U.S. patent application number 16/085848 was filed with the patent office on 2019-05-23 for targeting tumor cells with chemotherapeutic agents conjugated to anti-matriptase antibodies by in vivo cleavable linking moietie.
The applicant listed for this patent is Georgetown University, Rutgers, The State University of New Jersey. Invention is credited to Joseph R. Bertino, Chen-Yong Lin, Siang-Yo Lin, Zoltan Szekely.
Application Number | 20190151464 16/085848 |
Document ID | / |
Family ID | 66534135 |
Filed Date | 2019-05-23 |
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United States Patent
Application |
20190151464 |
Kind Code |
A1 |
Lin; Siang-Yo ; et
al. |
May 23, 2019 |
TARGETING TUMOR CELLS WITH CHEMOTHERAPEUTIC AGENTS CONJUGATED TO
ANTI-MATRIPTASE ANTIBODIES BY IN VIVO CLEAVABLE LINKING
MOIETIES
Abstract
The present invention relates to anti-matriptase antibodies and
immunoconjugates of anti-matriptase antibodies with cytotoxic
agents and the use thereof for killing or inhibiting the growth of
matriptase-expressing cancer cells, such as those of multiple
myeloma and breast cancers. In particular, immunoconjugates
comprising an anti-matriptase monoclonal antibody and anticancer
agents such as auristatin, including monomethyl auristatin E (MMAE)
and monomethyl auristatin F (MMAF) are introduced, which have
potent antitumor activity in vivo. Moreover, importantly; there was
no weight loss or other evidence of toxicity in the animals,
indicating that no significant free drug was released into the
circulation from the conjugate. The present invention also provides
compositions comprising these new immunoconjugates and use of them
for treatment of malignancies comprising cells that express
matriptase. In addition, administration of an anti-matriptase
antibody or immunoconjugates of an anti-matriptase antibody and a
cytotoxic agent in combination with administration of an
immunomodulatory agent, such as thalidomide or an analog thereof,
provides a more effective treatment of these cancers.
Inventors: |
Lin; Siang-Yo; (East
Brunswick, NJ) ; Bertino; Joseph R.; (Branford,
CT) ; Lin; Chen-Yong; (Falls Church, VA) ;
Szekely; Zoltan; (New Brunswick, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rutgers, The State University of New Jersey
Georgetown University |
New Brunswick
Washington |
NJ
DC |
US
US |
|
|
Family ID: |
66534135 |
Appl. No.: |
16/085848 |
Filed: |
March 17, 2017 |
PCT Filed: |
March 17, 2017 |
PCT NO: |
PCT/US17/22993 |
371 Date: |
September 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15075008 |
Mar 18, 2016 |
9849192 |
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16085848 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/6803 20170801; A61K 31/4439 20130101; A61K 47/6809
20170801; A61K 47/6811 20170801; A61K 31/40 20130101; A61K 47/65
20170801; C07K 16/30 20130101; C07K 16/40 20130101; C07K 2317/73
20130101; A61K 47/6871 20170801; A61P 35/00 20180101; A61K 47/60
20170801 |
International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/40 20060101 C07K016/40; A61K 31/4439 20060101
A61K031/4439; A61K 31/40 20060101 A61K031/40; A61K 47/60 20060101
A61K047/60; A61P 35/00 20060101 A61P035/00 |
Claims
1. An immunoconjugate selectively targeting cancer cells that
express matriptase, comprising an anti-matriptase antibody or
antigen-binding fragment thereof, and a cytotoxic agent, wherein
said cytotoxic agent is selected from the group consisting of
monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and
auristatin PE, wherein the anti-matriptase antibody or
antigen-binding fragment thereof is specific for activated
matriptase.
2. The immunoconjugate of claim 1, wherein the anti-matriptase
antibody comprises M69 monoclonal antibody or antigen-binding
portion thereof.
3. The immunoconjugate of claim 1, wherein the immunoconjugate
further comprises a linker comprising a cleavable linking
moiety.
4. (canceled)
5. The immunoconjugate of claim 3, wherein the cleavable linking
moiety comprises a Val-Cit linking moiety or a Phy-Lys linking
moiety, cleavable by Capthesin B.
6. The immunoconjugate of claim 1, wherein the linker is covalently
bound to a lysine residue on the anti-matriptase antibody.
7. The immunoconjugate of claim 1, wherein the linker is
PEG-containing.
8. The immunoconjugate of claim 1, wherein the immunoconjugate
further comprises a linker, and the linker comprises a first
linking component and a second linking component.
9. The immunoconjugate of claim 8, wherein the first linking
component is covalently bound to a lysine residue on the
anti-matriptase antibody.
10. The immunoconjugate of claim 8, wherein the first linking
component is bound to the second linking component through a
triazole moiety.
11. The immunoconjugate of claim 8, wherein the second linking
component comprises a cleavable linking moiety.
12. The immunoconjugate of claim 11, wherein the cleavable linking
moiety comprises a Val-Cit linking moiety or a Phy-Lys linking
moiety, cleavable by Capthesin B.
13. The immunoconjugate of claim 8, wherein at least one of the
first linking component and the second linking component is
PEG-containing.
14. (canceled)
15. The immunoconjugate of claim 8, wherein the cytotoxic agent is
bound to the second linking component.
16. A method of treating a malignancy comprising cells that express
matriptase, the method comprising administering to a subject in
need of such a treatment a therapeutically effective amount of a
composition comprising the immunoconjugate of claim 1.
17. The method of claim 16 wherein the malignancy comprises a
hematological malignancy selected from the group consisting of
acute lymphoblastic leukemia (ALL), acute myelogenous leukemia
(AML), chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma (SLL), chronic myelogenous leukemia (CML), acute monocytic
leukemia (AMOL)), Hodgkin's lymphomas, Non-Hodgkin's lymphomas,
Burkitt's lymphoma (BL), diffuse large B-cell lymphoma (DLBL),
Mantle cell lymphoma (MCL), multiple myeloma (MM), and an
epithelial malignancy selected from the group consisting of
prostate, breast, brain, kidney, lung, colon, bladder, skin,
thyroid, ovary tumors, and mesothelioma.
18. (canceled)
19. (canceled)
20. (canceled)
21. The method of claim 16, further comprising co-administering a
therapeutically effective amount of an immunomodulatory agent that
activates matriptase.
22. The method of claim 21, wherein the immunomodulatory agent
comprises thalidomide or a thalidomide analog.
23. An immunoconjugate comprising an anti-matriptase antibody or an
antigen-binding portion thereof that is specific for activated
matriptase, covalently bound to a cytotoxic agent through a linker,
the cytotoxic agent being selected from the group consisting of
monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and
auristatin PE, and the linker comprising: a first linking component
bound to the anti-matriptase antibody or an antigen-binding portion
thereof through a lysine residue located on the anti-matriptase
antibody or an antigen-binding portion thereof; and a second
linking component comprising a cleavable linking moiety, the second
linking component being bound to the first linking component
through a triazole moiety and to the cytotoxic agent through the
cleavable linking moiety, wherein the cleavable linking moiety
comprises a Val-Cit linking moiety or a Phy-Lys linking moiety,
cleavable by Capthesin B, wherein the first linking component
comprises between about 3 and about 7 poly(ethylene glycol) (PEG)
units between the amide bond between the lysine residue on the
anti-matriptase antibody or an antigen-binding portion thereof and
the triazole moiety, and wherein the second linking component
comprises between about 3 and about 7 poly(ethylene glycol) (PEG)
units between the triazole moiety and the cleavable linking
moiety.
24. The immunoconjugate of claim 23, wherein the anti-matriptase
antibody or an antigen-binding portion thereof comprises M69 or an
antigen-binding portion thereof.
25. The immunoconjugate of claim 24, wherein the cytotoxic agent
comprises monomethyl auristatin E (MMAE).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. patent
application Ser. No. 15/075,008, which is a continuation-in-part
under 35 U.S.C. .sctn. 120 of U.S. patent application Ser. No.
13/510,801, filed May 18, 2012, which is a National Stage Entry of
PCT/US10/57235, filed Nov. 18, 2010, which in turn claims priority
under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
61/293,030, filed Jan. 7, 2010, and U.S. Provisional Application
No. 61/262,373, filed Nov. 18, 2009. All of the foregoing
references are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to new immunoconjugates
comprising an anticancer agent and a monoclonal antibody and use of
such immunoconjugates for killing or inhibiting the growth of
matriptase expressing cancer cells, including but not limited to
hematological malignancies and epithelial cancers, such as those of
multiple myeloma and breast cancers. Thus, the present invention
also relates to new methods of treating matriptase-expressing
cancers.
BACKGROUND OF THE INVENTION
[0003] Matriptase is a type II transmembrane serine protease
expressed by cells of epithelial origin, including breast and
prostate tumor cells. Matriptase is characterized by an N-terminal
transmembrane domain and multiple extracellular domains, in
addition to the conserved extracellular domain (Lin C. Y., et al.,
J. Biol. Chem., 1999, 274(26): 18237-18242.). Matriptase is a
zymogen that needs to be activated by proteolytic cleavage to
become a two-chain active enzyme. Under normal physiological
conditions, there is excessive amount of endogenous inhibitor
called HGF activator inhibitor-I (HAI-1) that binds to matriptase,
tightly regulating the protease activity (Lin, C. Y., et al., J.
Biol, Chem., 1999, 274: 18231-18236; Oberst, M. D., et al., J.
Biol. Chem., 2003, 278:26773-26779.). Besides exerting an
inhibitory function, HAI-1 also plays a critical role in
activation, proper expression and intracellular trafficking of
matriptase (Oberst, M. D., et al., J. Biol. Chem., 2003, 278:
26773-26779; Oberst, M. D., et al., Am. J. Physiol. Cell Physiol.,
2005, 289:C462-C470.).
[0004] Matriptase is known to proteolytically activate the
hepatocyte growth factor (HGF) and the urokinase plasminogen
activator (uPA) and protease-activated receptor in vitro (Lee, S.
L., et al., J. Biol. Chem., 2000, 275:36720-36725; Suzuki, M., et
al., J. Biol. Chem., 2004, 279:14899-14908.). Both HGF and uPA have
been implicated for their roles in cellular invasion and metastasis
and in cellular motility (Trusolino, L. and Comoglio, P. M., Nat.
Rev. Cancer, 2002, 2:289-300; Sidenius, N. and Blasi, F., Cancer
Metastasis Rev., 2003, 22:205-222.). The cognate receptor for HGF
is Met, a receptor tyrosine kinase. Upon the binding of HGF, Met
can trigger multiple signaling pathways, including Ras-MAPK, PI3K,
Src and Stat3, which eventually leads to invasive growth. High
levels of Met combined with overexpression of matriptase are
associated with a poor outcome for patients with breast cancer.
[0005] Studies of matriptase levels in several solid
epithelial-derived tumors including breast, prostate and ovarian
carcinomas have been performed in the past few years (for a review,
see Uhland, K., Cell. Mol. Life Sci., 2007, 63(24):2968-78). A
tissue microarray from patients with breast carcinoma showed that
high-level expression of Met, matriptase and HAI-1 are associated
with poor patient outcome (Kang, J. Y., et al., Cancer Res., 2003,
63: 1 101-1105.). In prostate tumors, increased levels of
matriptase with decreased expression of HAI-1 were associated with
increasing tumor grade (Saleem, M., et al., Cancer Epidemiol.
Biomarkers Prev., 2006: 15, 217-227.). Overexpression of matriptase
was also found in 82% of stage III and in 55% stage III/VI of
patients with ovarian cancer.
[0006] Given the significant roles of matriptase in tumor
initiation, progression and metastases, several inhibitors for this
protease have been investigated in animal models for their
anticancer activity. Reduced tumor growth and metastasis formation
by matriptase inhibitor, ecotin, has been shown in a PC-3 prostate
carcinoma xenograft model (Takeuchi, N., et al., Proc. Natl. Acad.
Sci. USA, 1999, 96, 11054-11061.). CVS-3983, another matriptase
inhibitor, also reduced tumor size in a mouse model of
androgen-independent prostate cancer (Galkin, A. V., et al.,
Prostate, 2004, 61, 228-235).
[0007] Monomethyl auristatin E ("MMAE") is an FDA-approved
synthetic antineoplastic agent that has shown promising use in
chemotherapy based treatments. MMAE is a potent antimitotic
compound but it exhibits high levels of cytotoxicity when
administered alone, limiting its clinical value as a stand-alone
compound. Monomethyl auristatin F ("MMAF"), also known as
desmethyl-auristatin F, is an experimental synthetic
anti-neoplastic agent that, like MMAE, is an antimitotic agent.
Like MMAE, MMAF exhibits high levels of cytotoxicity when
administered alone. Therefore, there is an urgent need for
therapeutically effective antibody-based treatments that utilize
MMAF and MMAE-bound immunoconjugates.
SUMMARY OF THE INVENTION
[0008] The present invention provides new therapeutic agents for
the treatment of hematologic cancers, including acute lymphoblastic
leukemia (ALL), acute myelogenous leukemia (AML), chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
chronic myelogenous leukemia (CML), acute monocytic leukemia
(AMOL)), Hodgkin's lymphomas, Non-Hodgkin's lymphomas, Burkitt's
lymphoma (BL), diffuse large B-cell lymphoma (DLBL), Mantle cell
lymphoma (MCL), and multiple myeloma (MM), as well as new
therapeutic agents for the treatment of various epithelial cancers,
including prostate, breast, brain, kidney, lung, colon, bladder,
skin, thyroid, and ovary tumors, and mesothelioma to meet the
foregoing need.
[0009] Matriptase, a membrane-bound serine type II protease, is
expressed in cell lines of multiple myeloma (MM) and other
hematologic and epithelial cancer cells. The present invention
provides an anti-matriptase antibody conjugated to a cytotoxic
agent, for example, the auristatins MMAE and MMAF, for selectively
targeting matriptase expressing cells. Antibodies conjugated with
potent anticancer drugs to target matriptase-expressing cancer
cells have various advantages. For example, the selective delivery
of the chemotherapeutic agent to tumor cells overexpressing
matriptase results in less toxicity toward the normal tissues.
Moreover, since levels of matriptase mRNA have been found in tumors
in other organs, including kidney, lung, colon, bladder, pancreas,
prostate, skin, breast, thyroid and ovary, these immunoconjugates
are useful for treating these cancers.
[0010] In one embodiment, the present invention provides an
immunoconjugate of an anti-matriptase antibody and a cytotoxic
agent. In some embodiments, the cytotoxic agent is selected from
doxorubicin (DOX), auristatin, including monomethyl auristatin E
(MMAE), monomethyl auristatin F (MMAF), calicheamicin, and ricin.
In some embodiments, the cytotoxic agent is MMAE. In some
embodiments, the cytotoxic agent is MMAF. In some embodiments the
cytotoxic agent is covalently linked to the anti-matriptase
antibody. In some embodiments the anti-matriptase antibody is M69.
Therefore in some embodiments the immunoconjugate is MMAE
covalently linked to M69. In other embodiments the immunoconjugate
is MMAF covalently linked to M69.
[0011] In one embodiment, the present invention also provides for
linkers for use in immunoconjugates. In some embodiments, the
linkers comprise cleavable linking moieties. In some embodiments,
the cleavable linking moieties comprise Val-Cit moieties or Phy-Lys
moieties, cleavable by Capthesin B. In some embodiments, the
linkers are conjugated directly to a surface of an antibody. In
some embodiments, the linkers are covalently to a lysine side
chain. In some embodiments, the linkers comprise a first linking
component and a second linking component. In some embodiments, the
first linking component is bound to the second linking component
through a triazole moiety. In some embodiments, the first linking
component is bound to a surface of an antibody. In some
embodiments, the second linking component comprises a cleavable
linking moiety. In some embodiments, the second linking component
comprises a therapeutic agent. In some embodiments, the linker is
PEG-based. In some embodiments, the first linking component is
PEG-based. In some embodiments, the second linking component is
PEG-based. In some embodiments, the antibody comprises an
anti-matriptase antibody. In some embodiments, the anti-matrtiptase
antibody comprises M69. In some embodiments, the therapeutic agent
comprises a cytotoxic agent. In some embodiments, the cytotoxic
agent is selected from doxorubicin (DOX), auristatin, including
monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF),
calicheamicin, and ricin. In some embodiments, the cytotoxic agent
is MMAE. In some embodiments, the cytotoxic agent is MMAF.
[0012] Further, it has been discovered in accordance with the
present invention that thalidomide, an immunomodulatory agent,
markedly induces activation of matriptase in myeloma cells. Thus,
the present invention provides for administration of
immunoconjugates targeting active matriptase in combination with
administration of immunomodulatory agents, including but not
limited to thalidomide and its analogs, for the treatment of MM and
other cancers. Thalidomide analogs are known in the art and
include, for example, lenalidomide, CC-3052, CC-4047, CC-5103,
IMiD3, EM12, and ENMD0995.
[0013] Thus, in one aspect the present invention provides a method
of treating a hematological malignancy, comprising administering to
a subject in need of such a treatment a composition comprising a
therapeutically effective amount of an anti-matriptase antibody. In
some embodiments, the anti-matriptase antibody is M69.
[0014] In another aspect the present invention provides a method of
treating a hematological malignancy by administering to a subject
in need of such a treatment a therapeutically effective amount of a
composition containing an anti-matriptase antibody in combination
with a therapeutically effective amount of an immunomodulatory
agent that activates matriptase. In some embodiments the
anti-matriptase antibody is M69. In some embodiments, the
immunomodulatory agent is thalidomide or its analogs.
[0015] In another aspect the present invention provides a method of
treating a malignancy in which malignant cells express matriptase
by administering to a subject in need of such a treatment a
therapeutically effective amount of a composition containing an
immunoconjugate between an anti-matriptase antibody and a cytotoxic
agent. In some embodiments the cytotoxic agent is selected from
doxorubicin (DOX), auristatin, including monomethyl auristatin E
(MMAE), monomethyl auristatin F (MMAF), and auristatin PE,
calicheamicin, and ricin. In some embodiments, the cytotoxic agent
is MMAE. In some embodiments, the cytotoxic agent is MMAF. In some
embodiments the cytotoxic agent is covalently linked to the
anti-matriptase antibody. In some embodiments the anti-matriptase
antibody is M69. Therefore in some embodiments the immunoconjugate
is MMAE covalently linked to M69. In other embodiments the
immunoconjugate is MMAF covalently linked to M69.
[0016] In another aspect the present invention provides a method of
treating a malignancy in which malignant cells express matriptase
by administering to a subject in need of such a treatment a
therapeutically effective amount of a composition containing an
immunoconjugate between an anti-matriptase antibody and a cytotoxic
agent in combination with a therapeutically effective amount of an
immunomodulatory agent that activates matriptase. In some
embodiments the cytotoxic agent is selected from doxorubicin (DOX),
auristatin, including monomethyl auristatin E (MMAE), monomethyl
auristatin F (MMAF), and auristatin PE, calicheamicin, and ricin.
In some embodiments, the cytotoxic agent is MMAE. In some
embodiments, the cytotoxic agent is MMAF In some embodiments the
cytotoxic agent is covalently linked to the anti-matriptase
antibody. In some embodiments the anti-matriptase antibody is M69.
Therefore in some embodiments the immunoconjugate is MMAE
covalently linked to M69. In other embodiments the immunoconjugate
is MMAF covalently linked to M69. In some embodiments, the
immunomodulatory agent is thalidomide or its analogs. Therefore, in
some embodiments, the method comprises administration of a
therapeutically effective amount of M69-MMAE immunoconjugates in
combination with thalidomide or a thalidomide analog. In other
embodiments, the method comprises administration of a
therapeutically effective amount of M69-MMAF immunoconjugates in
combination with thalidomide or a thalidomide analog. In other
embodiments, the method comprises administration of a
therapeutically effective amount of M69-auristatin PE
immunoconjugates in combination with thalidomide or a thalidomide
analog.
[0017] In another aspect the present invention provides an
immunoconjugate selectively targeting cancer cells that express
matriptase, comprising an anti-matriptase antibody and a cytotoxic
agent.
[0018] In another aspect the present invention provides a
composition containing an immunoconjugate selectively targeting
cancer cells that express matriptase according to any of the
embodiments described above.
[0019] In another aspect the present invention provides a method of
diagnosing a hematological malignancy by contacting a test sample
containing hematological cells from a mammal with an
anti-matriptase antibody and detecting the formation of a complex
between the antibody and matriptase, wherein formation of a complex
is indicative of a malignancy.
[0020] In another aspect the present invention provides a method of
inhibiting the growth of a hematopoietic cell that expresses
matriptase by treating the hematopoietic cell with an
anti-matriptase antibody or an immunoconjugate according to any of
the embodiments described above.
[0021] In another aspect the present invention provides an assay
kit for detecting expression of matriptase in mammalian tissues or
cells, containing an immunoconjugate according to any of the
embodiments described above.
[0022] In another aspect the present invention provides a kit for
treatment of a malignancy in which malignant cells express
matriptase, the kit comprising an immunoconjugate according to any
of the embodiments described above.
[0023] In another aspect the present invention provides use of an
immunoconjugate according to any of the embodiments described above
for treatment of a malignancy in which malignant cells express
matriptase.
[0024] In another aspect the present invention provides use of an
immunoconjugate according to any of the embodiments described above
for manufacture of a medicament for treatment of a malignancy in
which malignant cells express matriptase.
[0025] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-D represent Western analyses of matriptase and
HAI-1 in human lymphoma and myeloma cells. The cell lines in panel
(A) include Hs 445 (lane 2), HuT 78 (lane 3), Farage (lane 4), Raji
(lane 5), Daudi (lane 6), Namalwa (lane 7), Ramos (lane 8), ST486
(lane 9), SU-DHL-4 (lane 10), SU-DHL-6 (lane 11), OCI-LY-3 (lane
12), RPMI-8226 (lane 13). The cell lines in panel (B) include HL-60
(lane 2), Reh (lane 3), Jurkat (lane 4), SUP-T1 (lane 5), CCRF-CEM
(lane 6), CCRF-HSB-2 (lane 7), MOLT-3 (lane 8), MOLT-4 (lane 9),
CCRF-SB (lane 10), RS4-11 (lane 11), THP-1 (lane 12), U937 (lane
13). Panel (C) shows the assessment of levels of matriptase in
three MM cell lines as indicated. GAPDH served as an internal
control. Panel (D) shows the tissue sections of paraffin-embedded
multiple myeloma stained with the matriptase mAb M24, the HAI-1mAb
M19, and the activated matriptase M69, respectively as
indicated.
[0027] FIG. 2 illustrates cytotoxic effects of M24-DOX on MM cells.
MM cells (1.times.105/well) in 24-well plates were treated with or
without M24-DOX at varying concentration for 48 hours. Cell number
was counted by Vi-Cell counter (Beckman). Error bars represent SEM.
Statistical comparisons: *, significantly lower than control,
p<0.036. Plotted values represent duplicates from three separate
experiments.
[0028] FIGS. 3A and B represent internalization of M24-DOX within
cells. Following exposure of MM.1S cells to M24-DOX for varying
periods of time, and the cells were fixed and stained. The
immunoconjugate was visualized by fluorescent microscopy. DAPI was
used for nuclear staining.
[0029] FIGS. 4A-C illustrate reduced cytotoxicity of M24-DOX to
normal marrow mesenchymal cells. Bone marrow-derived mesenchymal
stromal cells (5.times.104/well, 24-well plates) were treated with
M24-DOX or free DOX at varying concentrations for 4 days. Cell
numbers were determined by Vi-Cell counter. (A) shows that M24-DOX
lacks cytotoxicity to stromal cells; (B) shows reduced cytotoxicity
to cardiomyocytes; and (C) shows comparison of M24-DOX with DOX and
control.
[0030] FIG. 5 illustrates activation of matriptase in MM cells
induced by thalidomide. MM cells (MM.15) were treated with varying
concentrations of thalidomide as indicated for 24 hours followed by
Western analyses to detect the activation of matriptase using a
specific monoclonal antibody (M69) to the active form of the
enzyme. The latent form of the protease was also detected using a
mAb (M24) to the inactive form enzyme. GAPDH served as control.
[0031] FIGS. 6A and B illustrate that thalidomide sensitizes MM
cells to M69-DOX, but not M24-DOX: (A) comparison of treatment of
MM cells by M69-DOX alone with the M69-DOX/thalidomide combination
treatment; and (B) comparison of treatment of MM cells by M24-DOX
alone with the M24-DOX/thalidomide combination treatment.
[0032] FIG. 7 illustrates activation of matriptase in MM cells
induced by thalidomide. MM cells (MM.15) were treated with varying
concentrations of thalidomide as indicated for 24 hours followed by
Western analyses to detect the activation of matriptase using a
specific monoclonal antibody (M69) to the active form of the
enzyme. The latent form of the protease was also detected using a
mAb (M24) to the inactive form enzyme. GAPDH served as control.
[0033] FIG. 8 illustrates the M69-DOX conjugate targeting active
matriptase inhibited breast tumor growth in vivo, comparing
treatment by M69-DOX at 10 mg/Kg and 20 mg/Kg doses with treatment
by DOX at 2 mg/Kg dose and the control.
[0034] FIG. 9 illustrates reduction of the tumor weight by M69-DOX
at 10 mg/Kg and 20 mg/Kg doses in comparison with treatment by DOX
at 2 mg/Kg dose and the control.
[0035] FIG. 10 illustrates the average weights of the mice during
the treatment by M69-DOX at 20 mg/Kg and 2 mg/Kg doses or DOX at 2
mg/Kg dose in comparison with the control.
[0036] FIG. 11 illustrates the conjugation between MMAE and M69
utilizing lysine side chains with PEG.sub.5-DBCO.
[0037] FIG. 12 illustrates an MALDI-TOF analysis of the
immunoconjugate M69-MMAE after applying the chemical steps depicted
in FIG. 11.
[0038] FIG. 13 illustrates how matriptase-MMAE conjugate inhibits
growth of the TNBC MDA-MB-468 without causing weight loss or signs
of toxicity. Mice were inoculated s.c. with 10 million tumor cells
in the right flank. When the tumor was palpable (100-200 mm.sup.3)
mice were randomized into two groups (n=6) and treated at the times
indicated by the arrows with the immunoconjguate administered i.p.
5 mg (M69-MMAE)/kg (square shapes, bottom line). Control mice
received saline (diamond shapes, top line).
[0039] FIG. 14 illustrates IC50s of M69-MMAE immunoconjugates
toward matriptase-positive and negative cells in prostate cancer
cells and non-small cell lung (NSCL) cancer cells
[0040] FIG. 15A-C illustrates in vivo efficacy of M69-MMAE
immunoconjugates toward triple-negative breast cancer (MDA-MB-468)
(A) non-small cell lung (NSCL) cancer cells (H322) (B), and
prostate cancer cells (DU145) (C). Diamond shape represent control,
square shaped line represent M69-MMAE at 5 mg/mL, and for (B) the
triangle shaped line represents M69-MMAE at 1 mg/mL. Arrows
indicate times treated with the immunoconjugate.
[0041] FIG. 16 illustrates that DU145 and PC3 cells were exposed to
M69-MMAE at varying concentrations as indicated for 72 hours under
pH7.4 (diamond shape) or pH6.5 (square shape), HCl-acidified,
medium. Viability was measured as percentages of viability of cells
in control wells with respective medium.
[0042] FIG. 17 illustrates that Taxotere-resistant and -sensitive
prostate cancer cells, PC3R (diamond shaped) or PC3 (square
shaped), were treated with M69-MMAE at varying concentrations as
indicated. Viability was measured as percentages of viability of
cells in control wells.
[0043] FIG. 18 illustrates overall design of the ADC assembly used
in Examples 11-13. Octagons represent DBCO units, arrow represent
copper-free click chemistry.
[0044] FIG. 19 illustrates the chemical synthesis of
Azido-PEG.sub.4-Val-Cit-PABA-MMAE construct used in Examples
11-13.
DETAILED DESCRIPTION OF THE INVENTION
[0045] While matriptase is mainly produced by normal epithelial and
a variety of epithelial-derived carcinoma cells, it has been
discovered in accordance with the present invention that this
membrane-bound protease is also present in various cells of
hematological malignancies. The present invention provides methods
of treating hematological malignancies by administering antibodies
to matriptase. The invention further provides immunoconjugates of
an anti-matriptase antibody and a cytotoxic agent, and methods of
using the immunoconjugates for treatment of malignancies in which
cells express matriptase. In one preferred embodiment of this
invention, an auristatin, e.g. MMAE, MMAF, or auristatin PE is
conjugated with a monoclonal antibody to matriptase for targeting
myeloma cells. In some embodiments, the antibody is M69. Thus, in
some embodiments, the immunoconjugate is M69-auristatin, e.g.
M69-MMAE, M69-MMAF, and/or M69-auristatin PE.
[0046] Thus, in a first aspect the present invention provides a
method of treating a hematological malignancy by administering to a
subject in need of such a treatment a composition containing a
therapeutically effective amount of an anti-matriptase
antibody.
[0047] In one embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the hematological malignancy is a cancer comprising cells that
express matriptase.
[0048] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the hematological malignancy is selected from leukemias, lymphomas,
and myelomas.
[0049] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the hematological malignancy is selected from acute lymphoblastic
leukemia (ALL), acute myelogenous leukemia (AML), chronic
lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),
chronic myelogenous leukemia (CML), acute monocytic leukemia
(AMOL), Hodgkin's lymphomas, Non-Hodgkin's lymphomas, Burkitt's
lymphoma (BL), diffuse large B-cell lymphoma (DLBL), Mantle cell
lymphoma (MCL), and multiple myeloma.
[0050] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the hematological malignancy is multiple myeloma.
[0051] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the hematological malignancy is a cancer in which the malignant
cells express matriptase.
[0052] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is a monoclonal antibody (mAb). In
some embodiments, the monoclonal antibody is M69. In further
embodiments, the anti-matriptase antibody is covalently linked to a
cytotoxic compound. In yet further embodiments, a cytotoxic
compound is auristatin, including MMAE, MMAF, and/or auristatin PE.
Thus, in some embodiments, the present invention provides a method
of treating a hematological malignancy by administering a
therapeutically effective amount of an anti-matriptase antibody
conjugated to MMAE, MMAF, and/or auristatin PE, and in further
embodiments the anti-matriptase antibody is M69.
[0053] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is selected from chimeric antibodies,
humanized antibodies, and human antibodies.
[0054] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the composition further includes a pharmaceutically acceptable
carrier.
[0055] In a second aspect the present invention provides a method
of treating a hematological malignancy by administering to a
subject in need of such a treatment a therapeutically effective
amount of a composition containing an anti-matriptase antibody in
combination with a therapeutically effective amount of an
immunomodulatory agent that activates matriptase. In some
embodiments, the immunomodulatory agent is thalidomide or a
thalidomide analog.
[0056] In one embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the malignancy is multiple myeloma.
[0057] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is an antibody specific for activated
matriptase.
[0058] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is a mAb M69.
[0059] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is an antigen-binding fragment.
[0060] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is selected from the group consisting
of chimeric antibodies, humanized antibodies, and human
antibodies.
[0061] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the anti-matriptase antibody is a monoclonal antibody, and the
immunomodulatory agent is thalidomide or a thalidomide analog.
[0062] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the immunomodulatory agent is administered to the subject for a
sufficient amount of time so that matriptase is activated prior to
administration of the composition comprising the anti-matriptase
antibody.
[0063] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
the composition further comprises a pharmaceutically acceptable
carrier.
[0064] In a third aspect the present invention provides a method of
treating a malignancy comprising cells that express matriptase, the
method comprising administering to a subject in need of such a
treatment a therapeutically effective amount of a composition
comprising an immunoconjugate between an anti-matriptase antibody
and a cytotoxic agent.
[0065] In one embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the hematological malignancy is a
cancer comprising cells that express matriptase.
[0066] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the malignancy is a cancer.
[0067] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the malignancy is a matriptase-positive
malignant B cell lymphoma or an epithelial carcinoma.
[0068] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the malignancy is a matriptase-positive
malignant B cell lymphoma selected from the group consisting of
Mantle cell lymphoma (MCL), Burkitt's lymphoma (BL), and diffuse
large B-cell lymphoma (DLBL).
[0069] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the malignancy is an epithelial
carcinoma selected from the group consisting of prostate, breast,
brain, kidney, lung, colon, bladder, and ovary as well as other
tumor types, including thyroid tumor, skin tumor, and
mesothelioma.
[0070] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the malignancy is selected from the
group consisting of acute lymphoblastic leukemia (ALL), acute
myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL),
small lymphocytic lymphoma (SLL), chronic myelogenous leukemia
(CML), acute monocytic leukemia (AMOL), Hodgkin's lymphomas,
Non-Hodgkin's lymphomas, Mantle cell lymphoma (MCL) and multiple
myeloma.
[0071] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the malignancy is multiple myeloma.
[0072] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the anti-matriptase antibody is a
monoclonal antibody (mAb). In some embodiments, the monoclonal
antibody is M69.
[0073] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the anti-matriptase antibody is an
antigen-binding fragment. In some embodiments, the antigen-binding
fragment is an antigen binding fragment of M69.
[0074] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the anti-matriptase antibody is
selected from the group consisting of chimeric antibodies,
humanized antibodies, and human antibodies.
[0075] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the cytotoxic agent is selected from
toxins, antibiotics, and compounds comprising radioactive
isotopes.
[0076] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the cytotoxic agent is selected from
doxorubicin (DOX), auristatin, including MMAE, MMAF, and auristatin
PE, calicheamicin, and ricin.
[0077] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy comprising cells that
express matriptase, wherein the cytotoxic agent is doxorubicin
(DOX).
[0078] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the cytotoxic agent is
auristatin.
[0079] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the cytotoxic agent is MMAE.
[0080] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the cytotoxic agent is MMAF.
[0081] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the cytotoxic agent is auristatin
PE.
[0082] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the malignancy is a cancer
refractory to the treatment of doxorubicin.
[0083] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the malignancy is a cancer
refractory to the treatment of auristatin.
[0084] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the malignancy is a cancer
refractory to the treatment of MMAE.
[0085] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the malignancy is a cancer
refractory to the treatment of MMAF.
[0086] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the malignancy is a cancer
refractory to the treatment of auristatin PE.
[0087] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunoconjugate is M24-DOX or
M69-DOX.
[0088] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunoconjugate is
M24-auristatin or M69-auristatin.
[0089] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunoconjugate is M24-MMAE
or M69-MMAE.
[0090] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunoconjugate is M24-MMAF
or M69-MMAF.
[0091] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunoconjugate is
M24-auristatin PE or M69-auristatin PE.
[0092] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the cytotoxic agent is coupled to
the anti-matriptase antibody through a covalent bond.
[0093] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the subject is a mammalian
patient.
[0094] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the subject is a human.
[0095] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the composition further comprises
a pharmaceutically acceptable carrier.
[0096] In a fourth aspect the present invention provides a method
of treating a malignancy in which the malignant cells express
matriptase, the method comprising administering to a subject in
need of such a treatment a therapeutically effective amount of a
composition comprising an immunoconjugate between an
anti-matriptase antibody and a cytotoxic agent in combination with
a therapeutically effective amount of an immunomodulatory agent
that activates matriptase.
[0097] In one embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the malignancy is multiple
myeloma.
[0098] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
an antibody specific for activated matriptase.
[0099] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is a
monoclonal antibody (mAb). In some embodiments, the monoclonal
antibody is M69.
[0100] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
an antigen-binding fragment. In some embodiments, the
antigen-binding fragment is an antigen binding fragment of M69.
[0101] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
selected from the group consisting of chimeric antibodies,
humanized antibodies, and human antibodies.
[0102] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
mAb M69, and the cytotoxic agent is doxorubicin (DOX).
[0103] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
mAb M69, and the cytotoxic agent is auristatin.
[0104] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
mAb M69, and the cytotoxic agent is MMAE.
[0105] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
mAb M69, and the cytotoxic agent is MMAF.
[0106] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the anti-matriptase antibody is
mAb M69, and the cytotoxic agent is auristatin PE.
[0107] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunomodulatory agent is
thalidomide or a thalidomide analog.
[0108] In another embodiment of this aspect, the present invention
provides a method of treating a malignancy in which the malignant
cells express matriptase, wherein the immunomodulatory agent is
administered to the subject for a sufficient amount of time so that
matriptase is activated prior to administration of the composition
comprising the immunoconjugate.
[0109] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein:
(a) the malignancy is multiple myeloma; (b) the immunoconjugate
comprises an anti-matriptase antibody and DOX; and (c) the
immunomodulatory agent is thalidomide or a thalidomide analog.
[0110] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
(a) the immunoconjugate comprises an anti-matriptase antibody and
auristatin; and (b) the immunomodulatory agent is thalidomide or a
thalidomide analog.
[0111] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
(a) the immunoconjugate comprises an anti-matriptase antibody and
MMAE; and (b) the immunomodulatory agent is thalidomide or a
thalidomide analog.
[0112] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
(a) the immunoconjugate comprises an anti-matriptase antibody and
MMAF; and (b) the immunomodulatory agent is thalidomide or a
thalidomide analog.
[0113] In another embodiment of this aspect, the present invention
provides a method of treating a hematological malignancy, wherein
(a) the immunoconjugate comprises an anti-matriptase antibody and
auristatin PE; and (b) the immunomodulatory agent is thalidomide or
a thalidomide analog.
[0114] In a fifth aspect the present invention provides an
immunoconjugate selectively targeting cancer cells that express
matriptase, comprising an anti-matriptase antibody and a cytotoxic
agent.
[0115] In one embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the anti-matriptase antibody recognizes
an antigen expressed on the surface of the cancer cells.
[0116] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the anti-matriptase antibody is a
monoclonal antibody (mAb). In some embodiments, the monoclonal
antibody is M69.
[0117] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the anti-matriptase antibody is an
antigen-binding fragment. In some embodiments, the antigen-binding
fragment is an antigen binding fragment of M69.
[0118] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the anti-matriptase antibody is
selected from the group consisting of chimeric antibodies,
humanized antibodies, and human antibodies.
[0119] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the anti-matriptase antibody is a
monoclonal antibody, and the cytotoxic agent is selected from the
group consisting of doxorubicin (DOX), auristatin, including MMAE,
MMAF, and auristatin PE, calicheamicin, and ricin.
[0120] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the conjugate is formed through a
covalent bond between the anti-matriptase antibody and the DOX
moiety.
[0121] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled with up to 15 molecules of DOX.
[0122] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled with about 5-10 molecules of DOX.
[0123] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled about 7 molecules of DOX.
[0124] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein the conjugate is formed through a
covalent bond between the anti-matriptase antibody and an
auristatin moiety.
[0125] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled with up to 10 molecules of auristatin,
including MMAE, MMAF, and/or auristatin PE.
[0126] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled with about 5-10 molecules of auristatin,
including MMAE, MMAF, and/or auristatin PE.
[0127] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled about 3-5 molecules of auristatin, including
MMAE, MMAF, and/or auristatin PE.
[0128] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled about 3 molecules of auristatin, including
MMAE, MMAF, and/or auristatin PE.
[0129] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, wherein one molecule of the anti-matriptase
antibody is coupled about 1-3 molecules of auristatin, including
MMAE, MMAF, and/or auristatin PE.
[0130] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, the immunoconjugate having reduced or no
cardiotoxicity in comparison with the cytotoxic agent when
administered in the absence of the anti-matriptase antibody.
[0131] In another embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, the immunoconjugate having minimized or no
adverse effects on bone marrow-derived mesenchymal stromal cells
which do not express matriptase.
[0132] In another aspect the present invention provides a
composition comprising an immunoconjugate selectively targeting
cancer cells that express matriptase according to any of the
embodiments described above in the fifth aspect.
[0133] In one embodiment of this aspect, the present invention
provides an immunoconjugate selectively targeting cancer cells that
express matriptase, further comprising a pharmaceutically
acceptable carrier.
[0134] In another aspect the present invention provides an
immunoconjugate comprising a linker. In some embodiments, the
antibody comprises an anti-matriptase antibody. In some
embodiments, the anti-matriptase antibody comprises M69. In some
embodiments, the immunoconjugate comprising a linker comprises a
therapeutic agent. In some embodiments, the therapeutic agent is
any therapeutic agent according to the present disclosure. In some
embodiments, the therapeutic agent comprises auristatin. In some
embodiments, the therapeutic agent comprises MMAE.
[0135] In some embodiments, the linker comprises a cleavable
linking moiety. In some embodiments, the cleavable linking moiety
comprises a Cit-Val linking moiety. In some embodiments, the
cleavable linker comprises a Phe-Lys linking moiety. In some
embodiments, the cleavable linking moiety is cleavable by Capthesin
B.
[0136] In some embodiments, the linker is conjugated to a surface
of an antibody. In some embodiments, the antibody comprises an
anti-matriptase antibody. In some embodiments, the anti-matriptase
antibody comprises M69. In some embodiments, the linker is
covalently bound to an exposed amino acid residue. In some
embodiments, the exposed amino acid residue comprises a cysteine.
In further embodiments, the linker is covalently bound to the
cysteine through sulfhydryl-maleimide coupling. In some
embodiments, the exposed amino acid residue comprises a lysine. In
further embodiments, the linker is covalently bound to the lysine
through an amide linkage formed by acylation.
[0137] In some embodiments, the linker comprises a first linking
component and a second linking component. In some embodiments, the
first linking component is conjugated to a surface of an antibody
according to any aspect of the present disclosure. In some
embodiments, the first linking component is linked to the second
linking component. In some embodiments, the first linking component
is linked to the second linking component through click chemistry.
In some embodiments, the first linking component is linked to the
second linking component through a triazole moiety. In some
embodiments, the second linking component comprises a cleavable
linking moiety according to any aspect of the present disclosure.
In some embodiments, the second linking component comprises a
therapeutic agent according to any aspect of the present
disclosure. In some embodiments, the therapeutic agent comprises a
cytotoxic agent. In some embodiments, the cytotoxic agent is
selected from doxorubicin (DOX), auristatin, including monomethyl
auristatin E (MMAE), monomethyl auristatin F (MMAF), calicheamicin,
and ricin. In some embodiments, the cytotoxic agent is MMAE. In
some embodiments, the cytotoxic agent is MMAF.
[0138] In some embodiments, the linker is PEG-containing. In some
embodiments, a first linking component is PEG-containing. In some
embodiments, a second linking component is PEG-containing. In some
embodiments, a first linking component and a second linking
component are PEG-containing.
[0139] In another aspect, the present invention provides for an
immunoconjugate comprising an anti-matriptase antibody covalently
bound to a first linking component through a lysine side chain, the
first linking component being PEG-containing, the first linking
component bound to a second linking component through a triazole
moiety, the second linking component having a cleavable linking
moiety and a therapeutic agent, and the second linking component
being PEG-containing. In some embodiments, the anti-matriptase
antibody comprises M69. In some embodiments, the first linking
component contains between 1 and 10 PEG units. In some embodiments,
the second linking component contains between 1 and 10 PEG units.
In some embodiments, the cleavable linking moiety comprises a
Val-Cit moiety. In some embodiments, the cleavable linking moiety
comprises a Phe-Lys moiety. In some embodiments, the therapeutic
agent comprises a cytotoxic agent. In some embodiments, the
cytotoxic agent is selected from doxorubicin (DOX), auristatin,
including monomethyl auristatin E (MMAE), monomethyl auristatin F
(MMAF), calicheamicin, and ricin. In some embodiments, the
cytotoxic agent is MMAE. In some embodiments, the cytotoxic agent
is MMAF.
[0140] In another aspect the present invention provides a method of
diagnosing a hematological malignancy comprising contacting a test
sample containing hematological cells from a mammal with an
anti-matriptase antibody and detecting the formation of a complex
between the antibody and matriptase, wherein formation of a complex
is indicative of a malignancy.
[0141] In another aspect the present invention provides a method of
inhibiting the growth of a hematopoietic cell that expresses
matriptase, the method comprising treating the hematopoietic cell
with an anti-matriptase antibody or an immunoconjugate according to
any of the embodiments described above.
[0142] In another aspect the present invention provides an assay
kit for detecting expression of matriptase in mammalian tissues or
cells, comprising an immunoconjugate according to any of the
embodiments described above.
[0143] In another aspect the present invention provides a kit for
treatment of a malignancy comprising cells that express matriptase,
the kit comprising an immunoconjugate according to any of the
embodiments described above.
[0144] In another aspect the present invention provides use of an
immunoconjugate according to any of the embodiments described above
for treatment of a malignancy comprising cells that express
matriptase.
[0145] In another aspect the present invention provides use of an
immunoconjugate according to any of the embodiments described above
for manufacture of a medicament for treatment of a malignancy
comprising cells that express matriptase.
Definitions
[0146] As used herein, the term "biological sample" refers to a
specimen comprising body fluids, cells or tissue from a subject,
preferably a human subject. The sample can also be body fluid that
has come into contact, either naturally or by artificial methods
(e.g. surgical means), with a malignant cell or cells of a
pre-malignant lesion.
[0147] As used herein, the term "expression of matriptase," or the
like, refers to any biological sample comprising one or more cells
which express a form or forms of matriptase.
[0148] As used herein, the term "subject" refers to an animal,
preferably mammalian, and most preferably human.
[0149] As used herein, the term "antibody" refers to complete,
intact antibodies, and Fab fragments and F(ab), fragments thereof.
Complete, intact antibodies include monoclonal antibodies such as
murine monoclonal antibodies (mAb), chimeric antibodies, humanized
antibodies, and human antibodies.
[0150] An "antibody fragment" can be prepared by known methods, for
example, as disclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and
4,331,647 and references contained therein. Another form of an
antibody fragment is a peptide coding for a single
complementarity-determining region (CDR). A CDR is a segment of the
variable region of an antibody that is complementary in structure
to the epitope to which the antibody binds and is more variable
than the rest of the variable region. CDR peptides can be obtained
by constructing genes encoding the CDR of an antibody of interest.
Such genes are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of
antibody-producing cells.
[0151] As used herein, the term "immunoconjugate" refers to a
conjugate of an antibody component with a molecule or a therapeutic
or diagnostic agent. The therapeutic or diagnostic agent can
comprise a radioactive or non-radioactive label. The antibodies
that used to prepare immunoconjugates include, but is not limited
to, monoclonal antibodies, chimeric antibodies, humanized
antibodies, and human antibodies. Here a preferred immunoconjugate
is a conjugate between a matriptase monoclonal antibody and a
cytotoxic agent, and a more preferred immunoconjugate is one
comprising a matriptase monoclonal antibody and a FDA-approved
anticancer agent, including but not limited to doxorubicin (DOX),
auristatin, calicheamicin, or ricin.
[0152] As used herein, the term "cytotoxic agent" refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
chemotherapeutic agents, such as methotrexate, adriamicin, vinca
alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents, enzymes and fragments thereof such as
nucleolytic enzymes, antibiotics, and 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. The
term also encompasses compounds comprising one or more radioactive
isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212,
P32, as well as radioactive isotopes of Lu).
[0153] As used herein, the term "doxorubicin" (or "DOX") refers to
an anthracycline antibiotic with a systematic (IUPAC) chemical name
of
(8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,-
11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5-
,12-dione.
[0154] As used herein, the term "auristatin" includes, but is not
limited to, the antimitotic agents monomethyl auristatin E
("MMAE"). monomethyl auristatin F ("MMAF"), also known as
desmethyl-auristatin F. and auristatin PE. MMAE has a systematic
(IUPAC) chemical name of
(S)--N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-
-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-m-
ethyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butana-
mido)butanamide. MMAF has a systematic (IUPAC) chemical name of
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)--N,3-dimethyl-2-((S)-3-methyl--
2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolid-
in-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid.
Auristatin PE has a systematic (IUPAC) chemical name of
2-[[(2S)-2-(dimethylamino)-3-methylbutanoyl]amino]-N-[(3R,4S,5S)-3-methox-
y-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-(2-phenylethylamino)propyl-
]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl]-N,3-dimethylbutanamide.
[0155] As used herein, the term "carrier" refers to a molecule or
higher-ordered structure that is capable of associating with a
therapeutic or diagnostic agent to facilitate delivery of the agent
to a targeted cell. Carriers may include molecules such as lipids
or polymers, such as amphiphilic lipids or carbohydrates, or
higher-ordered structures, such as micelles, liposomes, and
nanoparticles.
[0156] The immunoconjugates or compositions disclosed herein can be
formulated according to known methods and may include one or more
pharmaceutically suitable excipients, one or more additional
ingredients, or combinations thereof.
[0157] The immunoconjugates or compositions disclosed herein can be
formulated for intravenous administration via, for example, bolus
injection or continuous infusion. Formulations for injection can be
presented in unit dosage form, e.g., in ampules or in multi-dose
containers, with an added preservative. The compositions can take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and can contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0158] The immunoconjugate or compositions may also be administered
to a mammal subcutaneously or even by other parenteral routes.
Moreover, the administration may be by continuous infusion or by
single or multiple boluses. In general, the dosage of an
administered immunoconjugate will vary depending upon such factors
as the patient's age, weight, height, sex, general medical
condition and previous medical history.
[0159] Additional pharmaceutical methods may be employed to control
the duration of action of the therapeutic or diagnostic conjugate
or naked antibody. Control release preparations can be prepared
through the use of polymers to complex or adsorb the
immunoconjugate or naked antibody.
[0160] An antibody preparation is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient mammal, including a reduction in the
number of cancer cells, a reduction in the size of a tumor, or an
inhibition in the growth of a tumor. In particular, an antibody
preparation is physiologically significant if its presence invokes
an antitumor response or mitigates the signs and symptoms of an
autoimmune disease state. A physiologically significant effect
could also be the evocation of a humoral and/or cellular immune
response in the recipient mammal
[0161] It will be appreciated that actual preferred amounts of a
pharmaceutical composition used in a given therapy will vary
depending upon the particular form being utilized, the particular
compositions formulated, the mode of application the particular
site of administration, the patient's weight, general health, sex,
etc., the particular indication being treated, etc. and other such
factors that are recognized by those skilled in the art including
the attendant physician or veterinarian. Optimal administration
rates for a given protocol of administration can be readily
determined by those skilled in the art using conventional dosage
determination tests.
[0162] Preparation of Immunoconjugates
[0163] The immunoconjugates described herein can be prepared by
known methods of linking antibodies with lipids, carbohydrates,
protein, or other molecules. For example, the binding molecules
described herein can be conjugated with one or more of the carriers
described herein (e.g., lipids, polymers, liposomes, micelles, or
nanoparticles) to form an immunoconjugate, and the immunoconjugate
can incorporate a therapeutic or diagnostic agent either
covalently, non-covalently, or otherwise. Further, any of the
binding molecules described herein can be further conjugated with
one or more therapeutic or diagnostic agents described herein, or
additional carriers. Generally, one therapeutic or diagnostic agent
may be attached to each binding molecule but more than one
therapeutic agent or diagnostic agent can be attached to the same
binding molecule. Further, the therapeutic agents do not need to be
the same but can be different therapeutic agents.
[0164] For example, to synthesize an immunoconjugate of an antibody
and DOX, DOX may be first reacted with succinimidyl
4[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), and the
antibody against matriptase may be reacted with
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), followed by
coupling of these two intermediates. Additional linkers that may be
used to prepare immunoconjugates are discussed infra in the section
titled "Linkers (Cleavable and Non-cleavable)".
[0165] Methods of Treatment
[0166] The present invention encompasses use of the matriptase
antibodies or immunoconjugates or compositions comprising the
matriptase antibodies or immunoconjugates as the primary
composition for treatment of a malignancy comprising cells that
express matriptase. The malignancy includes, but is not limited to,
solid tumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple
myeloma, a B-cell malignancy and/or a T-cell malignancy. The solid
tumor is selected from the group consisting of a melanoma,
carcinoma and sarcoma and the carcinoma is selected from the group
consisting of a renal carcinoma, lung carcinoma, intestinal
carcinoma, stomach carcinoma and melanoma. The B-cell malignancy is
selected from the group consisting of indolent forms of B-cell
lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic
leukemias, acute lymphatic leukemias, and multiple myeloma, B-cell
disorders and other diseases. In particular, the compositions
described herein are particularly useful for treatment of various
autoimmune as well as indolent forms of B-cell lymphomas,
aggressive forms of B-cell lymphomas, chronic lymphatic leukemias,
acute lymphatic leukemias, multiple myeloma, and Waldenstrom's
macroglobulinemia. The methods of treatment comprise administering
to a mammal in need of such treatment a therapeutically effective
amount of a composition comprising the antibody or
immunoconjugate.
[0167] Antibodies
[0168] Antibodies against matriptase ("anti-matriptase antibodies")
can be made by methods known in the art and disclosed, for example,
by Lin C. Y., et al., J. Biol. Chem., 1999, 274 (26):18237-18242.
Antibodies against matriptase include antibodies that are specific
for latent matriptase, antibodies that are specific for activated
matriptase, and antibodies that recognize both latent and activated
matriptase.
[0169] The antibodies and immunogenic portions thereof of this
invention are administered at a concentration that is
therapeutically effective to prevent or treat any of the
aforementioned disease states. To accomplish this goal, the
antibodies may be formulated using a variety of acceptable
excipients known in the art. Typically, the antibodies are
preferably administered by injection, either intravenously or
intraperitoneally. Methods to accomplish this administration are
known to those of ordinary skill in the art. The compositions may
also be topically or orally administered, or be capable of
transmission across mucous membranes.
[0170] Before administration to patients, formulants may be added
to the antibodies. A liquid formulation is preferred. For example,
these formulants may include oils, polymers, vitamins,
carbohydrates, amino acids, salts, buffers, albumin, surfactants,
or bulking agents. Preferably carbohydrates include sugar or sugar
alcohols, such as mono-, di- or polysaccharides, or water soluble
glucans. The saccharides or glucans can include fructose, dextrose,
lactose, glucose, mannose, sorbose, xylose, maltose, sucrose,
dextran, pullulan, dextrain, alpha- and beta-cyclodextrin, soluble
starch, hydroxyethyl starch and carboxymethylcellulose, or mixtures
thereof.
[0171] Additionally, antibodies can be chemically modified by
covalent conjugation to a polymer to increase their circulating
half-life. Preferred polymers, and methods to attach them to
peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337;
4,495,285; and 4,609,546. Preferred polymers are polyoxyethylated
polyols and polyethylene glycol (PEG). Water soluble
polyoxyethylated polyols are also useful in the present invention.
They include polyoxyethylated sorbitol, polyoxyethylated glucose,
polyoxyethylated glycerol (POG), etc.
[0172] The anti-matriptase antibodies of the present invention may
comprise chimeric or humanized antibodies. Both chimeric and
humanized antibodies comprise antibodies that are originally
derived from non-human species which have been chemically modified
to increase the similarity to antibody variants which are produced
naturally in humans, and consequentially less immunogenic. Both
chimeric and humanized antibodies are typically recombinantly
produced and expressed in mammalian cell culture, e.g. through CHO
cells. Such techniques are known to one of ordinary skill in the
art. The primary difference between chimeric anti-matriptase
antibodies and humanized anti-matriptase antibodies is that
chimeric anti-matriptase antibodies typically comprise a
substitution of the Fc region of the monoclonal antibody with a
human Fc region, whereas humanized antibodies are identical to a
human immunoglobulin (typically referred to as a human antibody
"scaffold") except the complementarity determining regions (CDRs)
are "swapped in" from the original mAb.
[0173] Another drug delivery system for increasing circulatory
half-life is the liposome. Methods of preparing liposome delivery
systems are discussed in Gabizon et al., Cancer Res. 42: 4734-9
(1982); Szoka et al., Annu. Rev. Biophys. Bioeng. 9: 467-508
(1980); Szoka et al., Meth. Enzymol. 149: 143-7 (1987); and Langne
et al., Pol. J. Pharmacol. 51: 211-22 (1999). Other drug delivery
systems are known in the art.
[0174] Linkers (Cleavable and Non-Cleavable)
[0175] The anti-matriptase antibodies of the present disclosure,
e.g. M69, may be covalently linked to a therapeutic or diagnostic
agent, e.g. DOX, auristatins, (including but not limited to
MMAE/MMAF), thus forming an immunoconjugate. An overview of
immunoconjugate linker-based technology is found in Jain N et al.
Current ADC Linker Chemistry, Pharm Res. 2015 November;
32(11):3526-40, hereby incorporated by reference in its entirety.
The covalent linkages in such immunoconjugates may comprise a
cleavable linking moiety, for example, a Val-Cit linker, which is
cleavable by Cathepsin B inside the lysosome. A commercially
available Val-Cit linker was modified for use in Example 10 infra.
Other cleavable linking moieties may comprise a Phe-Lys linker,
which is also cleavable by Cathespin B. Some of the simplest
cleavable linking moieties include disulfide (S--S) bridges, which
are cleavable in a reductive (i.e. intracellular) environment.
However, cleavable linking moieties such as Val-Cit linkers provide
more specificity than, for example, disulfide bridges, which may be
subject to indiscriminate cleavage, and thus present a superior
option, although any such cleavable linking moiety is to be
considered within the scope of the present invention. An overview
of cleavable linking moieties which may be suitable for the present
invention is provided in Leriche et al., Cleavable linkers in
chemical biology Bioorg Med Chem. 2012 Jan. 15; 20(2):571-82,
hereby incorporated by reference in its entirety. Alternatively,
the linker may non-cleavable. Non-cleavable linkers are more
diverse than cleavable linkers, and may comprise any linking moiety
that is resistant to cleavage in an intracellular environment. For
example, a specific non-cleavable linker which may be of interest
comprises a SMCC linker, which is found in the FDA approved
immunoconjugate trastuzumab emtansine (trade name Kadcyla), and is
also explored in Example 2, infra, for preparation of M69-DOX
immunoconjugates.
[0176] The linkers (and the therapeutic agents bound to said
linkers) of the present disclosure may be directly conjugated to
the antibody, i.e. covalently linked to the immunoglobulin. The
covalent linkage may occur directly to one of the amino acids
comprising the immunoglobulin backbone, ideally located within one
of the constant domains, as opposed to within the variable domains.
Such amino acids may be naturally occurring (e.g. a naturally
occurring lysine or cysteine residue) or, the immunoglobulin may be
artificially mutated (e.g. a non-naturally occurring lysine or
cysteine residue) in order to provide an optimal binding site with
minimal steric hindrance for the linker to bind to. Alternatively,
the linker can be bound to a chemical moiety (e.g. bound to an
N-glycan) found on post-translationally modified
immunoglobulins.
[0177] The majority of immunoconjugates utilize direct attachment
of the linker to a cysteine residue located in the antibody
backbone. These linkers are known as "maleimide-type linkers" and
utilize the reactivity of maleimide with sulfhydryl side chains
found in cysteine (Cys). The two most common types of maleimide
linkers include maleimidocaproyl (mc) and maleimidomethyl
cyclohexane-1-carboxylate (mcc) linkers. A downside of maleimide
linkers has been chemical instability in plasma, including a
retro-Michael reaction which results in premature loss of the
drug-linker from the immunoconjugate. A different type of direct
conjugation, and one used in Example 10 infra, is direct
conjugation to the .epsilon.-amino side chain of lysine (Lys) side
chain via acylation, creating an amide bond between the linker and
the antibody in which the linker consists of an activated carboxyl
group instead an azide functional group.
[0178] The linker and the therapeutic agent may be conjugated to
the antibody using two-step conjugation technology as described
herein. This two-step conjugation technology may utilize "click
chemistry," especially copper-free click chemistry. Use of click
chemistry in antibody conjugation may provide several unique
benefits, for example, UV based detection of the forming click ring
system for reaction monitoring and ADR determination. The linker
may be initially comprised of two distinct linking components, for
example (but not necessarily), one linking component containing an
acylating agent (such as, for example, a carboxylic acid, acyl
halide, or anhydride) that conjugates (i.e. acylates) to a lysine
side chain located in the immunoglobulin backbone and further
containing a strained alkyne (e.g. DBCO), and a second linking
component containing an azide (N.sub.3) group and the therapeutic
agent, such that when the first component and the second component
are brought within proximity to one another, the strained alkyne
reacts with the azide group (forming a triazole moiety), linking
the first linking component to the second linking component. Either
the first linking moiety or the second linking moiety may contain
the strained alkyne group and/or the azide group, and vice versa;
i.e. the first linking moiety may comprise a strained alkyne and
the second linking moiety an azide group, or the first linking
moiety may comprise an azide group and the second linking moiety
may comprise an a strained alkyne. Accordingly, when bound through
"click chemistry" the linkers of the present disclosure may
comprise a triazole moiety, e.g. a triazole moiety as formed
between the reaction of DBCO with N.sub.3 as illustrated in FIG.
11.
[0179] Thus in some embodiments, the overall structure of the
immunoconjugate would comprise mAb--first linking component--second
linking component--therapeutic agent, the first linking component
being conjugated to the second linking component via a triazole
moiety (formed by click chemistry), and the first linking component
being conjugated to an amino acid residue (e.g. cysteine or
preferably lysine as described herein) located in the
immunoglobulin backbone. In such embodiments, incorporation of a
cleavable linking moiety into either of the first and/or second
linking components, e.g. a Val-Cit linker, is possible and is shown
in FIG. 11. Ideally, the cleavable linking moiety would be located
in the second linking component (i.e. the linking component
containing the therapeutic agent, e.g. MMAE and/or MMAF).
[0180] The linkers in the immunoconjugates of the present
disclosure may be PEG-based linkers. For example, and as shown in
FIG. 11, in some embodiments both the first linking component and
the second linking component (and thus the overall linker) contain
poly(ethylene) glycol (PEG) repeats. PEG-based linkers may provide
a distinct number of advantages over linkers not containing PEG.
For example, PEG-based linkers may provide greater flexibility, so
that, for example, in an instance where the linker is conjugated to
an amino acid residue that is in close proximity to the
antigen-binding domain (e.g. the hypervariable region), there would
be a reduced chance that the linker/therapeutic agent would bind or
interact with the surface elements of the antigen/receptor, thus
making the overall immunoconjugate more therapeutically effective.
Additionally, in such embodiments where linker is generated through
click chemistry (e.g. using DBCO and N.sub.3), while not wishing to
be bound by theory, the use of PEG in the linking components may
act as a spacer to prevent non-specific interactions (pi-pi
interactions) between the tetracyclic ring structure found in the
triazole moiety and any exposed aromatic side chains, especially
tryptophan side chains. The PEG-based linkers of the present
disclosure may comprise repeats of anywhere between 1-20 PEG units
total, e.g. 1-10 PEG units per each of the first linking component
and second linking component in such embodiments that utilize a
first linking component and a second linking component. More
typically, 3-7 PEG units per each of the first linking component
and second linking components. By way of example, Example 10 infra
shows an M69-MMAE immunoconjugate that contains 5 PEG units for the
first linking component and 4 for the second linking component.
[0181] Auristatins, MMAE, MMAF
[0182] Auristatins, including MMAE, represent a class of very
potent cytotoxic agents. Without wishing to be bound by theory, it
is believed that auristatins like MMAE inhibit cell division by
blocking the polymerization of tubulin, which is critical for
proper cell division. Thus, compounds such as MMAE, MMAF, and
auristatin PE are classified as "antimitotic agents" for inhibiting
mitosis. The potency of such cytotoxic agents is significant. MMAE,
for example, is 100 to 1000 times more potent than DOX, although
DOX is suitable for use in this invention. Because of this potent
toxicity, MMAE is not suitable for administration as a drug by
itself. However, when coupled to an antibody such as matriptase
antibodies, e.g. M69, the cytotoxic payload is capable of being
directed to specific malignant cells.
[0183] As stated above, the antibodies and compositions of this
invention are used preferably to treat human patients to prevent or
treat any of the above-defined disease states. The preferred route
of administration is parenteral. In parenteral administration, the
compositions of this invention will be formulated in a unit dosage
injectable form such as a solution, suspension or emulsion, in
association with a pharmaceutically acceptable parenteral vehicle.
Such vehicles are inherently nontoxic and non-therapeutic. Examples
of such vehicles are saline, Ringer's solution, dextrose solution,
and Hanks' solution. Non-aqueous vehicles such as fixed oils and
ethyl oleate may also be used.
[0184] The anti-matriptase immunoconjugates disclosed herein,
including mAb-auristatin and mAb-Dox conjugates represent examples
for developing anticancer agents specifically targeting cancer
cells. Anti-matriptase mAbs can be linked to other types of
cytotoxic agent such as anti-tubulin compounds that would work
better with certain types of tumors, or can be coupled to lyposomal
or nanoparticles for targeted delivery of any types of agents,
including peptides, shRNA/siRNA and very toxic compounds of little
potential in cancer treatment due to severe side effects.
[0185] The following non-limiting examples further illustrate
certain aspects of the invention.
EXAMPLES
Example 1
[0186] Expression of Matriptase in Multiple Myeloma and B-Cell
Lymphoma Cells
[0187] A panel of 24 cell lines of human hematopoietic malignancies
was analyzed by Western blotting for matriptase as well as its
endogenous inhibitor HGF activator inhibitor-1 or HAI-1 (FIG. 1).
Cell lysates were prepared from 24 hematopoietic cancer cells
(lanes 2-14) and T-47D breast cancer cells (lane 1). Equal amounts
of proteins were resolved by SDS-PAGE. Levels of matriptase and
HAI-1 were assessed by immunoblot analysesusing an anti-matriptase
mAb and anti-HAI-1 mAb as indicated (FIGS. 1A and 1B).
[0188] Matriptase was expressed in the majority of the B-cell
lymphomas as well as in multiple myeloma cells, indicating that the
protease is an important marker for these cancers. In contrast,
HAI-1 levels in lymphoma and myeloma appear to be much lower than
those in carcinomas, and HAI-1 expression is often lost in highly
aggressive lymphomas (Table 1).
[0189] To further verify the expression of matriptase in multiple
myeloma cells, six cases of paraffin-embedded MM specimens were
examined. Matriptase was positive in four cases, among which two
were HAI-1 positive and the other two were negative (FIG. 1D).
[0190] The levels of matriptase in cell lines of multiple myeloma
were assessed (FIG. 1C). All three MM cells produced the
protease.
Example 2
[0191] Preparation of DOX-Immunoconjugate
[0192] Monoclonal antibodies against matriptase (M24 or M69,
obtained from C Y Lin, University of Maryland, Baltimore, Md.; Lin
C. Y., et al., J. Biol. Chem., 1999, 274 (26):18237-18242; Chen, Y.
W., et al. J. Biol. Chem., 2010, 285 (41):31755-31762) was
conjugated to DOX (Sigma) by using Protein-Protein Coupling kit
(Invitrogen) according to the manufacture's instruction with some
minor modification. Briefly, DOX was reacted with crosslinker SMCC
(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) at a
molar ratio (MR) between 1:3 and 1:1.5 (DOX:SMCC) for 1 hour at
room temperature with constant stiffing. About 200 mg M24 mAb was
reacted with SPDP (Succinimidyl 3-(2-pyridyldithio)-propionate) for
1 hour at room temperature followed by exchange of phosphate buffer
using Centrifugal Filter Device (Ultrafree, Millipore).
Alternatively, the mAb can be separated from free SPDP by gel
filtration with Sephadex G50. DOX maleimide derivative was then
conjugated to thiolated M24 or M69 mAb for 3 hours at room
temperature followed by dialysis in the phosphate buffer to remove
the free DOX. Gel filtration with Sephadex G50 can also be used to
remove the uncoupled DOX if the scale of preparation is larger than
one (1) gram of proteins. The molar ratio of protein to DOX is
determined through measuring DOX by fluorescence and protein
concentration.
[0193] Since DOX emits fluorescence by UV excitation, the coupling
of DOX to the antibody was measured, based on the fluorescence
generated by the immunoconjugate. M24-DOX at 1 .mu.g/ml
approximately has the same intensity of fluorescence as that of DOX
at 25 nM. Hence, 1 molecule of the mAB is coupled with nearly 7
molecules of DOX.
TABLE-US-00001 TABLE 1 Summary of matriptase and HAI-1 expression
in human lymphoma and leukemia cells Cell line Cell Type
Disease/Source Viruses* Matriptase HAI-1 HL-60 Promyeloblast AML -
- +/- Reh Lymphoblast ALL - - + + + Jurkat T-Lymphoblast ALL - - +
SUP-T1 T-Lymphoblast LL - + + + - CCRF-CEM T-Lymphoblast ALL - -
+/- CCRF-HSB-2 T-Lymphoblast ALL - - + + MOLT-3 T-Lymphoblast ALL -
- - MOLT-4 T-Lymphoblast ALL - - CCRF-SB B-Lymphoblast ALL EBV+ -
+/- RS4; 11 B-Lymphoblast ALL - - +/- THP-1 Monocyte Acute - + +
+/- Monocytic Leukemia U-937 Monocyte Histiocytic - - - Lymphoma Hs
445 B-Lymphoblast HL EBV+ - +/- HuT 78 Cutaneous T Sezary - - + +
lymphocyte Syndrome Farage B-Lymphoblast EBV- EBV+ +/- -
transformed B cell lymphoma RPMI 8266 B-Lymphoblast MM - + + + + +
Daudi B-Lymphoblast BL EBV+ + + + + - Namalwa B-Lymphoblast BL
EBV+, + + + + - SMRV+ Raji B-Lymphoblast BL EBV+ + + - Ramos
B-Lymphoblast BL - + + + + - ST486 B-Lymphoblast BL - + + + + -
SU-DHL-4 B-Lymphoblast DLBL - + + + + +/- SU-DHL-6 B-Lymphoblast
DLBL - + + + + +/- OCI-LY-3 B-Lymphoblast DLBL - + + + + +/-
Abbreviation: AML, acute myelocytic leukemia; ALL, acute
lymphocytic leukemia; BL, Burkitt's lymphoma; DLBL, diffuse large
B-cell lymphoma; EBV, Epstein-Barr virus; HL, Hodgkin's lymphoma;
LL, lymphoblastic leukemia; MM, multiple myeloma; SMRV, squirrel
monkey retrovirus.
[0194] Abbreviation: AML, acute myelocytic leukemia; ALL, acute
lymphocytic leukemia; BL, Burkitt's lymphoma; DLBL, diffuse large
B-cell lymphoma; EBV, Epstein-Barr virus; HL, Hodgkin's lymphoma;
LL, lymphoblastic leukemia; MM, multiple myeloma; SMRV, squirrel
monkey retrovirus.
Example 3
Cytotoxicity of M24-DOX Toward Multiple Myeloma Cells
[0195] To assess the cytotoxic effect of the immunoconjugate, MM
cells were treated with the conjugated DOX at various
concentrations for 48 hours. The immunoconjugate inhibited cell
proliferation in a dose-dependent manner. The EC50 of the
immunoconjugate was 5 .mu.g/ml (protein). This cytotoxic effect was
also observed with treatment of free DOX at 200 nM. As the
concentration of M24-DOX at 5 .mu.g/ml is equivalent to 250 nM of
free DOX based on the intensity of fluorescence, the potency of the
conjugated DOX is therefore similar to the free drug. Exposure of
the cells to unmodified antibody had no effect on cell growth,
demonstrating that the M24-DOX induced cytotoxic effect is mediated
through DOX activity. Two other MM cell lines including OPM-2 and
RPMI-8226 were also tested for the cytotoxic activity of M24-DOX as
shown in FIG. 2 where MM cells (1.times.10.sup.5/well) in 24-well
plates were treated with or without M24-DOX at varying
concentrations for 48 hours. Cell number was counted by Vi-Cell
counter (Beckman).
[0196] Both types of cells also responded to the drug in a similar
fashion as MM.1S did.
Example 4
Nuclear Localization of M24-DOX.
[0197] To demonstrate internalization of the immunoconjugate of DOX
in MM.1S cells, fluorescent microscopy was used to trace the
subcellular localization of M24-DOX after exposure of the cells to
the drug for various periods of time. Following exposure of MM.1S
cells to M24-DOX for varying periods of time, the cells were fixed
and stained. The immunoconjugate was visualized by fluorescent
microscopy. DAPI was used for nuclear staining.
[0198] Trace amounts of nuclear fluorescence were detected in a
small number of cells after 3 min exposure of the M24-DOX. Both the
number of fluorescence stained cells and intensity of signal
localizing in the nuclei were significantly increased with
incubation times longer than 30 min (FIG. 3A). The pictures with
higher magnification revealed swelling of nuclei of cells exposed
to the drug for 60 min or longer, indicative of typical DOX-induced
apoptosis (FIG. 3B). These data indicate that following binding to
the antigen, the immunoconjugate was rapidly internalized and
significant nuclear localization of DOX was observed after 10 min
of treatment.
Example 5
Tolerance of Bone Marrow-Derived Mesenchymal Stem Cells (MSCs)
Toward M24-DOX.
[0199] Human bone marrow-derived MSCs, which lack matriptase
expression, were used as a control to determine the cytotoxic
selectivity of M24-DOX as shown in FIG. 4 where bone marrow-derived
mesenchymal stromal cells (5.times.10.sup.4/well, 24-well plates)
were treated with M24-DOX or free DOX at varying concentrations for
4 days. Cell numbers were determined by Vi-Cell counter.
[0200] The MSCs were insensitive to the immunoconjugate at a
concentration as high as 20 .mu.g/ml, whereas free DOX was able to
significantly inhibit cell proliferation at 5 nM. Expression of
matriptase is mainly restricted to epithelial cells in normal
tissues. Absence of this protease in cardiomyocytes indicates that
M24-DOX will exhibit significantly reduced cardiotoxicity.
Example 6
Targeting Active Matriptase
[0201] DOX was also conjugated with another matriptase mAb (M69)
that specifically binds to the active two-chains of the enzyme.
While active matriptase levels were found in most breast tumors,
the active form of the protease is barely detectable in the normal
tissues of the mammary gland (FIG. 5). These data indicate that the
DOX-conjugate with M69, referred to as M69-DOX, offers additional
selectivity in targeting tumors expressing activated matriiptase
with regard to toxicity toward normal tissues. As epithelial
tissues express matriptase, the toxicity of M24-DOX toward the
healthy tissues is a concern. For instance, a previous
DOX-conjugate called BR96 targeting Lewis-Y antigen in carcinoma
cells failed in the phase II trial due to considerable
gastrointestinal (GI) toxicity (Tolcher, A. W., et al., J. Clin.
Oncol., 1999, 17: 478-484) In contrast to the expression of latent
form of matriptase in epithelial tissues, the level of the active
form of this protease is hardly detectable by immunohistochemistry.
Hence, M69-DOX is less toxic to epithelia.
Example 7
Enhanced Cytotoxic Effects of M69-DOX by Thalidomide
[0202] Thalidomide is a therapeutic for multiple myeloma treatment,
and the combination of thalidomide and dexamethasone is one of the
most common regimens for treatment of patients with multiple
myeloma, with an response rate of up to 60-70% (Denz, U., Eur. J.
Cancer., 2006, 42:1591-1600; Gieseler, F., et al., Thromb.
Haemost., 2008, 99: 1001-1007). The efficacy of both DOX-conjugates
in combination with current MM therapeutics such as thalidomide was
tested. M69-DOX by itself alone was less potent than M24-DOX.
Surprisingly, inhibition of cell proliferation by M69-DOX treatment
was significantly augmented by thalidomide, whereas the inhibitory
activity of M24-DOX was not altered (FIG. 6A-B). This enhanced
inhibition by M69-DOX combined with thalidomide appeared to be
synergistic, as thalidomide alone had no effect on MM cell
proliferation (FIG. 6A). Therefore these studies show that
thalidomide resulted in enhanced inhibition of proliferation.
Example 8
Matriptase Activation Induced by Thalidomide in MM Cells
[0203] To examine the increase in cytotoxicity that resulted with
the thalidomide and M69-DOX conjugate we measured the effect of
thalidomide on expression of activated matriptase. Activation of
matriptase in MM cells was markedly enhanced after 24-hour
treatment with 50 .mu.M thalidomide (FIG. 8) Thalidomide is known
to exert antiangeiogenic activity by interfering with the
interaction between MM and bone marrow stromal cells, at least in
part, through inhibition of VGEF production. While thalidomide
appears to have little effect on the cell proliferation of MM cells
(FIG. 7A-B), its ability to activate matriptase is an important
novel finding, suggesting a novel approach to exploit the response
of MM cells to thalidomide treatment by targeting active
matriptase-positive myeloma cells with the M69-DOX conjugate.
Example 9
Inhibition of Breast Tumor Growth by M69-DOX Targeting Active
Matriptase In Vivo
[0204] To assess the efficacy of M69-DOX to suppress tumor growth
in vivo, a human breast xenograft tumor model was used. NOD/SCID
mice bearing breast tumors (MDA-MB468) were treated twice weekly
with M69-DOX at either 20 or 10 mg/kg, as well as treated with DOX
at 2 mg/kg. While the tumor sizes of the mice in the untreated
control group were markedly increased at the third week, tumor
growth in the mice treated with M69-DOX at either dose and treated
with DOX was significantly abolished during the entire treatment
course (FIG. 8). As a single mAb was attached with 7 molecules of
DOX, a dose at 10 mg/kg of M69-DOX is equivalent to 0.035 mg/kg of
DOX administered. These results demonstrate strong potency and
efficacy of DOX conjugate targeting activated matriptase to treat
breast tumors in vivo.
[0205] Reduced Weights of Tumor Masses in Mice Treated with
M69-DOX. At the end of the treatment course, the tumor masses were
collected from the experimental mice, and were weighed. The average
weights of tumors from the groups treated with M69-DOX at either
dose as well as treated with DOX were reduced to about 50% of the
one from the untreated control group (FIG. 9). Significant
reduction in tumor weights by M69-DOX further confirms the efficacy
of site-directed delivery of DOX to tumor cells expressing
activated matriptase.
Minimal Toxicity by M69-DOX Treatment
[0206] The average weights of mice treated with either M69-DOX or
DOX were not markedly reduced (less than 15% loss) compared to
those from untreated control group, indicating that the M69-DOX at
such doses was well tolerated (FIG. 10). The dosage at 20 mg/kg of
M69-DOX did not lead to noticeable toxicity, indicating that the
drug dose can be escalated to reach the maximum tolerant dose.
Example 10
Coupling of MMAE Via a Releasable Linker to a M69 mAb to Form a
Conjugate: M69-MMAE.
[0207] MMAE was coupled to M69 to form an immunoconjugate as
described here. The releasable linker technology was based on
Seattle Genetics' Valine-Citrulline-PABA linker, the original
Cathepsin B cleavable linker, as described in Bioconjugate Chem.
2002, 13:855-869; Blood 2003, 102:1458-1465; Nature Biotech., 2003,
21:778-784, all of which are hereby incorporated by reference in
their entirety. The Valine-Citrulline-p-aminobenzylalcohol
carbamate linker was modified by (i) a two-stage PEGylation, (ii)
conjugation via copper-free click chemistry, and (iii) acylating
lysine side chains of the mAb instead of cysteine
sulfhydryl-maleimide coupling. The overall design of the ADC
assembly is depicted in FIG. 18. Chemical synthesis of
Azido-PEG.sub.4-Val-Cit-PABA-MMAE construct was as depicted in FIG.
19. The present design improved this linker platform using
copper-free click chemistry using DBCO and N.sub.3, enabling the
performance of crucial drug loading steps in a stoichiometrically
controlled manner under very mild conditions. Lysine side chains
were utilized to conjugate the linker-drug ligand onto the surface
of the antibodies instead of using temporarily reduced sulfhydryl
groups of cysteine residues (FIG. 11). Since this approach does not
affect disulfide brides between cysteines, the anti-matriptase
antibodies remain structurally intact during the conjugation
procedure, eliminating the problem with loss of activity by
misfolded/dissociated antibody chains. Furthermore, lysine side
chains are readily available on the surface of the mAb, so there
was no need to reduce Cys-Cys disulfide bridges eliminating the
problem of structural destabilization of the mAb and mismatched
disulfide bond formation upon oxidation following maleimide
conjugation. Appropriate analytical procedures (HR-MALDI-TOF mass
spectrometry) demonstrated that recent batches of immunoconjugates
meeting industrial standards (FIG. 11). A further advancement was
achieved by using PEG spacers in the drug-linker part of the
conjugate on both precursors of the click chemistry that
facilitates the use of even very hydrophobic drug molecules. The
PEG chains were long enough to keep the mAb and the drug separate,
decreasing the probability of unspecific drug and antibody
interactions, and the PEG.sub.5-PEG.sub.4 configuration of the
linker compensated the hydrophobicity of tetracyclic system
resulted by the copper-free click chemistry. The cytotoxic compound
selected was monomethyl auristatin E, or MMAE. The flexibility and
length of the PEGylated linker provide high accessibility for the
Cathepsin B to cleave and release MMAE. The conjugation reactions
were monitored by MALDI-TOF mass spectrometry showing 7000 Da
increase of the average MW that corresponds to 3.5 drugs linked to
each mAb molecule.
[0208] To demonstrate the cytotoxic potency of M69-MMAE, the in
vitro cytotoxic efficacy of the immunoconjugates toward a triple
negative breast cancer, MDA-MB468, was assessed. The breast cancer
cells were sensitive to the MMAE-linked immunoconjugate with an
IC50 of 3.4 .mu.g/ml. As each M69 molecule contained an average of
3.5 molecules of MMAE (FIG. 11), the sensitivity to MMAE in the
conjugate was 1.6 nM. This experiment also demonstrated that the
immunoconjugate is internalized and toxin released
intracellularly.
Example 11
In Vitro Cytotoxicity of M69-MMAE Immunoconjugates
[0209] To evaluate the in vitro cytotoxicity of the M69-MMAE
immunoconjugates as described in Example 10, tumor cells such as
TNBC (MDA-MB-468, MDA-MB-231 and BT549), prostate (DU145 and PC3),
pancreas (PANC1 and MiaPacal), NSCL (H322 and H1299), Ovary
(OVCAR5), stomach (AGS), and Mantle cells lymphoma (Mino, Jeko,
Mayer and Z138) cancers were exposed to M69-MMAE immunoconjugates
with various concentrations as indicated. After 72 hours of
continuous exposure to the immunoconjugate, cytotoxicity was
assessed by MTS (Promega) assay as follows: MTS colorimetric assay
(CellTiter 96 from Promega) was performed according to the
manufacturer's protocols. Cancer cells were plated in 96-well
plates at 8000 cells/well, and were exposed to a graded titration
of M69-MMAE for 72 hours at 37.degree. C. The percentage viability,
relative to untreated control wells, was plotted versus
immunoconjugate concentration. Results of each study are the
average of quintuplicate determinations. The values of IC50 of
M69-MMAE against these cells were summarized in Table 2 below.
IC50s of M69-MMAE toward the indicated tumor cells were determined
by a MTS colorimetric method with 96-well plates as described. The
IC50 values were presented as means (.mu.g/mL).+-.SD of the ADC as
well as equivalent nM of MMAE concentration considering that the
ADC has a stoichiometric ration of 3.5 MMAE/mAb.
[0210] These cancer cells were sensitive to the immunoconjugate
with IC50s ranging from low single digit to tens .mu.g/ml of the
conjugate. While gastric cancer cells, AGS, were very sensitive to
the MMAE conjugate with IC50 as low as 1.5 .mu.g/mL, Mantle cell
lymphoma cells, Mayer, required a higher concentration (30
.mu.g/mL) of the conjugate to reach IC50. As about three molecules
of MMAE are attached to each mAb, the IC50 values were also
presented as carried MMAE concentration as indicated. IC50s of
mariptase-negative cells such BT549 and H1299 were also obtained to
demonstrate selective cytotoxicity of the conjugate (FIG. 14). The
ratios of IC50s of matriptase-positive and -negative cells were
quite significant ranging from 18 to 48 (H1299 vs H322, and BT549
vs MDA-MB231), suggesting that the degree of selectivity of
M69-MMAE should offer benefit of minimized side effects.
TABLE-US-00002 TABLE 2 IC50s of M69-MMAE toward various cancer
cells IC50s (.mu.g/mL), IC50s (nM), Cancer Type Cell Line M69-MMAE
conc. MMAE conc. Breast MDA-MB231 2.6 .+-. 0.09 59.2 MDA-MB468 3.4
.+-. 0.30 77.5 BT549 125.0 .+-. 10.25 2847.5 Prostate DU145 8.0
.+-. 3.4 182.2 PC3 6.8 .+-. 0.65 154.9 Non-small cell H322 6.3 .+-.
0.20 143.5 Lung H1299 113 .+-. 5.20 2574.1 Ovary Ovcar5 16.0 .+-.
0.4 364.5 Pancreatic PANC1 12.0 .+-. 0.06 273.4 MiaPacal 8.0 .+-.
0.83 182.2 Stomach AGS 1.5 .+-. 0.03 34.2 Mantle Cell Mino 6.2 .+-.
0.18 141.2 Lymphoma JeKo-1 13.9 .+-. 3.9 316.6 Z138 19.0 .+-. 1.9
432.8 Maver 30.0 .+-. 2.8 683.4
Example 12
In Vivo Xenograph Studies Involving M69-MMAE Immunoconjugates
[0211] A pilot xenograft study was conducted using the M69-MMAE
immunoconjugate as described in Example 10. A toxicity study in
nude mice with the mouse conjugate (N=6), administered i.p. weekly
.times.3, at 10 mg/kg was conducted prior to testing the chimeric
M69-MMAE conjugate. There was no weight loss or signs of toxicity,
indicating that the construct was stable and did not target normal
tissues. Based on this information, mice bearing the human triple
negative breast tumor MDA-MB-468 were treated with M69-MMAE
immunoconjugates. FIG. 13 shows that the conjugate has potent
anticancer activity against this triple negative breast cancer,
importantly; there was no weight loss or other evidence of toxicity
in the animals, indicating that no significant free drug was
released into the circulation from the conjugate. As this is a
mouse antibody in the mouse, an antibody response to matriptase
would not be expected. FIG. 14 shows the results of median tumor
volume (in mm.sup.3) for the control mice and those mice treated
with M69-MMAE immunoconjugates; a significant increase in tumor
size was observed in the control group relative to those treated
with M69-MMAE.
[0212] Based on these results, further in vivo xenograft studies
were warranted. These additional xenograft studies were performed
to assess the in vivo efficacy of M69-MMAE toward various types of
cancer. TNBC (MDA-MB-468 and MDA-MB-231), prostate cancer cells
(DU145), and NSCL cancer cells (H322) were harvested in the
exponential growth phase with trypsin solution and washed with PBS.
Viable cells (7.times.10.sup.6) in 100 .mu.l of PBS suspension were
mixed with 100 .mu.l of Matrigel (BD Biosciences) and injected
subcutaneously at right flank of the animals Once the tumors were
palpable, the mice were randomized into treatment groups of six
mice each. The animal received intraperitoneal injection of 5 mg/kg
M69-MMAE or 1 mg/kg M69-MMAE twice weekly. Tumor size was measured
with digital calipers and was calculated using the formula
4/3.pi..times.length.times.width.sup.2. The results showed
effective regression of the triple-negative breast tumors
(MDA-MB468 and MDA-MB-231) by the treatment of M69-MMAE (FIG. 15A).
Tumor growth of both NSCL and prostate cancer mice was also greatly
abated compared the one of the vehicle controls (FIG. 15B,C). The
antitumor activity of M69-MMAE remains effective at the dose as low
as 1 mg/kg (FIG. 15B). Surprisingly, there was no weight loss or
other evidence of toxicity in the animals, indicating that no
significant free drug was released into the circulation from the
conjugate (FIG. 15A).
Example 13
Prostate Cancer Cells are More Sensitive to M69-MMAE Under Acidic
Extracellular pH
[0213] Activation of matriptase is induced by acidosis. Thus,
cancer cells expressing activated matriptase are more sensitive to
the cytotoxic effect of the M69-MMAE immunoconjugates as described
in Example 10 under acidic condition due to increased antigen
levels. This was proven by an experiment wherein both DU145 and PC3
prostate cancer cells become more sensitive to the immunoconjugate
with HCl-acidified medium at a pH 6.5. The IC50s were significantly
reduced from 8 to 1 .mu.g/ml, and from 6.8 to less than 2 .mu.g/mL
for DU145 and PC3 cells respectively (FIG. 16). The exposures to
acidic condition were only maintained about 30 min, as the
carbonate buffering of the medium gradually brought up the pH after
1 hour in CO2 incubator.
[0214] Tumor microenviroments are characterized by acidic
extracellular pH (pHe) due to accumulation of lactic acid resulted
from high glycolic activity of cancer cells regardless of
oxygenation condition, a phenomena also called "Warburg effect".
Accumulating evidence show that high lactate levels is a main
driving force for invasive and metastatic progression of tumor as
well as tumor recurrences. Thus, M69-MMAE allow selective delivery
of a potent toxin to cancer cells, such as prostate cancer cells,
that express activated matriptase in an acidic environment.
Example 14
[0215] Taxatere-resistant prostate cancer cells (PC3R) are
sensitive to M69-MMAE
[0216] To determine in vitro efficacy of the M69-MMAE
immunoconjugates as described in Example 10 to chemo-resistant
cancer cells, taxtere-resistant cells were treated with the
conjugate. Despite the resistance to mitotic inhibition, PC3R cells
were as sensitive as the parental PC cells to MMAE conjugate (FIG.
17). This highlights how M69-MMAE is an effective treatment for
patients with chemo-resistant prostate cancer.
[0217] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the scope and spirit of the present invention. Therefore, it
should be understood that various embodiments of the invention
described herein are illustrative only and not intended to limit
the scope of the invention. All references cited herein are
incorporated by reference in their entirety.
* * * * *