.
[0441] CD79b-MC-vc-PAB-MMAE ADC was dosed once intravenously at 2
mg/kg, iv, once. GA101 dosed 30 mg/kg, ip, once. CHP was dosed
cyclophosphamide, 30 mg/kg, iv, once+doxorubicine, 2.475 mg/kg, iv,
once+prednisone, 0.15 mg/kg, po, qdx5. Bendamustine was dosed 30
mg/kg, iv, once.
[0442] As described above, tumors were measured 1-2 times a week
throughout the study using UltraCal-IV calipers and tumor volume
was calculated using following formula: tumor volume
(mm.sup.3)=0.5a.times.b.sup.2, wherein a and b are the long and
short diameters of the tumor, respectively.
[0443] In this study, as shown in FIG. 3A, treatment with
anti-CD79b-MC-vc-PAB-MMAE ADC combined well with G-CHP (or G-Benda)
with better efficacy than the anti-CD79b-MC-vc-PAB-MMAE ADC or
G-CHP (or G-Benda) alone. The combinations were well-tolerated
based on minimal changes in animal body weights during the
treatment period.
Example 5
Anti-CD79b Immunoconjugate in Combination Therapy in Lymphoma
[0444] The combination efficacy of anti-CD79b-MC-vc-PAB-MMAE ADC
(DCDS4501A; huMA79bv28-MC-vc-PAB-MMAE) with various combination
therapies was evaluated in a tumor xenograft model of TMD8
(ABC-DLBCL).
[0445] Female C.B-17 SCID mice (13 weeks old from Charles River
Laboratories; Hollister, Calif.) were each inoculated in the flank
with 5 million TMD8 (ABC-DLBCL) cells. When the xenograft tumors
reached desired volume, animals were randomized and received
treatments (Day 0 of the study). There were seven treatment groups
(1) vehicle, (2) GA101, (3) anti-CD79b-MC-vc-PAB-MMAE, (4)
lenalidomide, (5) GA101+anti-CD79b-MC-vc-PAB-MMAE, (6)
GA101+lenalidomide, and (7)
GA101+lenalidomide+anti-CD79b-MC-vc-PAB-MMAE. CD79b-MC-vc-PAB-MMAE
ADC was dosed once intravenously at 2 mg/kg, iv, once. GA101 dosed
1 mg/kg, ip, qwx3. Lenalidomide was administered at 20 mg/kg, po,
(qdx5)x3.
[0446] As described above, tumors were measured 1-2 times a week
throughout the study using UltraCal-IV calipers and tumor volume
was calculated using following formula: tumor volume
(mm.sup.3)=0.5a.times.b.sup.2, wherein a and b are the long and
short diameters of the tumor, respectively.
[0447] Literature (Br J Haematol 2013 Zhang et al.) reported that
lenalidomide preferentially suppresses the growth of the activated
B-cell-like (ABC) subtype, with minimal effect on non-ABC-DLBCL
cells. In this study using ABC-DLBCL, as shown in FIG. 3B,
treatment with lenalidomide monotherary showed little efficacy in
this model. Further, combining lenalidomide with GA101 did not
provide additional efficacy over GA101 alone. However, use of
anti-CD79b-MC-vc-PAB-MMAE ADC alone or in combination in the ABC
subtype showed strong efficacy with tumor regression. In addition,
all treatments were well tolerated.
Example 6
Anti-CD79b Immunoconjugate in Combination Therapy in Lymphoma
[0448] The combination efficacy of anti-CD79b-MC-vc-PAB-MMAE ADC
(DCDS4501A; huMA79bv28-MC-vc-PAB-MMAE) with various combination
therapies was evaluated in a tumor xenograft model of WSU-DLCL2
(DLBCL).
[0449] Female C.B-17 SCID mice (13 weeks old from Charles River
Laboratories; Hollister, Calif.) were each inoculated in the flank
with 20 million WSU-DLCL2 (DLBCL) cells. When the xenograft tumors
reached desired volume, animals were randomized and received
treatments (Day 0 of the study). There were twelve treatment groups
(1) vehicle, (2) GA101, (3) Bcl2i (GDC-199), (4) PI3Ki (GDC-032),
(5) anti-CD79b-MC-vc-PAB-MMAE ADC, (6)
GA101+anti-CD79b-MC-vc-PAB-MMAE, (7) GA101+Bcl2i (GDC-199), (8)
GA101+PI3Ki (GDC-032), (9) GA101+Bcl2i
(GDC-199)+anti-CD79b-MC-vc-PAB-MMAE, (10) GA101+PI3Ki
(GDC-032)+anti-CD79b-MC-vc-PAB-MMAE, (11) Rituximab, and (12)
Rituximab+anti-CD79b-MC-vc-PAB-MMAE.
[0450] CD79b-MC-vc-PAB-MMAE ADC was dosed once intravenously at 2
mg/kg, iv, once. GA101 dosed 30 mg/kg, ip, once. Bcl2 inhibitor,
GDC-199, was dosed at 100 mg/kg, po, qdx21. PI3K inhibitor, GDC-032
was dosed at 10 mg/kg, po, qdx21. Rituximab was dosed at 30 mg/kg,
ip, once.
[0451] As described above, tumors were measured 1-2 times a week
throughout the study using UltraCal-IV calipers and tumor volume
was calculated using following formula: tumor volume
(mm.sup.3)=0.5a.times.b.sup.2, wherein a and b are the long and
short diameters of the tumor, respectively. Results are shown in
FIG. 4. In this study, the Bcl2 inhibitor, GDC-0199, PI3K
inhibitor, GDC-0032, and anti-CD20 (GA101 or Rituximab) monotherapy
at the doses tested had little effect on the tumor growth. However,
efficacy became more apparent when combining Bcl2 inhibitor
GDC-0199 with anti-CD20, GA101. Furthermore, among all the
different treatments evaluated the triple combination of
anti-CD79b-vcMMAE, GA101 and Bcl2 inhibitor displayed the greatest
efficacy, causing complete tumor remission.
Example 7
Anti-CD79b Immunoconjugate in Combination with Venetoclax
[0452] This study will evaluate the efficacy, safety, and
pharmacokinetics of the combination of obinutuzumab (GA101 or G)
plus polatuzumab vedotin (anti-CD79b(huMA79b.v23)-MC-vc-PAB-MMAE
ADC (DCDS4501A) or pola) plus a selective Bcl2 inhibitor (ABT-199
(i.e., venetoclax, GDC-0199,
4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-
-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfony-
l)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, V, and/or
CAS#1257044-40-8) (G+pola+V) in patients with relapse or refractory
(R/R) follicular lymphoma (FL) or diffuse large B cell lymphoma
(DLBCL).
[0453] Efficacy Objectives:
[0454] Response will be determined on the basis of positron
emission tomography and computed tomography (PET-CT) scans or CT
scans alone, using Revised Lugano Response Criteria for Malignant
Lymphoma, hereinafter referred to as Lugano 2014 criteria. Response
will be determined by an Independent Review Committee (IRC) and by
the investigator. The primary efficacy objective for this study is
to evaluate the efficacy of G+Pola+V on the basis of the following
endpoint: Complete response (CR) at end of induction (EOI), as
determined by the IRC on the basis of PET-CT scans.
[0455] The secondary efficacy objective for this study is to
evaluate the efficacy of G+Pola+V on the basis of the following
endpoints: CR at EOI, as determined by the investigator on the
basis of PET-CT scans, CR at EOI, as determined by the investigator
on the basis of CT scans alone, Objective response (defined as a CR
or partial response [PR]) at EOI, as determined by the IRC and by
the investigator on the basis of PET-CT scans, Objective response
(defined as a CR or PR) at EOI, as determined by the IRC and by the
investigator on the basis of CT scans alone, Best response of CR or
PR during the study, as determined by the investigator on the basis
of CT scans alone.
[0456] The exploratory efficacy objective for this study is to
evaluate the long-term efficacy of G+Pola+V on the basis of the
following endpoints: for patients who have positive PET scans at
EOI: CR at 12 months, as determined by the IRC and by the
investigator on the basis of PET-CT scans, progression-free
survival, defined as the time from initiation of study treatment to
first occurrence of disease progression or relapse, as determined
by investigator on the basis of CT scans alone, or death from any
cause, event-free survival, defined as the time from initiation of
study treatment to any treatment failure, including disease
progression or relapse, as determined by investigator on the basis
of CT scans alone, initiation of new anti-lymphoma therapy, or
death from any cause, whichever occurs first, disease-free
survival, defined, among patients who achieve a CR, as the time
from the first occurrence of a documented CR to relapse, as
determined by the investigator on the basis of CT scans alone, or
death from any cause, whichever occurs first, overall survival, and
defined as the time from initiation of study treatment to death
from any cause.
[0457] All patients enrolled in the dose-escalation phase will
receive induction treatment, administered in 21-day cycles. When
study treatments are given on the same day, they will be
administered sequentially in the following order: venetoclax,
obinutuzumab, and polatuzumab vedotin.
Cycle 1:
[0458] Venetoclax 400, 600, or 800 mg by mouth (PO) once daily on
Days 1-21 [0459] Obinutuzumab 1000 mg IV on Days 1, 8, and 15
[0460] Polatuzumab vedotin 1.4 or 1.8 mg/kg intravenously (IV) on
Day 1
Cycles 2-6:
[0460] [0461] Venetoclax 400, 600, or 800 mg PO once daily on Days
1-21 [0462] Obinutuzumab 1000 mg IV on Day 1 [0463] Polatuzumab
vedotin 1.4 or 1.8 mg/kg IV on Day 1
[0464] After completion of induction treatment, patients with FL
will continue to receive daily venetoclax treatment (during Month
1) until response is assessed at EOI. Venetoclax will be
discontinued if response assessments at EOI indicate that a patient
is not eligible for post-induction treatment (referred to as
maintenance). Patients who achieve a CR, PR, or SD at EOI will
receive maintenance treatment with obinutuzumab and venetoclax.
Polatuzumab vedotin will not be given as maintenance treatment.
Maintenance treatment will continue until disease progression or
unacceptable toxicity for up to 24 months. When study treatments
are given on the same day, venetoclax will be administered prior to
obinutuzumab.
[0465] Treatments will be administered as follows: [0466]
Venetoclax 400, 600, or 800 mg PO once daily for 8 months (Months
1-8) [0467] Obinutuzumab 1000 mg IV on Day 1 of every other month
(i.e., every 2 months) for 24 months, starting with Month 2 (e.g.,
Months 2, 4, 6, 8, etc.).
[0468] A 3+3 dose-escalation schema will be used. The obinutuzumab
dose will remain fixed at 1000 mg during the dose-escalation phase.
The starting doses in Cohort 1 are 1.4 mg/kg for polatuzumab
vedotin and 400 mg for venetoclax. In Cohorts 2-6, dose escalation
of polatuzumab vedotin and venetoclax will proceed in increments
that parallel the magnitude of dose increases tested in ongoing
Phase Ib trials. For polatuzumab vedotin, there are 2 possible dose
levels: 1.4 or 1.8 mg/kg. For venetoclax, there are 3 possible dose
levels: 400, 600, or 800 mg. Intrapatient dose escalation is not
allowed.
[0469] All patients enrolled in the expansion phase will receive
induction treatment, administered in 21-day cycles. When study
treatments are given on the same day, they will be administered
sequentially in the following order: venetoclax, obinutuzumab, and
polatuzumab vedotin.
Cycle 1:
[0470] Venetoclax at the RP2D (mg) PO once daily on Days 1-21
[0471] Obinutuzumab 1000 mg IV on Days 1, 8, and 15 [0472]
Polatuzumab vedotin at the RP2D (mg/kg) IV on Day 1
Cycles 2-6:
[0472] [0473] Venetoclax at the RP2D (mg) PO once daily on Days
1-21 [0474] Obinutuzumab 1000 mg IV on Day 1 [0475] Polatuzumab
vedotin at the RP2D (mg/kg) IV on Day 1
[0476] After completion of induction treatment, patients will
continue to receive daily venetoclax treatment (during Month 1)
until response is assessed at EOI. Venetoclax will be discontinued
if response assessments at EOI indicate that a patient is not
eligible for post-induction treatment. Patients with DLBCL who
achieve a CR or PR at EOI will receive post-induction treatment
(referred to as consolidation) with obinutuzumab and venetoclax,
and patients with FL who achieve a CR, PR, or SD at EOI will
receive post-induction treatment (referred to as maintenance) with
obinutuzumab and venetoclax. Polatuzumab vedotin will not be given
as post-induction treatment. Post-induction treatment will continue
until disease progression or unacceptable toxicity for up to 8
months for consolidation treatment or 24 months for maintenance
treatment. When study treatments are given on the same day,
venetoclax will be administered prior to obinutuzumab.
[0477] Diffuse Large B-Cell Lymphoma:
[0478] Consolidation treatment consisting of the following,
administered for 8 months (Months 1-8): [0479] Venetoclax at the
RP2D (mg) PO once daily for 8 months (Months 1-8) [0480]
Obinutuzumab 1000 mg IV on Day 1 of every other month (i.e., every
2 months), starting with Month 2 (i.e., Months 2, 4, 6, and 8)
[0481] Follicular Lymphoma: Maintenance treatment consisting of the
following, administered for 24 months (Months 1-24): [0482]
Venetoclax at the RP2D (mg) PO once daily for 8 months (Months 1-8)
[0483] Obinutuzumab 1000 mg IV on Day 1 of every other month (i.e.,
every 2 months) for 24 months, starting with Month 2 (e.g., Months
2, 4, 6, 8, etc.).
[0484] Inclusion Criteria:
[0485] Patients must meet the following criteria for study entry:
signed Informed Consent Form, age.gtoreq.18 years, Eastern
Cooperative Oncology Group Performance Status of 0, 1, or 2, for
patients enrolled in the dose-escalation phase: R/R FL after
treatment with at least 1 prior chemoimmunotherapy regimen that
included an anti-CD20 monoclonal antibody and for which no other
more appropriate treatment option exists, as determined by the
investigator, for patients enrolled in the expansion phase: B-cell
lymphoma classified as either of the following: --R/R FL after
treatment with at least 1 prior chemoimmunotherapy regimen that
included an anti-CD20 monoclonal antibody and for which no other
more appropriate treatment option exists, as determined by the
investigator--R/R DLBCL after treatment with at least 1 prior
chemoimmunotherapy regimen that included an anti-CD20 monoclonal
antibody, with no curative option as determined by the
investigator, histologically documented CD20-positive non-Hodgkin's
lymphoma as determined by the local laboratory,
fluorodeoxyglucose-avid lymphoma (i.e., PET-positive lymphoma), at
least one bi-dimensionally measurable lesion (>1.5 cm in its
largest dimension by CT scan or magnetic resonance imaging),
availability of a representative tumor specimen and the
corresponding pathology report for retrospective central
confirmation of the diagnosis of FL or DLBCL. If the archival
tissue is unavailable or unacceptable, a pretreatment core,
excisional, or incisional tumor biopsy is required. Cytological or
fine-needle aspiration samples are not acceptable. If the patient
received anti-lymphoma treatment between the time of the most
recent available biopsy and initiation of study treatment, a
core-needle biopsy is strongly recommended.
[0486] Exclusion Criteria:
[0487] Patients who meet any of the following criteria will be
excluded from study entry: known CD20-negative status at relapse or
progression, prior allogeneic stem cell transplant (SCT),
completion of autologous SCT within 100 days prior to Day 1 of
Cycle 1, prior standard or investigational anti-cancer therapy as
specified: --Radioimmunoconjugate within 12 weeks prior to Day 1 of
Cycle 1, --Monoclonal antibody or antibody-drug conjugate therapy
within 4 weeks prior to Day 1 of Cycle 1, and--Radiotherapy,
chemotherapy, hormonal therapy, or targeted small-molecule therapy
within 2 weeks prior to Day 1 of Cycle 1, clinically significant
toxicity (other than alopecia) from prior therapy that has not
resolved to Grade.ltoreq.2 (per NCI CTCAE v4.0) prior to Day 1 of
Cycle 1, current Grade>1 peripheral neuropathy, .cndot.CNS
lymphoma or leptomeningeal infiltration, treatment with systemic
corticosteroids >20 mg/day prednisone or equivalent, patients
who are receiving corticosteroids .ltoreq.20 mg/day prednisone or
equivalent must be documented to be on a stable dose for at least 4
weeks prior to Day 1 of Cycle 1. If corticosteroid treatment is
urgently required for lymphoma symptom control prior to the start
of study treatment, up to 100 mg/day of prednisone or equivalent
can be given for a maximum of 5 days, but all tumor assessments
must be completed prior to start of corticosteroid treatment.
History of severe allergic or anaphylactic reaction to humanized or
murine monoclonal antibodies known sensitivity or allergy to murine
products or any component of the obinutuzumab, polatuzumab vedotin,
or venetoclax formulations, active bacterial, viral, fungal, or
other infection, caution should be exercised when considering the
use of obinutuzumab in patients with a history of recurring or
chronic infections, requirement for warfarin treatment (because of
potential drug-drug interactions that may increase the exposure of
warfarin), treatment with the following agents within 7 days prior
to the first dose of venetoclax: --Strong CYP3A inhibitors such as
fluconazole, ketoconazole, and clarithromycin and--Strong CYP3A
inducers such as rifampin and carbamazepine, consumption of
grapefruit, grapefruit products, Seville oranges (including
marmalade that contains Seville oranges), or star fruit within 3
days prior to the first dose of venetoclax, clinically significant
history of liver disease, including viral or other hepatitis,
current alcohol abuse, or cirrhosis, positive for hepatitis B
surface antigen, total hepatitis B core antibody, or hepatitis C
virus antibody at screening, known history of HIV positive status,
for patients with unknown HIV status, HIV testing will be performed
at screening if required by local regulations, history of
progressive multifocal leukoencephalopathy, vaccination with a live
virus vaccine within 28 days prior to Day 1 of Cycle 1, history of
other malignancy that could affect compliance with the protocol or
interpretation of results, with the exception of the following:
--Curatively treated carcinoma in situ of the cervix,
good-prognosis ductal carcinoma in situ of the breast, basal- or
squamous-cell skin cancer, Stage I melanoma, or low-grade,
early-stage localized prostate cancer--Any previously treated
malignancy that has been in remission without treatment for
.gtoreq.2 years prior to enrollment, evidence of any significant,
uncontrolled concomitant disease that could affect compliance with
the protocol or interpretation of results, including significant
cardiovascular disease (such as New York Heart Association Class
III or IV cardiac disease, myocardial infarction within the
previous 6 months, unstable arrhythmia, or unstable angina) or
significant pulmonary disease (such as obstructive pulmonary
disease or history of bronchospasm), major surgical procedure other
than for diagnosis within 28 days prior to Day 1 of Cycle 1, or
anticipation of a major surgical procedure during the course of the
study, inadequate hematologic function (unless due to underlying
lymphoma), defined as follows: --Hemoglobin<9 g/dL,
--ANC<1.5.times.10.sup.9/L, and--Platelet
count<75.times.10.sup.9/L.
Example 8
Anti-CD79b Immunoconjugate in Combination with Lenalidomide
[0488] This study will evaluate the safety, efficacy, and
pharmacokinetics of induction treatment consisting of obinutuzumab
(GA101 or G) in combination with polatuzumab vedotin
(anti-CD79b(huMA79b.v23)-MC-vc-PAB-MMAE ADC (DCDS4501A) or pola)
and lenalidomide (Len) (G+Pola+Len) in patients with relapsed or
refractory follicular lymphoma (FL) or diffuse large B-cell
lymphoma (DLBCL), followed by post induction treatment with
obinutuzumab in combination with lenalidomide in patients with FL
who achieve a complete response (CR), partial response (PR), or
stable disease at end of induction (EOI) and in patients with DLBCL
who achieve a CR or PR at EOI. Specific objectives and
corresponding endpoints for the study are outlined below.
[0489] Response will be determined on the basis of positron
emission tomography (PET) and computed tomography (CT) scans or CT
scans alone, using Revised Lugano Response Criteria for Malignant
Lymphoma, hereinafter referred to as the Lugano 2014 criteria.
Response will be determined by an Independent Review Committee
(IRC) and by the investigator.
[0490] Primary Efficacy Objective:
[0491] The primary efficacy objective for this study is to evaluate
the efficacy of induction treatment with G+Pola+Len on the basis of
the following endpoint: CR at EOI, as determined by the IRC on the
basis of PET-CT scans.
[0492] Secondary Efficacy Objectives:
[0493] The secondary efficacy objective for this study is to
evaluate the efficacy of induction treatment with G+Pola+Len on the
basis of the following endpoints: CR at EOI, as determined by the
investigator on the basis of PET-CT scans, CR at EOI, as determined
by the investigator on the basis of CT scans alone, Objective
response (defined as a CR or PR) at EOI, as determined by the IRC
and by the investigator on the basis of PET-CT scans, Objective
response (defined as a CR or PR) at EOI, as determined by the IRC
and by the investigator on the basis of CT scans alone, Best
response of CR or PR during the study, as determined by the
investigator on the basis of CT scans alone.
[0494] Exploratory Efficacy Objective:
[0495] The exploratory efficacy objective for this study is to
evaluate the long-term efficacy of G+Pola+Len on the basis of the
following endpoints: for patients who have positive PET scans at
EOI: CR at 12 months, as determined by the IRC and by the
investigator on the basis of PET-CT scans, PFS, defined as the time
from initiation of study treatment to first occurrence of disease
progression or relapse, as determined by investigator on the basis
of CT scans alone, or death from any cause, EFS, defined as the
time from initiation of study treatment to any treatment failure,
including disease progression or relapse, as determined by
investigator on the basis of CT scans alone, initiation of new
anti-lymphoma therapy, or death from any cause, whichever occurs
first, disease-free survival, defined, among patients achieving a
CR, as the time from the first occurrence of a documented CR to
relapse, as determined by the investigator on the basis of CT scans
alone, or death from any cause, whichever occurs first, and overall
survival, defined as the time from initiation of study treatment to
death from any cause.
[0496] Inclusion Criteria:
[0497] Patients must meet the following criteria for study entry:
signed Informed Consent Form, age.gtoreq.18 years, Eastern
Cooperative Oncology Group Performance Status of 0, 1, or 2. For
patients enrolled in the dose-escalation phase: relapsed or
refractory FL after treatment with at least one prior
chemoimmunotherapy regimen that included an anti-CD20 monoclonal
antibody and for which no other more appropriate treatment option
exists as determined by the investigator. For patients enrolled in
the expansion phase: lymphoma classified as either of the
following: relapsed or refractory FL after treatment with at least
one prior chemoimmunotherapy regimen that included an anti-CD20
monoclonal antibody and for which no other more appropriate
treatment option exists as determined by the investigator, relapsed
or refractory DLBCL after treatment with at least one prior
chemoimmunotherapy regimen in patients who are not eligible for
autologous stem-cell transplantation or who have experienced
disease progression following treatment with high-dose chemotherapy
plus autologous stem-cell transplantation, histologically
documented CD20-positive B-cell lymphoma as determined by the local
laboratory, fluorodeoxyglucose-avid lymphoma (i.e., PET-positive
lymphoma), at least one bi-dimensionally measurable lesion (>1.5
cm in its largest dimension by CT scan or magnetic resonance
imaging), availability of a representative tumor specimen and the
corresponding pathology report for retrospective central
confirmation of the diagnosis of FL or DLBCL. If the archival
tissue is unavailable or unacceptable, a pretreatment core-needle,
excisional, or incisional tumor biopsy is required. Cytological or
fine-needle aspiration samples are not acceptable.
[0498] Exclusion Criteria:
[0499] Patients who meet any of the following criteria will be
excluded from study entry: known CD20-negative status at relapse or
progression, central nervous system lymphoma or leptomeningeal
infiltration, prior allogeneic stem-cell transplantation (SCT),
completion of autologous SCT within 100 days prior to Day 1 of
Cycle 1, history of resistance to lenalidomide or response duration
of <1 year (for patients who had a response to a prior
lenalidomide-containing regimen), prior standard or investigational
anti-cancer therapy as specified: Lenalidomide, fludarabine, or
alemtuzumab within 12 months prior to Day 1 of Cycle 1,
radioimmunoconjugate within 12 weeks prior to Day 1 of Cycle 1,
monoclonal antibody or antibody-drug conjugate therapy within 4
weeks prior to Day 1 of Cycle 1, radiotherapy, chemotherapy,
hormonal therapy, or targeted small-molecule therapy within 2 weeks
prior to Day 1 of Cycle 1, clinically significant toxicity (other
than alopecia) from prior therapy that has not resolved to
Grade.ltoreq.2 (per NCI CTCAE, Version 4.0) prior to Day 1 of Cycle
1, treatment with systemic immunosuppressive medications,
including, but not limited to, prednisone, azathioprine,
methotrexate, thalidomide, and anti-tumor necrosis factor agents
within 2 weeks prior to Day 1 of Cycle 1. Treatment with inhaled
corticosteroids and mineralocorticoids is permitted, if
corticosteroid treatment is urgently required for lymphoma symptom
control prior to the start of study treatment, up to 100 mg/day of
prednisone or equivalent can be given for a maximum of 5 days, but
all tumor assessments must be completed prior to initiation of
corticosteroid treatment, history of severe allergic or
anaphylactic reaction to humanized or murine monoclonal antibodies,
known sensitivity or allergy to murine products or any component of
obinutuzumab, polatuzumab vedotin, or lenalidomide formulations,
history of erythema multiforme, Grade.gtoreq.3 rash, or
desquamation (blistering) following prior treatment with
immunomodulatory derivatives such as thalidomide and lenalidomide,
active bacterial, viral, fungal, or other infection, caution should
be exercised when considering the use of obinutuzumab in patients
with a history of recurring or chronic infections, positive for
hepatitis B surface antigen, total hepatitis B core antibody, or
hepatitis C virus antibody at screening, known history of HIV
positive status, history of progressive multifocal
leukoencephalopathy, vaccination with a live virus vaccine within
28 days prior to Day 1 of Cycle 1, history of other malignancy that
could affect compliance with the protocol or interpretation of
results, with the exception of the following: curatively treated
carcinoma in situ of the cervix; good-prognosis ductal carcinoma in
situ of the breast; basal- or squamous-cell skin cancer; Stage I
melanoma; or low-grade, early-stage localized prostate cancer, any
previously treated malignancy that has been in remission without
treatment for .gtoreq.2 years prior to enrollment, contraindication
to treatment for TE prophylaxis, Grade.gtoreq.2 neuropathy,
evidence of any significant, uncontrolled concomitant disease that
could affect compliance with the protocol or interpretation of
results, including significant cardiovascular disease (such as New
York Heart Association Class III or IV cardiac disease, myocardial
infarction within the previous 6 months, unstable arrhythmia, or
unstable angina) or significant pulmonary disease (such as
obstructive pulmonary disease or history of bronchospasm), major
surgical procedure other than for diagnosis within 28 days prior to
Day 1 of Cycle 1 or anticipation of a major surgical procedure
during the course of the study, inadequate hematologic function
(unless due to underlying lymphoma), defined as follows:
Hemoglobin<9 g/dL, ANC<1.5.times.10.sup.9/L, Platelet
count<75.times.10.sup.9/L, any of the following abnormal
laboratory values (unless due to underlying lymphoma): calculated
creatinine clearance <60 mL/min (using the Cockcroft-Gault
formula), AST or ALT>2.5.times. upper limit of normal (ULN),
serum total bilirubin >1.5.times.ULN (or >3.times.ULN for
patients with Gilbert syndrome), INR or PT>1.5.times.ULN in the
absence of therapeutic anticoagulation, or PTT or
aPTT>1.5.times.ULN in the absence of a lupus anticoagulant.
[0500] Obinutuzumab:
[0501] Induction-Patients will receive obinutuzumab 1000 mg
intravenously on Days 1, 8, and 15 of Cycle 1 and on Day 1 of each
subsequent 28-day cycle for up to 6 cycles. Post-Induction--For
consolidation treatment, patients with DLBCL will receive
obinutuzumab 1000 mg intravenously on Day 1 of every other month
for approximately 6 months of additional treatment. For maintenance
treatment, patients with FL will receive obinutuzumab 1000 mg
intravenously on Day 1 of every other month for approximately 24
months of additional treatment.
[0502] Polatuzumab Vedotin:
[0503] Induction-Patients will receive polatuzumab vedotin 1.4 or
1.8 mg/kg intravenously on Day 1 of each 28-day cycle for up to 6
cycles. .cndot.In the Phase Ib portion of the study, the total dose
of polatuzumab vedotin for each patient will depend on dose-level
assignment and the patient's weight on Day 1 of Cycle 1 (or within
96 hours before Day 1 of Cycle 1). In the Phase II portion of the
study, the total dose of polatuzumab vedotin for each patient will
depend on the RP2D established in the Phase Ib portion and the
patient's weight on Day 1 of Cycle 1 (or within 96 hours before Day
1 of Cycle 1). Post-Induction-No polatuzumab vedotin will be
administered.
[0504] Lenalidomide:
[0505] Induction-Patients will receive lenalidomide 10, 15, or 20
mg orally once daily on Days 1-21 of each 28-day cycle for up to 6
cycles. In the Phase Ib portion of the study, the dose of
lenalidomide for each patient will depend on dose-level assignment
on Day 1 of Cycle 1. In the Phase II portion of the study, the dose
of lenalidomide for each patient will depend on the RP2D
established in the Phase Ib portion of the study.
Post-Induction-Patients will receive lenalidomide 10 mg orally once
daily on Days 1-21 of each month. For consolidation treatment for
patients with DLBCL will receive lenalidomide 10 mg orally once
daily on Days 1-21 of each month (starting Month 1 and continuing
through Month 6) for approximately 6 months of additional
treatment. For maintenance treatment, patients with FL will receive
lenalidomide 10 mg orally once daily on Days 1-21 of each month
(starting Month 1 and continuing through Month 12) for
approximately 12 months of additional treatment.
[0506] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
TABLE-US-00003 SEQ ID NAME SEQUENCE NO Human CD79b RFIARKRGFT
VKMHCYMNSA SGNVSWLWKQ EMDENPQQLK 1 precursor; LEKGRMEESQ NESLATLTIQ
GIRFEDNGIY FCQQKCNNTS Acc. No. EVYQGCGTEL RVMGFSTLAQ LKQRNTLKDG
IIMIQTLLII NP_000617.1; LFIIVPIFLL LDKDDSKAGM EEDHTYEGLD IDQTATYEDI
signal VTLRTGEVKW SVGEHPGQE sequence = amino acids 1 to 28 Human
mature AR SEDRYRNPKG SACSRIWQSP RFIARKRGFT VKMHCYMNSA 2 CD79b,
SGNVSWLWKQ EMDENPQQLK LEKGRMEESQ NESLATLTIQ without GIRFEDNGIY
FCQQKCNNTS EVYQGCGTEL RVMGFSTLAQ signal LKQRNTLKDG IIMIQTLLII
LFIIVPIFLL LDKDDSKAGM sequence; EEDHTYEGLD IDQTATYEDI VTLRTGEVKW
SVGEHPGQE amino acids 29 to 229 VH of mMAb Gly Pro Glu Leu Val Lys
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys 3 anti-CD20 1 5 10 15
antibody B- Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp Met Asn Trp Val
Lys Leu Ly1 20 25 30 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg
Ile Phe Pro Gly Asp 35 40 45 Gly Asp Thr Asp Tyr Asn Gly Lys Phe
Lys Gly Lys Ala Thr Leu Thr 50 55 60 Ala Asp Lys Ser Ser Asn Thr
Ala Tyr Met Gln Leu Thr Ser Leu Thr 65 70 75 80 Ser Val Asp Ser Ala
Val Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly 85 90 95 Tyr Trp Leu
Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 VL
of mMAb Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser
Ser 4 anti-CD20 1 5 10 15 antibody B- Lys Ser Leu Leu His Ser Asn
Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Ly1 20 25 30 Gln Lys Pro Gly
Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn 35 40 45 Leu Val
Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr 50 55 60
Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 65
70 75 80 Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly
Gly Gly 85 90 95 Thr Lys Leu Glu Ile Lys Arg 100 GA101 HVR-H1 Gly
Tyr Ala Phe Ser Tyr 5 1 5 GA101 HVR-H2 Phe Pro Gly Asp Gly Asp Thr
Asp 6 1 5 GA101 HVR-H3 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr 7 1
5 10 GA101 HVR-L1 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile
Thr Tyr Leu Tyr 8 1 5 10 15 GA101 HVR-L2 Gln Met Ser Asn Leu Val
Ser 9 1 5 GA101 HVR-L3 Ala Gln Asn Leu Glu Leu Pro Tyr Thr 10 1 5
GA101 VH Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 11 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Ala Phe Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp
Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr
Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 GA101 VL Asp Ile Val Met Thr
Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 12 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30
Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val 115 GA101 Heavy
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 13
Chain 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe
Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp
Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn
Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245
250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370
375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 GA101 Light Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 14 Chain 1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145
150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 15 humanized B- 1 5 10 15 Ly1 antibody Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH2)
20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr
Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp
Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 16 humanized B- 1 5 10 15 Ly1 antibody Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH3) 20
25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn
Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Leu Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly
Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser 115 humanized B- QVQLVQSGAE VKKPGSSVKV SCKASGYAFS
YSWINWVRQA 17 Ly1 Heavy PGQGLEWMGR IFPGDGDTDY NGKFKGRVTI TADKSTSTAY
Chain MELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSSA STKGPSVFPL
APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV
PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK
DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV
LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL
VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM
HEALHNHYTQ KSLSLSPG humanized B- DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL
HSNGITYLYW 18 Ly1 Light YLQKPGQSPQ LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI
Chain SRVEAEDVGV YYCAQNLELP YTFGGGTKVE IKRTVAAPSV
FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL
SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC huMA79bv28 EVQLVESGGG
LVQPGGSLRL SCAASGYTFS SYWIEWVRQA 19 heavy chain PGKGLEWIGE
ILPGGGDTNY NEIFKGRATF SADTSKNTAY variable LQMNSLRAED TAVYYCTRRV
PIRLDYWGQG TLVTVSS region huMA79bv28 DIQLTQSPSS LSASVGDRVT
ITCKASQSVD YEGDSFLNWY 20 light chain QQKPGKAPKL LIYAASNLES
GVPSRFSGSG SGTDFTLTIS variable SLQPEDFATY YCQQSNEDPL TFGQGTKVEI KR
region huMA79bv28 GYTFSSYWIE 21 HVR H1 huMA79bv28
GEILPGGGDTNYNEIFKG 22 HVR H2 huMA79bv28 TRRVPIRLDY 23 HVR H3
huMA79bv28 KASQSVDYEGDSELN 24 HVR L1 huMA79bv28 AASNLES 25 HVR L2
huMA79bv28 QQSNEDPLT 26 HVR L3 huMA79bv28 EVQLVESGGGLVQPGGSLRLSCAAS
27 heavy chain (HC) framework region (FR) 1 huMA79bv28
WVRQAPGKGLEWI 28 HC FR2 huMA79bv28 RATFSADTSKNTAYLQMNSLRAEDTAVYYC
29 HC FR3 huMA79bv28 WGQGTLVTVSS 30 HC FR4 huMA79bv28
DIQLTQSPSSLSASVGDRVTITC 31 light chain (LC) FR1 huMA79bv28
WYQQKPGKAPKLLTY 32 LC FR2 huMA79bv28
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 33 LC FR3 huMA79bv28 FGQGTKVEIKR
34 LC FR4 huMA79bv28 DIQLTQSPSS LSASVGDRVT ITCKASQSVD YEGDSFLNWY 35
light chain QQKPGKAPKL LIYAASNLES GVPSRFSGSG SGTDFTLTIS (Ig.kappa.)
SLQPEDFATY YCQQSNEDPL TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL
NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV
THQGLSSPVT KSFNRGEC huMA79bv28 EVQLVESGGG LVQPGGSLRL SCAASGYTFS
SYWIEWVRQA 36 heavy chain PGKGLEWIGE ILPGGGDTNY NEIFKGRATF
SADTSKNTAY (IgG1) LQMNSLRAED TAVYYCTRRV PIRLDYWGQG TLVTVSSAST
KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY
SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV
FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPG huMA79bv28 EVQLVESGGG LVQPGGSLRL
SCAASGYTFS SYWIEWVRQA 37 A118C PGKGLEWIGE ILPGGGDTNY NEIFKGRATF
SADTSKNTAY cysteine LQMNSLRAED TAVYYCTRRV PIRLDYWGQG TLVTVSSCST
engineered KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS heavy chain
GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC (IgG1) NVNHKPSNTK
VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN
YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG
huMA79bv28 DIQLTQSPSS LSASVGDRVT ITCKASQSVD YEGDSFLNWY 38 V205C
QQKPGKAPKL LIYAASNLES GVPSRFSGSG SGTDFTLTIS cysteine SLQPEDFATY
YCQQSNEDPL TFGQGTKVEI KRTVAAPSVF engineered IFPPSDEQLK SGTASVVCLL
NNFYPREAKV QWKVDNALQS light chain GNSQESVTEQ DSKDSTYSLS STLTLSKADY
EKHKVYACEV (Ig.kappa.) THQGLSSPCT KSFNRGEC huMA79bv28 EVQLVESGGG
LVQPGGSLRL SCAASGYTFS SYWIEWVRQA 39 S400C PGKGLEWIGE ILPGGGDTNY
NEIFKGRATF SADTSKNTAY cysteine LQMNSLRAED TAVYYCTRRV PIRLDYWGQG
TLVTVSSAST engineered KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS
heavy chain GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC (IgG1)
NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT
CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK
CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDC DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
LSLSPGK VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 40 humanized B- 1 5 10 15 Ly1 antibody Ser Val Lys Val
Ser Cys Lys Val Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH4) 20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp
Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 41 humanized B- 1 5 10 15 Ly1 antibody Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH5) 20 25 30 Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu
Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ser 42 humanized B- 1 5 10 15 Ly1 antibody Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH6) 20 25 30 Trp Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 VH
of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
43 humanized B- 1 5 10 15 Ly1 antibody Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ala Phe Ser Tyr Ser (B-HH7) 20 25 30 Trp Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55
60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 VH of
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 44
humanized B- 1 5 10 15 Ly1 antibody Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Tyr Ser (B-HH8) 20 25 30 Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg
Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 VH of
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 45
humanized B- 1 5 10 15 Ly1 antibody Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Ser Tyr Ser (B-HH9) 20 25 30 Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg
Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 VH of
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 46
humanized B- 1 5 10 15 Ly1 antibody Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Tyr Ser (B-HL8) 20 25 30 Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg
Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 VH of
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 47
humanized B- 1 5 10 15 Ly1 antibody Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Ala Phe Ser Tyr Ser (B-HL10) 20 25 30 Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg
Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 48 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL11) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Glu Val Gln Leu Val Glu Ser
Gly Ala Gly Leu Val Lys Pro Gly Gly 49 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL12) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Lys Pro Gly Gly 50 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL13) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Lys Lys Pro Gly Gly 51 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL14) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Ser 52 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL15) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 53 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL16) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VH of Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 54 humanized B- 1 5 10 15 Ly1
antibody Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Tyr Ser (B-HL17) 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 VL of Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Thr Pro Gly 55 humanized B- 1 5 10 15 Ly1
antibody Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu
His Ser (B-KVI) 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met
Ser Asn Leu Val Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu
Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110
Arg Thr Val 115
Sequence CWU 1
1
551179PRTHomo sapiens 1Arg Phe Ile Ala Arg Lys Arg Gly Phe Thr Val
Lys Met His Cys Tyr 1 5 10 15 Met Asn Ser Ala Ser Gly Asn Val Ser
Trp Leu Trp Lys Gln Glu Met 20 25 30 Asp Glu Asn Pro Gln Gln Leu
Lys Leu Glu Lys Gly Arg Met Glu Glu 35 40 45 Ser Gln Asn Glu Ser
Leu Ala Thr Leu Thr Ile Gln Gly Ile Arg Phe 50 55 60 Glu Asp Asn
Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn Thr Ser 65 70 75 80 Glu
Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly Phe Ser 85 90
95 Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly Ile Ile
100 105 110 Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val Pro
Ile Phe 115 120 125 Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly Met
Glu Glu Asp His 130 135 140 Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr
Ala Thr Tyr Glu Asp Ile 145 150 155 160 Val Thr Leu Arg Thr Gly Glu
Val Lys Trp Ser Val Gly Glu His Pro 165 170 175 Gly Gln Glu
2201PRTHomo sapiens 2Ala Arg Ser Glu Asp Arg Tyr Arg Asn Pro Lys
Gly Ser Ala Cys Ser 1 5 10 15 Arg Ile Trp Gln Ser Pro Arg Phe Ile
Ala Arg Lys Arg Gly Phe Thr 20 25 30 Val Lys Met His Cys Tyr Met
Asn Ser Ala Ser Gly Asn Val Ser Trp 35 40 45 Leu Trp Lys Gln Glu
Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu 50 55 60 Lys Gly Arg
Met Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr 65 70 75 80 Ile
Gln Gly Ile Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln Gln 85 90
95 Lys Cys Asn Asn Thr Ser Glu Val Tyr Gln Gly Cys Gly Thr Glu Leu
100 105 110 Arg Val Met Gly Phe Ser Thr Leu Ala Gln Leu Lys Gln Arg
Asn Thr 115 120 125 Leu Lys Asp Gly Ile Ile Met Ile Gln Thr Leu Leu
Ile Ile Leu Phe 130 135 140 Ile Ile Val Pro Ile Phe Leu Leu Leu Asp
Lys Asp Asp Ser Lys Ala 145 150 155 160 Gly Met Glu Glu Asp His Thr
Tyr Glu Gly Leu Asp Ile Asp Gln Thr 165 170 175 Ala Thr Tyr Glu Asp
Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp 180 185 190 Ser Val Gly
Glu His Pro Gly Gln Glu 195 200 3112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys 1
5 10 15 Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp Met Asn Trp Val Lys
Leu 20 25 30 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Phe
Pro Gly Asp 35 40 45 Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys Gly
Lys Ala Thr Leu Thr 50 55 60 Ala Asp Lys Ser Ser Asn Thr Ala Tyr
Met Gln Leu Thr Ser Leu Thr 65 70 75 80 Ser Val Asp Ser Ala Val Tyr
Leu Cys Ala Arg Asn Val Phe Asp Gly 85 90 95 Tyr Trp Leu Val Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110
4103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile
Ser Cys Arg Ser Ser 1 5 10 15 Lys Ser Leu Leu His Ser Asn Gly Ile
Thr Tyr Leu Tyr Trp Tyr Leu 20 25 30 Gln Lys Pro Gly Gln Ser Pro
Gln Leu Leu Ile Tyr Gln Met Ser Asn 35 40 45 Leu Val Ser Gly Val
Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr 50 55 60 Asp Phe Thr
Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 65 70 75 80 Tyr
Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly 85 90
95 Thr Lys Leu Glu Ile Lys Arg 100 56PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Gly
Tyr Ala Phe Ser Tyr 1 5 68PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Phe Pro Gly Asp Gly Asp Thr
Asp 1 5 710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr 1 5 10
816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile
Thr Tyr Leu Tyr 1 5 10 15 97PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Gln Met Ser Asn Leu Val Ser
1 5 109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Ala Gln Asn Leu Glu Leu Pro Tyr Thr 1 5
11119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 12115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His
Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu
Val Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val
115 13448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
14219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90
95 Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 15119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 16119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 16Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Leu Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
17448PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325
330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
18219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90
95 Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 19117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser
Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn
Tyr Asn Glu Ile Phe 50 55 60 Lys Gly Arg Ala Thr Phe Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Arg Val Pro
Ile Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 20112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 20Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu 20 25 30 Gly Asp Ser Phe
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu
Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65
70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 2110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 21Gly Tyr Thr Phe Ser Ser
Tyr Trp Ile Glu 1 5 10 2218PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Gly Glu Ile Leu Pro Gly Gly
Gly Asp Thr Asn Tyr Asn Glu Ile Phe 1 5 10 15 Lys Gly
2310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr 1 5 10
2415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Lys Ala Ser Gln Ser Val Asp Tyr Glu Gly Asp Ser
Phe Leu Asn 1 5 10 15 257PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Ala Ala Ser Asn Leu Glu Ser
1 5 269PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Gln Gln Ser Asn Glu Asp Pro Leu Thr 1 5
2725PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25
2813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Ile 1 5 10 2930PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Arg Ala Thr Phe Ser Ala Asp Thr
Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 3011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 3123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Asp
Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys 20 3215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15
3332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 3411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 35218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr
Glu 20 25 30 Gly Asp Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Leu Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 36446PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 36Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile
Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Gly Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe 50
55 60 Lys Gly Arg Ala Thr Phe Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 37446PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro
Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe 50 55 60 Lys Gly Arg
Ala Thr Phe Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser Cys Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215
220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340
345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
38218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 38Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Ser Val Asp Tyr Glu 20 25 30 Gly Asp Ser Phe Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr
Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90
95 Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr 130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145
150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro 195 200 205 Cys Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 215 39447PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 39Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile
Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Gly Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe 50
55 60 Lys Gly Arg Ala Thr Phe Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Cys 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
440 445 40119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 40Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Val Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg
Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
41119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 41Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 42119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr
Ser 20 25 30 Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 43119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 43Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30 Trp Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
44119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 44Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Tyr Ser 20 25 30 Trp Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 45119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 46119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 46Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30 Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
47119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ala Phe Ser Tyr Ser 20 25 30 Trp Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 48119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
48Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 49119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 49Glu Val Gln Leu Val Glu
Ser Gly Ala Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30 Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
50119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 50Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Tyr Ser 20 25 30 Trp Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 51119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
51Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Lys Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 52119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 52Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30 Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45
Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
53119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 53Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Tyr Ser 20 25 30 Trp Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Phe
Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60 Lys Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 54119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
54Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr
Ser 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp
Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Val Phe
Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 55115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 55Asp Ile Val Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly
Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Ala Gln Asn 85 90 95 Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 110 Arg Thr Val 115
* * * * *