U.S. patent application number 12/620050 was filed with the patent office on 2010-07-22 for methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists.
Invention is credited to Gregory R. Mundy, Toshiyuki Yoneda.
Application Number | 20100183599 12/620050 |
Document ID | / |
Family ID | 46278871 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100183599 |
Kind Code |
A1 |
Mundy; Gregory R. ; et
al. |
July 22, 2010 |
Methods of treating multiple myeloma and myeloma-induced bone
resorption using integrin antagonists
Abstract
Antagonists of .alpha.4 integrin/.alpha.4 integrin ligand
adhesion, which inhibit the biological effects of such adhesion are
described and methods for their use are detailed. Such antagonists
are useful in suppressing bone destruction associated with multiple
myeloma. The homing of multiple myeloma cells to bone marrow and
their .alpha.4 integrin-dependent release of bone-resorbing
factors, resulting in bone destruction in patients with multiple
myeloma, is inhibited.
Inventors: |
Mundy; Gregory R.; (San
Antonio, TX) ; Yoneda; Toshiyuki; (San Antonio,
TX) |
Correspondence
Address: |
STEPTOE & JOHNSON LLP
1330 CONNECTICUT AVE., NW
WASHINGTON
DC
20036
US
|
Family ID: |
46278871 |
Appl. No.: |
12/620050 |
Filed: |
November 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10086217 |
Feb 21, 2002 |
7618630 |
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12620050 |
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09943659 |
Aug 31, 2001 |
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10086217 |
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09805840 |
Mar 13, 2001 |
7211252 |
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09943659 |
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PCT/US99/21170 |
Sep 13, 1999 |
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09805840 |
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60100182 |
Sep 14, 1998 |
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Current U.S.
Class: |
424/133.1 ;
424/144.1; 424/173.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/2836 20130101; C07K 14/70542 20130101; C07K 16/2842
20130101; C07K 16/2839 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/173.1; 424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00 |
Claims
1-50. (canceled)
51. A method for inhibiting osteoclastogensis, the method
comprising administering to an individual a therapeutically
effective amount of a composition comprising an antagonist of an
interaction between an .alpha.4 subunit-bearing integrin and a
ligand for an .alpha.4 subunit bearing integrin, wherein the
antagonist is an antagonist of VLA-4.
52. A method for inhibiting osteoclastogensis, the method
comprising administering to an individual a therapeutically
effective amount of a composition comprising an antagonist of an
interaction between an .alpha.4 subunit-bearing integrin and a
ligand for an .alpha.4 subunit bearing integrin and a compound.
53. The method according to claim 52, wherein the compound is a
chemotherapeutic agent.
54. The method according to claim 51, wherein the antagonist is an
anti-VLA4 antibody, or antigen-binding fragment thereof.
55. The method according to claim 52, wherein the antagonist is an
anti-VLA4 antibody, or antigen-binding fragment thereof.
56. The method according to claim 52, wherein the compound is
selected from the group consisting of melphalan, a bisphosphonate,
thalidomide, erythropoietin, an antagonist of IL-6 and an
antagonist of IL-15.
57. The method according to claim 55, wherein the compound is
melphalan.
58. The method according to claim 54, wherein the antibody or
antigen-binding fragment thereof, is a monoclonal antibody or
antigen-binding fragment thereof.
59. The method according to claim 55, wherein the antibody or
antigen-binding fragment thereof, is a monoclonal antibody or
antigen-binding fragment thereof.
60. The method according to claim 54, wherein the anti-VLA-4
antibody or antigen-binding fragment thereof is selected from the
group consisting of a human antibody, a chimeric antibody, a
humanized antibody and an antigen-binding Fab, Fab', F(ab').sub.2
or F(v) fragment of a human, chimeric or humanized antibody.
61. The method according to claim 55, wherein the anti-VLA-4
antibody or antigen-binding fragment thereof is selected from the
group consisting of a human antibody, a chimeric antibody, a
humanized antibody and an antigen-binding Fab, Fab', F(ab').sub.2,
or F(v) fragment of a human, chimeric or humanized antibody.
62. The method of claim 52, wherein the antagonist is administered
at a dosage that is lower when administered in combination with the
compound than when not administered in combination with the
compound.
63. The method of claim 52, wherein the compound is administered at
a dosage that is lower when administered in combination with the
antagonist than when not administered in combination with the
antagonist.
64. The method of claim 52, wherein the antagonist is administered
at a dosage that is lower when administered in combination with the
compound than when not administered in combination with the
compound; and wherein the compound is administered at a dosage that
is lower when administered in combination with the antagonist than
when not administered in combination with the antagonist.
65. The method of claim 54, wherein the anti-VLA4 antibody or
antigen-binding fragment thereof, binds the .alpha. chain of
VLA-4.
66. The method of claim 55, wherein the anti-VLA4 antibody or
antigen-binding fragment thereof, binds the .alpha. chain of
VLA-4.
67. The method of claim 54, wherein the anti-VLA4 antibody or
antigen-binding fragment thereof, is a B epitope specific
anti-VLA-4 antibody or antigen-binding fragment thereof.
68. The method of claim 55, wherein the anti-VLA4 antibody or
antigen-binding fragment thereof, is a B epitope specific
anti-VLA-4 antibody or antigen-binding fragment thereof.
69. The method of claim 54, wherein the anti-VLA4 antibody or
antigen-binding fragment thereof, is a humanized anti-VLA-4
antibody or antigen-binding fragment thereof.
70. The method of claim 55, wherein the anti-VLA4 antibody or
antigen-binding fragment thereof, is a humanized anti-VLA-4
antibody or antigen-binding fragment thereof.
Description
[0001] This utility application is a continuation-in-part of U.S.
application Ser. No. 09/943,659, filed Aug. 31, 2001, which is a
continuation-in-part of U.S. application Ser. No. 09/805,840, filed
Mar. 13, 2001, which is a continuation of PCT application number
PCT/US99/21170, filed Sep. 13, 1999, which claims benefit of U.S.
provisional application No. 60/100,182, filed Sep. 14, 1998. The
disclosures of U.S. application Ser. Nos. 09/943,659 and
09/805,840, PCT application number PCT/US99/21170 and U.S.
provisional application No. 60/100,182 are incorporated herein by
reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to a treatment for multiple
myeloma, and the release of bone-resorting factors by myeloma
cells, resulting in severe bone loss, which is the major
side-effect of myeloma in man. More particularly, this invention
relates to integrin antagonists, such as antagonists of alpha4
containing integrins, which inhibit the biological effects of such
adhesion, associated with homing of multiple myeloma cells to bone
marrow; their subsequent integrin-dependent survival; and their
integrin-dependent release of bone-resorbing factors, resulting in
bone destruction in patients with multiple myeloma.
BACKGROUND OF THE INVENTION
[0003] Multiple myeloma is a B-cell malignancy that has strong
predilection for colonizing the bone marrow and is associated with
severe osteoclastic bone resorption. Multiple myeloma is the second
most common hematologic malignancy, with 15,000 new cases diagnosed
each year and 30,000 to 40,000 myeloma patients in the U.S.
annually (Mundy and Bertolini 1986). Eighty percent of the patients
suffer from devastating osteolytic bone destruction caused by
increased osteoclast (OCL) formation and activity (Mundy and
Bertolini 1986). This bone destruction can cause excruciating bone
pain, pathologic fractures, spinal cord compression, and
life-threatening hypercalcemia. Because multiple myeloma cannot be
cured by standard chemotherapy or stem cell transplantation (Attal
et al., 1996), and because of the severe morbidity and potential
mortality associated with myeloma bone disease, treatment
strategies that control the myeloma growth itself, and in
particular the osteolytic bone destruction that occurs in these
patients, are vitally important.
[0004] However, the pathologic mechanisms responsible for the
increased osteoclast activity in patients with multiple myeloma are
unknown (Mundy, 1998). The bone lesions occur in several patterns.
Occasionally, patients develop discrete osteolytic lesions that are
associated with solitary plasmacytomas. Some patients have diffuse
osteopenia, which mimics the appearance of osteoporosis, and is due
to the myeloma cells being spread diffusely throughout the axial
skeleton. In most patients there are multiple discrete lytic
lesions occurring adjacent to nests of myeloma cells. Hypercalcemia
occurs as a consequence of bone destruction in about one-third of
patients with advanced disease. Rarely, patients with myeloma do
not have lytic lesions or bone loss, but rather have an increase in
the formation of new bone around myeloma cells. This rare situation
is known as osteosclerotic myeloma.
[0005] Osteolytic bone lesions are by far the most common skeletal
manifestations in patients with myeloma (Mundy, 1998). Although the
precise molecular mechanisms remain unclear, observations over 15
years have shown that: 1) The mechanism by which bone is destroyed
in myeloma is via the osteoclast, the normal bone-resorbing cell;
2) Osteoclasts accumulate on bone-resorbing surfaces in myeloma
adjacent to collections of myeloma cells and it appears that the
mechanism by which osteoclasts are stimulated in myeloma is a local
one; 3) It has been known for many years that cultures of human
myeloma cells in vitro produce several osteoclast activating
factors; including lymphotoxin-alpha (LT-.alpha.), interleukin-1
(IL-1), parathyroid-hormone related protein (PTHrP) and
interleukin-6 (IL-6); 4) Hypercalcemia occurs in approximately
one-third of patients with myeloma some time during the course of
the disease. Hypercalcemia is always associated with markedly
increased bone resorption and frequently with impairment in
glomerular filtration; 5) The increase in osteoclastic bone
resorption in myeloma is usually associated with a marked
impairment in osteoblast function. Alkaline phosphatase activity in
the serum is decreased or in the normal range, unlike patients with
other types of osteolytic bone disease, and radionuclide scans do
not show evidence of increased uptake, indicating impaired
osteoblast responses to the increase in bone resorption.
[0006] Although various mediators listed above have been implicated
in the stimulation of osteoclast activity in patients with multiple
myeloma, reports of factors produced by myeloma cells have not been
consistent, and some studies have been inconclusive due to the
presence of other contaminating cell types, including stromal cells
and macrophages, in the multiple myeloma cell population. IL-6 is a
major myeloma growth factor that enhances the growth of several
myeloma cell lines and freshly isolated myeloma cells from patients
(Bataille et al., 1989). IL-6 production can be detected in about
40% of freshly isolated myeloma cells by PCR, but only 1 in 150
patients studied show detectable IL-6 production by
immunocytochemistry or ELISA assays (Epstein 1992). The IL-6
receptors were only detected in 6 of 13 samples from patients with
multiple myeloma (Bataille et al., 1992). Furthermore, mature
myeloma cells have been reported to have a minimal proliferative
response to IL-6. Interleukin-11 (IL-11) has an IL-6-like activity
on plasmacytomas, but to date no one has demonstrated that myeloma
cells produce IL-11. Bataille and coworkers (1995) have shown that
perfusion of 5 patients with refractory myeloma with an antibody to
IL-6 decreased the size of the myeloma cell burden in only 2 of
these patients. IL-1 is an extremely potent bone resorbing agent
that induces hypercalcemia in animal models in the absence of renal
failure (Boyce et al., 1989). In contrast, hypercalcemia rarely
occurs in myeloma patients without renal failure. More importantly,
in highly purified myeloma cells, no IL-1 and only rare TNF-.alpha.
production can be detected, suggesting that other contaminating
cell types such as macrophages may be the source of IL-1 and
TNF-.alpha. (Epstein 1992). Similarly, LT-.alpha. is produced by
most human myeloma cell lines (Bataille et al., 1995) but does not
appear to be produced by myeloma cells in vivo (Alsina et al.,
1996). In addition to IL-1, TNF-.alpha., LT-.alpha., and IL-6,
myeloma cells produce a truncated form of M-CSF which is
biologically active, but M-CSF does not cause hypercalcemia or
induce osteoclast formation by itself in human marrow cultures
(MacDonald et al., 1986).
[0007] Thus, the role of any of these factors in osteolytic bone
disease in patients with myeloma has not been clearly demonstrated
in vivo, so that known cytokines clearly do not totally account for
the bone resorption seen in these patients.
Role of Adhesive Molecule
Interactions in Myeloma Bone Disease
[0008] Anderson and coworkers were the first group to demonstrate
the importance of adhesive interactions between myeloma cells and
cells in the marrow microenvironment both in the growth of myeloma
cells and the development of osteolytic bone disease. Multiple
myeloma cells express cell surface adhesion molecules, CD29
(VLA-4), LFA-1, and CD44 (Chauhan et al., 1995). These workers
suggested that myeloma cells localized to the marrow via specific
adhesion interactions between extracellular matrix proteins and
bone marrow stromal cells. They further showed that adhesion of
multiple myeloma cells to stromal cells triggered IL-6 secretion by
both normal and multiple myeloma bone marrow-derived stromal cells
and increased IL-6-mediated tumor cell growth. However, antibodies
to CD29, LFA-1 or CD44 did not decrease IL-6 production by marrow
stromal cells in response to myeloma cells, suggesting that another
ligand-receptor interaction triggered the IL-6 secretion by bone
marrow stromal cells binding to myeloma cells. Mere identification
of a possible adhesion pathway does not necessarily mean that the
pathway is important. In this case none of the implicated pathways
plays a role in IL-6 production.
[0009] Vanderkerken et al. (1997) also examined the phenotypic
adhesion profile of murine 5T2 cells and ST33 myeloma cells in a
model of murine myeloma. These investigators showed that these cell
lines expressed VLA-4, VLA-5, LFA-1, and CD44, and suggested that
these adhesive interactions might be important for myeloma cells to
bind to marrow stromal cells.
[0010] Nevertheless, despite many laboratory advances, the
fundamental mechanisms underlying increased osteoclastic bone
destruction in myeloma in vivo remain poorly understood. This is
reflected in the inability to easily translate the data on adhesive
interactions obtained in vitro to the in vivo setting. For example,
many in vitro studies implicate both the integrin VLA-4 and the
integrin LFA-1 in the adhesion of hematopoietic stem cells to bone
marrow stroma (reviewed in Papayannopoulou and Nakamoto, 1993).
These in vitro data would predict that either pathway, if blocked
in vivo, would result in peripheralization of hematopoietic stem
cells from marrow to peripheral blood. Yet, in a primate study,
while a monoclonal antibody (mAb) to VLA-4 effectively
peripheralized stem cells, a monoclonal antibody to the beta2
integrin chain of LFA-1 was without effect, despite increasing
neutrophil counts, thus demonstrating the efficacy of the mAb
(Papayannopoulou and Nakamoto, 1993). These data show that the in
vitro results were in fact unable to accurately predict in vivo
relevance.
[0011] It should be noted that the role of integrin VLA-4 has been
studied in metastasis of multiple tumors, including leukemias such
as lymphoma, with contradictory results. Thus, transfection of the
human alpha 4 chain into Chinese Hamster Ovary (CHO) cells resulted
in VLA-4 expression, and rendered these cells able to migrate to
bone marrow in vivo, a phenomenon inhibited by mAbs to VLA-4
(Matsuura et al., 1996). In contrast, transfection of lymphoma
cells with VLA-4 strongly inhibited metastasis to liver, lung and
kidney, and was without effect on homing and proliferation in
marrow (Gosslar et al., 1996). In addition, expression of VLA-4 on
highly metastatic murine melanoma cells strongly inhibited the
formation of pulmonary metastases in vivo (Qian et al., 1994), and
did not predispose melanoma to bone marrow metastasis.
[0012] In summary it is not clear on the basis of in vitro studies,
how to reliably predict in vivo relevance of adhesion pathways.
Furthermore, even when in vivo studies have been performed, the
resultant data are inconsistent. One major reason for the
perplexing inconsistencies in the field of multiple myeloma is that
currently available animal models are not good predictors of human
disease. In the case of multiple myeloma, human and murine myeloma
cell lines which can be grown in vitro rarely are associated with
bone destruction in vivo (Mundy 1998).
[0013] It would be highly desirable to identify compounds or
antagonists which inhibit production of these bone-resorbing
factors, thus halting progressive bone destruction and improving
the quality of life of patients with myeloma.
SUMMARY OF THE INVENTION
[0014] We have used a recently developed murine model of multiple
myeloma in which the mouse develops severe osteolysis with all the
hallmarks of human disease (Garrett 1997). Using this cell line and
animal model we have established that inhibition of the .alpha.4
integrin/.alpha.4 integrin ligand pathway in vivo leads to reduced
capacity for multiple myeloma cells to proliferate and/or survive.
We show that cell-cell attachment between myeloma cells and marrow
stromal cells via the VLA-4/VCAM-1 interaction is required for an
increase in the production of an activity which stimulates
osteoclastic bone resorption in the bone microenvironment in
vitro.
[0015] We propose that this interaction is critical to the homing
of myeloma cells to the marrow compartment, to their subsequent
survival and growth, to ultimately to the progression of
myeloma-induced osteolysis. We tested this in the animal model and
found that, in vivo, an antagonist of the alpha4 subunit-containing
integrin VLA-4 strongly inhibits the production of antibody of the
IgG2b subtype. This isotype is the same as that produced by the
5TGM1 cell line, and is an accurate surrogate for the number of
myeloma cells in the marrow compartment at any time. Thus, blockade
of the VLA-4 pathway strongly inhibits IgG2b production, and by
implication, the level of myeloma burden.
[0016] One aspect of the invention is a method for treating
multiple myeloma comprising administering to an individual a
therapeutically effective amount of a composition comprising an
antagonist of an interaction between an integrin with an .alpha.4
subunit (e.g., VLA-4) and a ligand for this integrin (e.g.,
VCAM-1). This antagonist can be an .alpha.4 integrin binding agent
or an .alpha.4 integrin ligand binding agent. Preferred agents are
anti-VLA4 or anti-.alpha.4.beta.7 antibody homologs (human
antibody, a chimeric antibody, a humanized antibody and fragments
thereof); anti-VCAM-1 antibody homologs (a human antibody; a
chimeric antibody, a humanized antibody and fragments thereof); and
a small molecule inhibitor of interactions of .alpha.4 subunit
containing integrins with their ligands. The composition can be
administered at a dosage so as to provide from about 0.1. to about
20 mg/kg body weight. In particular, the preferred agents can
antagonize an interaction: a) of both VLA-4 and .alpha.4.beta.7
collectively with their respective .alpha.4 ligands; or b) only of
VLA-4 with its .alpha.4 ligand; or c) only of .alpha.4.beta.7 with
its .alpha.4 ligand. One or more antagonists of an interaction
between an integrin with an .alpha.4 subunit and a ligand for this
integrin could be administered in combination with one or more
compounds that preferably are not an antagonist of an interaction
between an integrin with an .alpha.4 subunit and a ligand for this
integrin. Preferably, the compound to be administered in
combination with an antagonist of an interaction between an
integrin with an .alpha.4 subunit and a ligand for this integrin is
a chemotherapeutic agent. Preferably, that chemotherapeutic agent
is melphalan.
[0017] Another aspect of the invention is a method for inhibiting
bone resorption associated with tumors of bone marrow, the method
comprising administering to a mammal with said tumors an antagonist
of an interaction between an .alpha.4 subunit containing integrin
such as VLA-4 and a ligand for this .alpha.4 subunit containing
integrin, such as VCAM-1, in an amount effective to provide
inhibition of the bone resorption. This antagonist can be an
.alpha.4 integrin binding agent such as a VLA-4 binding agent or an
.alpha.4 integrin ligand binding agent such as a VCAM-1 binding
agent. Preferred agents are anti-VLA4 or and .alpha.4.beta.7
antibody homologs (human antibody, a chimeric antibody, a humanized
antibody and fragments thereof); anti-VCAM-1 antibody homologs (a
human antibody, a chimeric antibody, a humanized antibody and
fragments thereof); and a small molecule inhibitor of the
interaction of .alpha.4 subunit-containing integrins with their
respective .alpha.4 integrin ligands (e.g, the VCAM-1/VLA-4
interaction). The antagonist can be administered at a dosage so as
to provide from about 0.1 to about 20 mg/kg body weight. One or
more antagonists of an interaction between an integrin with an
.alpha.4 subunit and a ligand for this integrin could be
administered in combination with one or more compounds that
preferably are not an antagonist of an interaction between an
integrin with an .alpha.4 subunit and a ligand for this integrin.
Preferably, the compound to be administered in combination with an
antagonist of an interaction between an integrin with an .alpha.4
subunit and a ligand for this integrin is a chemotherapeutic agent.
Preferably, that chemotherapeutic agent is melphalan.
[0018] Yet another aspect of the invention is a method of treating
a subject having a disorder characterized by the presence of
osteoclastogenesis, the method comprising administering to the
subject an antagonist of an interaction between an .alpha.4 subunit
bearing integrin and a ligand for an .alpha.4 subunit-bearing
integrin, in an amount sufficient to suppress the
osteoclastogenesis. Similarly, the antagonist can be an .alpha.4
binding agent or an .alpha.4 ligand binding agent. Preferred agents
are anti-VLA4 or anti-.alpha.4.beta.7 antibody homologs (human
antibody, a chimeric antibody, a humanized antibody and fragments
thereof); anti-VCAM-1 antibody homologs (a human antibody, a
chimeric antibody, a humanized antibody and fragments thereof); and
a small molecule inhibitor of the interaction of .alpha.4
subunit-containing integrins with their respective .alpha.4
integrin ligands (e.g, the VCAM-1/VLA-4 interaction). The
composition can be administered at a dosage so as to provide from
about 0.1 to about 20 mg/kg body weight. One or more antagonists of
an interaction between an integrin with an .alpha.4 subunit and a
ligand for this integrin could be administered in combination with
one or more compounds that preferably are not an antagonist of an
interaction between an integrin with an .alpha.4 subunit and a
ligand for this integrin. Preferably, the compound to be
administered in combination with an antagonist of an interaction
between an integrin with an .alpha.4 subunit and a ligand for this
integrin is a chemotherapeutic agent. Preferably, that
chemotherapeutic agent is melphalan.
[0019] Unless stipulated otherwise, all references are incorporated
herein by reference.
[0020] Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising," will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1. Effect of Neutralizing Antibodies on TRAP-positive
Multinucleated OC-like Cell Formation in the Co-cultures of 5TGM1
cells and Bone Marrow Cells
[0022] A mixture of 5TGM1 cells (1 e 3) and marrow cells (1 e 6) in
suspension was plated in 48-well culture plates and cultured with
or without 10 .mu.g/ml anti-VCAM-1 antibody (VCAM-1 Ab),
anti-.alpha.4betal antibody (.alpha.4.beta.11Ab), anti-ICAM-1
antibody (ICAM-1 Ab) or rat IgG as a control. After 6 days of
culture, cultures were fixed and the number of TRAP-positive
multinucleated OC-like cells (TRAP(+) MNC) determined. Both VCAM-1
Ab and .alpha.4betal Ab inhibited TRAP(+) MNC formation, while
ICAM-1 Ab had no effect. Data are expressed as mean.+-.S.E. (n=3).
*=Significantly different from IgG control.
[0023] FIG. 2 Effect of 5TGM1 and ST2 Conditioned Media on Bone
Resorption in Organ Cultures of Fetal Rat Long Bones
[0024] Conditioned media (48 hours) obtained from ST2 alone, 5TGM1
alone, and co-cultures of ST2 and 5TGM1 were assayed for bone
resorbing activity in organ cultures of .sup.45calcium-labeled
fetal rat long bones. Labeled fetal rat long bones were cultured in
the presence of conditioned media (40%6v/v) or control medium for
120 hours. Data are expressed as percentage increase of calcium
release over than in the control medium. Release from conditioned
medium of ST2 stromal cells is shown as the open bar. Release from
5TGM1 is the hatched bar. Release from conditioned medium harvested
from co-culture of 5TGM1 and ST2 is the closed bar. Data are
expressed as mean.+-.S.E. (n=4). *=significantly different from ST2
alone. ***=significantly different from 5TGM1 alone.
[0025] FIG. 3 Effect of Recombinant Soluble VCAM-1 (sVCAM-1) on the
Production of Osteoclastogenic Activity by 5TGM1 Cells
[0026] Conditioned medium was harvested from 5TGM1 cells cultured
in the presence or absence of sVCAM-1 (1.times.10.sup.-8 to
1.times.10.sup.-7 Molar) for 24 hours. Osteoclastogenic activity of
these conditioned media was assayed in the mouse marrow cultures.
Bone marrow cells (1e6/well) were plated into 48-well plates, and
cultured in the presence of conditioned media (hatched bars) or
control medium (IMDM) containing the same concentrations of sVCAM-1
(open bars). After 6 days, cultures were fixed and the number of
TRAP-positive multinucleated OC-like cells (TRAP+MNC) was
determined. Conditioned medium from 5TGM1 cells treated with
1.times.10.sup.-7 MsVCAM-1 increased TRAP(+)MNC formation. Data are
expressed as mean.+-.S.E. (n=3). *=significantly different from
controls.
[0027] FIG. 4 Effect of mAb PS2 to VLA-4 on serum IgG2b elevation
in 5TGM1-bearing mice
[0028] Mice were injected with 1e5 5TGM1 cells, which were allowed
to colonize the bone marrow. Mice were split into two groups of
three, one serving as a control group, and the second treated on
days 8, 11, 14, 17, and 20 with 80 .mu.g mAb PS/2 (.about.4 mg/kg).
Levels of IgG2b, the antibody isotype produced by 5TGM1 myeloma
cells, were measured weekly from weeks 1 to 6. mAb treatment
strongly inhibited IgG2b production, indicative of inhibition of
myeloma cell survival and growth in vivo.
[0029] FIG. 5 Effect of mAb M/K-2.7 to VCAM-1 on serum IgG2b
elevation in 5TGM1-bearing mice
[0030] Mice were injected with 5TGM1 cells as described in FIG. 4,
which were allowed to colonize the bone marrow. Mice were split
into groups of four or five, one group serving as a control group
(open square), the second/third groups treated prophylactically at
80 .mu.g (open diamonds) and 160 .mu.g mAb (open circles) (.about.4
to 8 mg/kg), the fourth group treated therapeutically at 160 .mu.g
mAb (triangles). Levels of IgG2b, the antibody isotype produced by
5TGM1 myeloma cells, were measured. mAb treatment strongly
inhibited IgG2b production, indicative of inhibition of myeloma
cell survival and growth in vivo.
[0031] FIG. 6 Effect of anti-.alpha.4 Integrin Antibody on Survival
of Multiple Myeloma-bearing Mice
[0032] FIG. 7 depicts an experimental protocol of an in vivo
combination therapy experiment.
[0033] FIG. 8: Serum IgG.sub.2 Levels in 5TGM1-bearing Mice
[0034] FIG. 8 shows the results (serum IgG2b levels in mice bearing
5TGM-1 cells) of an in vivo combination therapy experiment.
[0035] Ctr=Control. Mel=melphalan. .alpha.4Ab=anti-.alpha.4Ab.
[0036] FIG. 9: Tumor Volume in Bone
[0037] FIG. 9 shows the results (tumor volume in bone) of an in
vivo combination therapy experiment.
[0038] Ctr=Control. Mel=melphalan. .alpha.4Ab=anti-.alpha.4Ab.
[0039] FIG. 10: Effect of PS/2 mAb Treatment on Spleen Weight
[0040] FIG. 10 shows the results (spleen weight) of an experiment
relating to PS/2 mAb administration in an in vivo model system for
multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0041] FIG. 11: Effect of PS/2 Treatment on Splenocytes in Mice
with 5TGM1 Myeloma
[0042] FIG. 11 shows the results (percentage of myeloma cells in
the spleen) of an experiment relating to PS/2 mAb administration in
an in vivo model system for multiple myeloma. The mice of this
model system were immuno-competent C57BL/KaLwRij mice.
[0043] FIG. 12: Effect of PS/2 mAb Treatment on Splenocyte FACS
Profile
[0044] FIG. 12 shows the representative FACS plots of the staining
for lineage markers and cytoplasmic mouse IgG2b in Splenocytes of
an experiment relating to PS/2 mAb administration in an in vivo
model system for multiple myeloma. Four panels are shown: disease
free; untreated; PS/2 treated and rat IgG2b treated. The mice of
this model system were immuno-competent C57BL/KaLwRij mice.
[0045] FIG. 13: Effect of PS/2 mAb Treatment on Spleen Tumor
Burden
[0046] FIG. 13 shows the results (spleen tumor burden) of an
experiment relating to PS/2 mAb administration in an in vivo model
system for multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0047] FIG. 14: Effect of PS/2 mAb Treatment on Bone Marrow Cells
in Mice with 5TGM1 Myeloma
[0048] FIG. 14 shows the results (effects on bone marrow cells) of
an experiment relating to PS/2 mAb administration in an in vivo
model system for multiple myeloma. The mice of this model system
were immuno-competent C57BL/KaLwRij mice.
[0049] FIG. 15: Effect of PS/2 mAb Treatment on Bone Marrow Tumor
Burden mIgG2b Positive Cells
[0050] FIG. 15 shows the results (bone marrow tumor burden) of an
experiment relating to PS/2 mAb administration in an in vivo model
system for multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0051] FIG. 16: Effect of PS/2 mAb Treatment on 5TGM1 Cells in the
Blood
[0052] FIG. 16 shows the results (effects on 5TGM1 myeloma cells in
peripheral blood) of an experiment relating to PS/2 mAb
administration in an in vivo model system for multiple myeloma. The
mice of this model system were immuno-competent C57BL/KaLwRij
mice.
[0053] FIG. 17A; FIG. 17B and FIG. 17C: Effect of PS/2 Treatment on
Blood Chemistry
[0054] FIGS. 17A, 17B and 17C show the results (effects on blood
chemistry: LDH levels (FIG. 17A); AST levels (FIG. 17B) and
phosphorus levels (FIG. 17C)) of an experiment relating to PS/2 mAb
administration in an in vivo model system for multiple myeloma. The
mice of this model system were immuno-competent C57BL/KaLwRij
mice.
[0055] LDH=lactate dehydrogenase. AST=aspartate
aminotransferase.
[0056] FIG. 18: Effect of PS/2 mAb on Circulation mIgG2b Levels
[0057] FIG. 18 shows the results (effects on circulating mIgG2b
levels at the conclusion of the experiment) of an experiment
relating to PS/2 mAb administration in an in vivo model system for
multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0058] FIG. 19: C57BL/KaLwRij Multiple Myeloma Model Acute
Treatment Protocols
[0059] FIG. 19 depicts an experimental acute treatment protocol of
an experiment relating to PS/2 mAb administration in an in vivo
model system for multiple myeloma. The mice of this model system
were immuno-competent C57BL/KaLwRij mice.
[0060] FIG. 20A and FIG. 20E: Effect of PS/2 Treatment on IgG2b
Positive Cells Acute Treatment Model
[0061] FIGS. 20A and 20B show the results (effects on IgG2b
positive cells; 1 day treatment (FIG. 20A) and 2 day treatment
(FIG. 20B)) of an acute treatment protocol of an experiment
relating to PS/2 mAb administration in an in vivo model system for
multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0062] FIG. 21: Effect of Anti-VLA4-Antibody PS/2 on Tumor Burden
in Spleen
[0063] FIG. 21 shows the results (effects on tumor burden in
spleen; 1 and 2 day treatments) of an acute treatment protocol of
an experiment relating to PS/2 mAb administration in an in vivo
model system for multiple myeloma. Three panels are shown. The
panels are, from left to right, total cells, IgG2b negative cells
and IgG2b positive cells. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0064] FIG. 22: Effect of 6 Days of PS/2 Treatment
[0065] FIG. 22 shows the results (effects on IgG2b positive cells;
6 day treatments) of an acute treatment protocol of an experiment
relating to PS/2 mAb administration in an in vivo model system for
multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0066] FIG. 23: Effect of 6 Days of PS/2 Treatment on Bone Marrow
cells
[0067] FIG. 23 shows the results (effects on tumor burden in bone
marrow; 6 day treatments) of an acute treatment protocol of an
experiment relating to PS/2 mAb administration in an in vivo model
system for multiple myeloma. The mice of this model system were
immuno-competent C57BL/KaLwRij mice.
[0068] BM cells=bone marrow cells.
[0069] FIG. 24: C57BL/KaLwRij Model of Multiple Myeloma Combination
Treatment with PS/2 and Melphalan
[0070] FIG. 24 depicts an experimental protocol of an in vivo
combination therapy experiment in immunocompetent C57BL/KaLwRij
mice.
[0071] rIgG2b isotype control=rat IGg2b isotype control.
[0072] FIG. 25: Effect of Treatment on mIgG2b Plasma Levels
[0073] FIG. 25 shows the results that melphalan and PS/2 mAb
together, but not either alone, reduced mIgG2b levels in an in vivo
combination therapy experiment in immunocompetent C57BL/KaLwRij
mice.
[0074] rIgG2b=rat IGg2b.
[0075] FIGS. 26A, 26B and 26C: Effect of Treatment on Myeloma Cells
at Day 27
[0076] FIGS. 26A, 26B and 26C show the results (effects of
treatment on myeloma cells at day 27: in blood (FIG. 26A); in
spleen (FIG. 26B) and in bone marrow (FIG. 26C)) of an in vivo
combination therapy experiment in immunocompetent C57BL/KaLwRij
mice.
[0077] FIGS. 27A and 27B: Effect of Treatment on Tumor Burden at
Day 27
[0078] FIGS. 27A and 27B shows the results (effects of treatment on
tumor burden at day 27: in spleen (FIG. 27A) and in bone marrow
(FIG. 27B)) of an in vivo combination therapy experiment in
immunocompetent C57BL/KaLwRij mice.
[0079] FIG. 28 shows PS/2 mAb plasma levels of an in vivo
combination therapy experiment in immunocompetent C57BL/KaLwRij
mice.
[0080] FIG. 29: C57BL/KaLwRij Model of Multiple Myeloma PS/2 and
Anti-VLA4 Small Molecule Long Term Treatment
[0081] FIG. 29 depicts an experimental protocol of an experiment
relating to BIO8809 administration in an in vivo model system
(immunocompetent C57BL/KaLwRij mouse model) for multiple
myeloma.
[0082] 8809=BIO8809. 9257=BIO9257.
[0083] FIGS. 30A, 30B and 30C: Effect of Long Term Treatment on
Myeloma Cells
[0084] FIGS. 30A, 30B and 30C show the results (effects of long
term treatment on myeloma cells: in blood (FIG. 30A), in spleen
(FIG. 30B) and in bone marrow (FIG. 30C)) of an experiment relating
to BIO8809 administration in an in vivo model system
(immunocompetent C57BL/KaLwRij mouse model) for multiple
myeloma.
[0085] 8809=BIO8809. 9257=BIO9257.
[0086] FIGS. 31A, 31B and 31C: Effect of Long Term Treatment on
Tumor Burden
[0087] FIGS. 31A, 31B and 31C show the results (effects of long
term treatment on tumor burden: in blood (FIG. 31A); in spleen
(FIG. 31B) and in bone marrow (FIG. 31C)) of an experiment relating
to BIO8809 administration in an in vivo model system
(immunocompetent C57BL/KaLwRij mouse model) for multiple
myeloma.
[0088] 8809=BIO8809. 9257=BIO9257.
[0089] FIG. 32: C57BL/KaLwRij Model of Multiple Myeloma PS/2 and
Anti-VLA4 Small Molecule Acute Treatment
[0090] FIG. 32 depicts an acute treatment protocol of an experiment
relating to BIO8809 administration in an in vivo model system
(immunocompetent C57BL/KaLwRij mouse model) for multiple
myeloma.
[0091] 8809=BIO8809. 9257=BIO9257.
[0092] FIGS. 33A, 33B and 33C: Effect of Acute Treatment on Myeloma
Cells
[0093] FIGS. 33A, 33B and 33C show the results (effects of acute
treatment on myeloma cells: in blood (FIG. 33A); in spleen (FIG.
33B) and in bone marrow (FIG. 33C)) of an experiment relating to
BIO8809 administration in an in vivo model system (immunocompetent
C57BL/KaLwRij mouse model) for multiple myeloma.
[0094] 8809=BIO8809. 9257=BIO9257.
[0095] FIGS. 34A and 34B: Effect of Acute Treatment on Tumor
Burden
[0096] FIGS. 34A and 34B show the results (effects of acute term
treatment on tumor burden: in spleen (FIG. 34A) and in bone marrow
(FIG. 34B)) of an experiment relating to BIO8809 administration in
an in vivo model system (immunocompetent C57BL/KaLwRij mouse model)
for multiple myeloma.
[0097] 8809=BIO8809. 9257=BIO9257.
[0098] FIG. 35 depicts an experimental protocol of an in vivo
combination therapy experiment relating to survival as measured by
the onset of hindleg paralysis.
[0099] FIG. 36: Inhibition of Hindleg Paralysis shows the results
of an in vivo combination therapy experiment relating to survival
as measured by the onset of hindleg paralysis.
[0100] Mel=melphalan.
DETAILED DESCRIPTION OF THE INVENTION
[0101] The invention relates to treatments for, among other things,
preventing multiple myeloma. More particularly, methods of the
invention relate to the use of antagonists of an interaction
between an integrin containing an .alpha.4 subunit and a ligand for
this integrin in the treatment of multiple myeloma. The term
"multiple myeloma" is intended to mean a medical condition in an
individual having a neoplastic disease of plasma cells, with the
neoplastic clone representing cells at different stages in the
plasma cell lineage from patient to patient (Mundy, 1998).
[0102] Alpha 4 .beta.1 integrin is a cell-surface receptor for
VCAM-1, fibronectin and possibly other molecules that bind with, or
otherwise interact with, alpha 4 .beta.1 integrin. In this regard,
such molecules that bind with, or otherwise interact with, alpha 4
subunit containing integrin are individually and collectively
referred to as ".alpha.4 ligand(s)"). The term .alpha.4.beta.1
integrin ("VLA-4" or ".alpha.4.beta.1, or ".alpha.4.beta.1
integrin", used interchangeably) herein thus refers to polypeptides
which are capable of binding to VCAM-1 and members of the
extracellular matrix proteins, most particularly fibronectin, or
homologs or fragments thereof, although it will be appreciated by
workers of ordinary skill in the art that other ligands for VLA-4
may exist and can be analyzed using conventional methods.
[0103] Nevertheless, it is known that the .alpha.4 subunit will
associate with other .beta. subunits besides .beta.1 so that we may
define the term ".alpha.4 integrin" as being those integrins whose
.alpha.4 subunit associates with one or another of the .beta.
subunits. A further example of an ".alpha.4" integrin is
.alpha.4.beta.7 (R. Lobb and M. Hemler, 1994). As used herein, the
term ".alpha.4 integrin(s)" means VLA-4, as well as integrins that
contain .beta.1, .beta.7 or any other .beta. subunit.
[0104] As discussed herein, the antagonists used in methods of the
invention are not limited to a particular type or structure of
molecule so that, for purposes of the invention, any agent capable
of binding to any integrin containing an .alpha.4 subunit such as
VLA-4 on the surface of VLA-4 bearing cells and/or .alpha.4.beta.7
integrin on the surface of .alpha.4.beta.7-bearing cells [see Lobb
and Hemler, J. Clin. Invest., 94: 1722-1728 (1994)] and/or to their
respective .alpha.4 ligands such as VCAM-1 and MadCAM,
respectively, on the surface of VCAM-1 and MadCAM bearing cells,
and which effectively blocks or coats VLA-4 (or .alpha.4.beta.7) or
VCAM-1 (or MadCAM) (i.e., a "an .alpha.4 integrin binding agent"
and ".alpha.4 integrin ligand binding agent" respectively), is
considered to be an equivalent of the antagonists used in the
examples herein.
[0105] An integrin "antagonist" includes any compound that inhibits
an .alpha.4 integrin(s) from binding with an .alpha.4 integrin
ligand and/or receptor. Anti-integrin antibody or antibody
homolog-containing proteins (discussed below) as well as other
molecules such as soluble forms of the ligand proteins for
integrins are useful. Soluble forms of the ligand proteins for
.alpha.4 integrins include soluble VCAM-1 or collagen peptides,
VCAM-1 fusion protein, or bifunctional VCAM-1/Ig fusion proteins.
For example, a soluble form of an .alpha.4 integrin ligand or a
fragment thereof may be administered to bind to integrin, and
preferably compete for an integrin binding site on cells, thereby
leading to effects similar to the administration of antagonists
such as anti-.alpha.4 integrin (e.g., .alpha.4.beta.7 antibodies
and/or VLA-4 antibodies). In particular, soluble .alpha.4 integrin
mutants that bind .alpha.4 integrin ligand but do not elicit
integrin-dependent signaling are included within the scope of the
invention. Such mutants can act as competitive inhibitors of wild
type integrin protein and are considered "antagonists". Other
antagonists used in the methods of the invention are "small
molecules", as defined below.
[0106] Included within the invention are methods using an agent
that antagonizes the action of more than one .alpha.4 integrin,
such as a single small molecule or antibody homolog that
antagonizes several .alpha.4 integrins such as VLA-4 and
.alpha.4.beta.7, or other combinations of .alpha.4 integrins. Also
included within the scope of the invention are methods using a
combination of different molecules such that the combined activity
antagonizes the action of more than one .alpha.4 integrin, such as
methods using several small molecules or antibody homologs that in
combination antagonize the .alpha.4 integrins VLA-4 and
.alpha.4.beta.7, or other combinations of integrins.
[0107] As discussed herein, certain integrin antagonists can be
fused or otherwise conjugated to, for instance, an antibody homolog
such as an immunoglobulin or fragment thereof and are not limited
to a particular type or structure of an integrin or ligand or other
molecule. Thus, for purposes of the invention, any agent capable of
forming a fusion protein (as defined below) and capable of binding
to .alpha.4 integrin ligands and which effectively blocks or coats
.alpha.4.beta.7 and/or VLA-4 integrin is considered to be an
equivalent of the antagonists used in the examples herein.
[0108] For the purposes of the invention an "antagonist of the
.alpha.4 integrin ligand/.alpha.4 integrin interaction" refers to
an agent, e.g., a polypeptide or other molecule, which can inhibit
or block .alpha.4 ligand (e.g., VCAM-1) and/or .alpha.4 integrin
(e.g., .alpha.4.beta.7 or VLA-4)-mediated binding or which can
otherwise modulate .alpha.4 ligand and/or .alpha.4 integrin
function, e.g., by inhibiting or blocking .alpha.4-ligand-mediated
.alpha.4 integrin signal transduction or .alpha.4 ligand mediated
.alpha.4 ligand signal transduction and which is effective in the
treatment of multiple myeloma, preferably in the same manner as are
anti-.alpha.4 integrin antibodies.
[0109] Specifically, an antagonist of the VCAM-1/VLA-4 interaction
is an agent which has one or more of the following properties: (1)
it coats, or binds to VLA-4 on the surface of a VLA-4 bearing cell
(e.g., a myeloma cell) with sufficient specificity to inhibit a
VLA-4-ligand/VLA-4 interaction, e.g., the VCAM-1/VLA-4 interaction
between bone stromal cells and myeloma cells; (2) it coats, or
binds to, VLA-4 on the surface of a VLA-4 bearing cell (i.e., a
myeloma cell) with sufficient specificity to modify, and preferably
to inhibit, transduction of a VLA-4-mediated signal e.g.,
VLA-4/VCAM-1-mediated signaling; (3) it coats, or binds to, a
VLA-4-ligand, (e.g., VCAM-1) on bone stromal cells with sufficient
specificity to inhibit the VLA-4/VCAM interaction; (4) it coats, or
binds to, a VLA-4-ligand (e.g., VCAM-1) on bone stromal cells with
sufficient specificity to modify, and preferably to inhibit,
transduction of VLA-4-ligand mediated VLA-4 signaling, e.g.,
VCAM-1-mediated VLA-4 signaling. In preferred embodiments the
antagonist has one or both of properties 1 and 2. In other
preferred embodiments the antagonist has one or both of properties
3 and 4. Moreover, more than one antagonist can be administered to
a patient, e.g., an agent which binds to VLA-4 can be combined with
an agent which binds to VCAM-1.
[0110] For example, antibodies or antibody homologs (discussed
below) as well as soluble forms of the natural binding proteins for
VLA-4 and VCAM-1 are useful. Soluble forms of the natural binding
proteins for VLA-4 include soluble VCAM-1 peptides, VCAM-1 fusion
proteins, bifunctional VCAM-1/Ig fusion proteins, fibronectin,
fibronectin having an alternatively spliced non-type III connecting
segment, and fibronectin peptides containing the amino acid
sequence EILDV or a similar conservatively substituted amino acid
sequence. Soluble forms of the natural binding proteins for VCAM-1
include soluble VLA-4 peptides, VLA-4 fusion proteins, bifunctional
VLA-4/Ig fusion proteins and the like. As used herein, a "soluble
VLA-4 peptide" or a "soluble VCAM-1 peptide" is an VLA-4 or VCAM-1
polypeptide incapable of anchoring itself in a membrane. Such
soluble polypeptides include, for example, VLA-4 and VCAM
polypeptides that lack a sufficient portion of their membrane
spanning domain to anchor the polypeptide or are modified such that
the membrane spanning domain is non-functional. These binding
agents can act by competing with the cell-surface binding protein
for VLA-4 or by otherwise altering VLA-4 function. For example, a
soluble form of VCAM-1 (see, e.g., Osborn et al. 1989, Cell, 59:
1203-1211) or a fragment thereof may be administered to bind to
VLA-4, and preferably compete for a VLA-4 binding site on myeloma
cells, thereby leading to effects similar to the administration of
antagonists such as small molecules or anti-VLA-4 antibodies.
[0111] In another example, VCAM-1, or a fragment thereof, which is
capable of binding to VLA-4 on the surface of VLA-4 bearing myeloma
cells, e.g., a fragment containing the two N-terminal domains of
VCAM-1, can be fused to a second peptide, e.g., a peptide which
increases the solubility or the in vivo life time of the VCAM-1
moiety. The second peptide can be a fragment of a soluble peptide,
preferably a human peptide, more preferably a plasma protein, or a
member of the immunoglobulin superfamily. In particularly preferred
embodiments the second peptide is IgG or a portion or fragment
thereof, e.g., the human IgG1 heavy chain constant region and
includes, at least the hinge, CH2 and CH3 domains.
[0112] Other antagonists useful in the methods of the invention
include, but are not limited to, agents that mimic the action of
peptides (organic molecules called "small molecules") capable of
disrupting the .alpha.4 integrin/.alpha.4 integrin ligand
interaction by, for instance, blocking VLA-4 by binding VLA-4
receptors on the surface of cells or blocking VCAM-1 by binding
VCAM-1 receptors on the surface of cells. These "small molecules"
may themselves be small peptides, or larger peptide-containing
organic compounds or non-peptidic organic, compounds. A "small
molecule", as defined herein, is not intended to encompass an
antibody or antibody homolog. Although the molecular weight of such
"small molecules" is generally less than 2000, we don't intend to
apply this figure as an absolute upper limit on molecular
weight.
[0113] For instance, small molecules such as oligosaccharides that
mimic the binding domain of a VLA-4 ligand and fit the receptor
domain of VLA-4 may be employed. (See, J. J. Devlin et al., 1990,
Science 249: 400-406 (1990), J. K. Scott and G. P. Smith, 1990,
Science 249: 386-390, and U.S. Pat. No. 4,833,092 (Geysen), all
incorporated herein by reference.) Conversely, small molecules that
mimic the binding domain of a VCAM-1 ligand and fit the receptor
domain of VCAM-1 may be employed.
[0114] Examples of other small molecules useful in the invention
can be found in Komoriya et al. ("The Minimal Essential Sequence
for a Major Cell Type-Specific Adhesion Site (CS1) Within the
Alternatively Spliced Type III Connecting Segment Domain of
Fibronectin Is Leucine-Aspartic Acid-Valine", J. Biol. Chem., 266
(23), pp. 15075-79 (1991)). They identified the minimum active
amino acid sequence necessary to bind VLA-4 and synthesized a
variety of overlapping peptides based on the amino acid sequence of
the CS-1 region (the VLA-4 binding domain) of a particular species
of fibronectin. They identified an 8-amino acid peptide,
Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two smaller overlapping
pentapeptides, Glu-Ile-Leu-Asp-Val and Leu-Asp-Val-Pro-Ser, that
possessed inhibitory activity against fibronectin-dependent cell
adhesion. Certain larger peptides containing the LDV sequence were
subsequently shown to be active in vivo (T. A. Ferguson et al.,
"Two Integrin Binding Peptides Abrogate T-cell-Mediated Immune
Responses In Vivo", Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76
(1991); and S. M. Wahl et al., "Synthetic Fibronectin Peptides
Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and
Recruitment", J. Clin. Invest., 94, pp. 655-62 (1994)). A cyclic
pentapeptide, Arg-Cys-Asp-TPro-Cys (wherein TPro denotes
4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to
fibronectin has also been described. (See, e.g., D. M. Nowlin et
al., "A Novel Cyclic Pentapeptide Inhibits Alpha4Beta1
Integrin-mediated Cell Adhesion", J. Biol, Chem., 268(27), pp.
20352-59 (1993); and PCT publication PCT/US91/04862). This
pentapeptide was based on the tripeptide sequence Arg-Gly-Asp from
FN which had been known as a common motif in the recognition site
for several extracellular-matrix proteins.
[0115] Examples of other small molecule VLA-4 inhibitors have been
reported, for example, in Adams et al., "Cell Adhesion Inhibitors",
PCT US97/13013, describing linear peptidyl compounds containing
beta-amino acids which have cell adhesion inhibitory activity.
International patent applications WO 94/15958 and WO 92/00995
describe cyclic peptide and peptidomimetic compounds with cell
adhesion inhibitory activity. International patent applications WO
93/08823 and WO 92/08464 describe guanidinyl-, urea- and
thiourea-containing cell adhesion inhibitory compounds. U.S. Pat.
No. 5,260,277 describes guanidinyl cell adhesion modulation
compounds.
[0116] Examples of small molecules that bind to or otherwise
interact with VLA-4 molecules (and/or that specifically inhibit the
binding of a ligand to VLA-4) and inhibit VLA-4 dependent cell
adhesion are disclosed in PCT publication WO 01/12186 A1, published
Feb. 22, 2001 (PCT application number PCT/US00/22285, filed Aug.
14, 2000), the disclosure of which is incorporated by reference
herein. A preferred small molecule is BIO-8809, shown below:
##STR00001##
[0117] Small molecules mimetic agents may be produced by
synthesizing a plurality of peptides semi-peptidic compounds or
non-peptidic, organic compounds, and then screening those compounds
for their ability to inhibit the .alpha.4 integrin/.alpha.4
integrin ligand interaction. See generally U.S. Pat. No. 4,833,092,
Scott and Smith, "Searching for Peptide Ligands with an Epitope
Library", Science, 249, pp. 386-90 (1990), and Devlin et al.,
"Random Peptide Libraries: A Source of Specific Protein Binding
Molecules", Science, 249, pp. 40407 (1990).
[0118] In other preferred embodiments, the agent that is used in
the method of the invention to bind to, including block or coat,
cell-surface .alpha.4 integrin and/or .alpha.4 integrin ligand is
an anti-VLA-4 and/or anti-.alpha.4.beta.7 monoclonal antibody or
antibody homolog. Preferred antibodies and homologs for treatment,
in particular for human treatment, include human antibody homologs,
humanized antibody homologs, chimeric antibody homologs, Fab, Fab',
F(ab').sub.2 and F(v) antibody fragments, and monomers or dimers of
antibody heavy or light chains or mixtures thereof. Monoclonal
antibodies against VLA-4 are a preferred binding agent in the
method of the invention.
[0119] As used herein, the term "antibody homolog" includes intact
antibodies consisting of immunoglobulin light and heavy chains
linked via disulfide bonds. The term "antibody homolog" is also
intended to encompass a protein comprising one or more polypeptides
selected from immunoglobulin light chains, immunoglobulin heavy
chains and antigen-binding fragments thereof which are capable of
binding to one or more antigens. The component polypeptides of an
antibody homolog composed of more than one polypeptide may
optionally be disulfide-bound or otherwise covalently
crosslinked.
[0120] Accordingly, therefore, "antibody homologs" include intact
immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as
subtypes thereof), wherein the light chains of the immunoglobulin
may be of types kappa or lambda.
[0121] "Antibody homologs" also include portions of intact
antibodies that retain antigen-binding specificity, for example,
Fab fragments, Fab' fragments, F(ab')2 fragments, F(v) fragments,
heavy chain monomers or dimers, light chain monomers or dimers,
dimers consisting of one heavy and one light chain, and the like.
Thus, antigen-binding fragments, as well as full-length dimeric or
trimeric polypeptides derived from the above-described antibodies
are themselves useful.
[0122] As used herein, a "humanized antibody homolog" is an
antibody homolog, produced by recombinant DNA technology, in which
some or all of the amino acids of a human immunoglobulin light or
heavy chain that are not required for antigen binding have been
substituted for the corresponding amino acids from a nonhuman
mammalian immunoglobulin light or heavy chain.
[0123] As used herein, a "chimeric antibody homolog" is an antibody
homolog, produced by recombinant DNA technology, in which all or
part of the hinge and constant regions of an immunoglobulin light
chain, heavy chain, or both, have been substituted for the
corresponding regions from another immunoglobulin light chain or
heavy chain. In another aspect the invention features a variant of
a chimeric molecule which includes: (1) a VLA-4 targeting moiety,
e.g., a VCAM-1 moiety capable of binding to antigen (i.e., VLA-4)
on the surface of VLA-4 bearing myeloma cells; (2) optionally, a
second peptide, e.g., one which increases solubility or in vivo
life time of the VLA-4 targeting moiety, e.g., a member of the
immunoglobulin superfamily or fragment or portion thereof, e.g., a
portion or a fragment of IgG, e.g., the human IgG1 heavy chain
constant region, e.g., CH2 and CH3 hinge regions; and a toxin
moiety. The VLA-4 targeting moiety can be any naturally occurring
VLA-4 ligand or fragment thereof, e.g., a VCAM-1 peptide or a
similar conservatively substituted amino acid sequence. A preferred
targeting moiety is a soluble VCAM-1 fragment, e.g., the N-terminal
domains 1 and 2 of the VCAM-1 molecule. The chimeric molecule can
be used to treat a subject, e.g., a human, at risk for disorder,
e.g., multiple myeloma, characterized by the presence of myeloma
cells bearing VLA-4, and preferably activated VLA-4.
[0124] As used herein, a "human antibody homolog" is an antibody
homolog produced by recombinant DNA technology, in which all of the
amino acids of an immunoglobulin light or heavy chain that are
derived from a human source.
Methods of Making Anti-VLA-4 Antibody Homologs
[0125] The technology for producing monoclonal antibody homologs is
well known. Briefly, an immortal cell line (typically myeloma
cells) is fused to lymphocytes (typically splenocytes) from a
mammal immunized with whole cells expressing a given antigen, e.g.,
VLA-4, and the culture supernatants of the resulting hybridoma
cells are screened for antibodies against the antigen. See,
generally, Kohler et al., 1975, Nature, 265: 295-297.
[0126] Immunization may be accomplished using standard procedures.
The unit dose and immunization regimen depend on the species of
mammal immunized, its immune status, the body weight of the mammal,
etc. Typically, the immunized mammals are bled and the serum from
each blood sample is assayed for particular antibodies using
appropriate screening assays. For example, anti-VLA-4 antibodies
may be identified by immunoprecipitation of 125I-labeled cell
lysates from VLA-4-expressing cells. (See, Sanchez-Madrid et al.,
1986, Eur. J. Immunol., 16: 1343-1349 and Hemler et al., 1987, J.
Biol. Chem., 262, 11478-11485). Anti-VLA-4 antibodies may also be
identified by flow cytometry, e.g., by measuring fluorescent
staining of Ramos cells incubated with an antibody believed to
recognize VLA-4 (see, Elices et al., 1990 Cell, 60: 577-584). The
lymphocytes used in the production of hybridoma cells typically are
isolated from immunized mammals whose sera have already tested
positive for the presence of anti-VLA-4 antibodies using such
screening assays.
[0127] Typically, the immortal cell line (e.g., a myeloma cell
line) is derived from the same mammalian species as the
lymphocytes. Preferred immortal cell lines are mouse myeloma cell
lines that are sensitive to culture medium containing hypoxanthine,
aminopterin and thymidine ("HAT medium"). Typically, HAT-sensitive
mouse myeloma cells are fused to mouse splenocytes using 1500
molecular weight polyethylene glycol ("PEG 1500"). Hybridoma cells
resulting from the fusion are then selected using HAT medium, which
kills unfused and unproductively fused myeloma cells (unfused
splenocytes die after several days because they are not
transformed). Hybridomas producing a desired antibody are detected
by screening the hybridoma culture supernatants. For example,
hybridomas prepared to produce anti-VLA-4 antibodies may be
screened by testing the hybridoma culture supernatant for secreted
antibodies having the ability to bind to a recombinant
a4-subunit-expressing cell line (see, Elices et al., supra).
[0128] To produce anti-VLA-4 antibody homologs that are intact
immunoglobulins, hybridoma cells that tested positive in such
screening assays were cultured in a nutrient medium under
conditions and for a time sufficient to allow the hybridoma cells
to secrete the monoclonal antibodies into the culture medium.
Tissue culture techniques and culture media suitable for hybridoma
cells are well known. The conditioned hybridoma culture supernatant
may be collected and the anti-VLA4 antibodies optionally further
purified by well-known methods.
[0129] Alternatively, the desired antibody may be produced by
injecting the hybridoma cells into the peritoneal cavity of an
unimmunized mouse. The hybridoma cells proliferate in the
peritoneal cavity, secreting the antibody which accumulates as
ascites fluid. The antibody may be harvested by withdrawing the
ascites fluid from the peritoneal cavity with a syringe.
[0130] Several mouse anti-VLA-4 monoclonal antibodies have been
previously described. See, e.g., Sanchez-Madrid et al., 1986,
supra; Hemler et al., 1987, supra; Pulido et al., 1991, J. Biol.
Chem., 266 (16), 10241-10245). These anti-VLA-4 monoclonal
antibodies such as HP 1/2 and other anti-VLA-4 antibodies (e.g.,
HP2/1, HP2/4, L25, P4C2, P4G9) capable of recognizing the P chain
of VLA-4 will be useful in the methods of treatment according to
the present invention. Anti VLA-4 antibodies that will recognize
the VLA-4 .alpha.4 chain epitopes involved in binding to VCAM-1 and
fibronectin ligands (i.e., antibodies which can bind to VLA-4 at a
site involved in ligand recognition and block VCAM-1 and
fibronectin binding) are preferred. Such antibodies have been
defined as B epitope-specific antibodies (B1 or B2) (Pulido et al.,
1991, supra) and are also anti-VLA-4 antibodies according to the
present invention.
[0131] Fully human monoclonal antibody homologs against VLA-4 are
another preferred binding agent which may block or coat VLA-4
antigens in the method of the invention. In their intact form these
may be prepared using in vitro-primed human splenocytes, as
described by Boerner et al., 1991, J. Immunol., 147, 86-95.
Alternatively, they may be prepared by repertoire cloning as
described by Persson et al., 1991, Proc. Nat. Acad. Sci. USA, 88:
2432-2436 or by Huang and Stollar, 1991, J. Immunol. Methods 141,
227-236. U.S. Pat. No. 5,798,230 (Aug. 25, 1998, "Process for the
preparation of human monoclonal antibodies and their use") who
describe preparation of human monoclonal antibodies from human B
cells. According to this process, human antibody-producing B cells
are immortalized by infection with an Epstein-Barr virus, or a
derivative thereof, that expresses Epstein-Barr virus nuclear
antigen 2 (EBNA2). EBNA2 function, which is required for
immortalization, is subsequently shut off, which results in an
increase in antibody production.
[0132] In yet another method for producing fully human antibodies,
U.S. Pat. No. 5,789,650 (Aug. 4, 1998, "Transgenic non-human
animals for producing heterologous antibodies") describes
transgenic non-human animals capable of producing heterologous
antibodies and transgenic non-human animals having inactivated
endogenous immunoglobulin genes. Endogenous immunoglobulin genes
are suppressed by antisense polynucleotides and/or by antiserum
directed against endogenous immunoglobulins. Heterologous
antibodies are encoded by immunoglobulin genes not normally found
in the genome of that species of non-human animal. One or more
transgenes containing sequences of unrearranged heterologous human
immunoglobulin heavy chains are introduced into a non-human animal
thereby forming a transgenic animal capable of functionally
rearranging transgenic immunoglobulin sequences and producing a
repertoire of antibodies of various isotypes encoded by human
immunoglobulin genes. Such heterologous human antibodies are
produced in B-cells which are thereafter immortalized, e.g., by
fusing with an immortalizing cell line such as a myeloma or by
manipulating such B-cells by other techniques to perpetuate a cell
line capable of producing a monoclonal heterologous, fully human
antibody homolog.
[0133] Large nonimmunized human phage display libraries may also be
used to isolate high affinity antibodies that can be developed as
human therapeutics using standard phage technology (Vaughan et al.,
1996). Yet another preferred binding agent which may block or coat
VLA-4 antigens in the method of the invention is a humanized
recombinant antibody homolog having anti-VLA-4 specificity.
Following the early methods for the preparation of chimeric
antibodies, a new approach was described in EP 0239400 (Winter et
al.) whereby antibodies are altered by substitution of their
complementarity determining regions (CDRs) for one species with
those from another. This process may be used, for example, to
substitute the CDRs from human heavy and light chain Ig variable
region domains with alternative CDRs from murine variable region
domains. These altered Ig variable regions may subsequently be
combined with human Ig constant regions to created antibodies which
are totally human in composition except for the substituted murine
CDRs. Such CDR-substituted antibodies would be predicted to be less
likely to elicit an immune response in humans compared to chimeric
antibodies because the CDR-substituted antibodies contain
considerably less non-human components. The process for humanizing
monoclonal antibodies via CDR "grafting" has been termed
"reshaping". (Riechmann et al., 1988, Nature 332, 323-327;
Verhoeyen et al., 1988, Science 239, 1534-1536).
[0134] Typically, complementarity determining regions (CDRs) of a
murine antibody are transplanted onto the corresponding regions in
a human antibody, since it is the CDRs (three in antibody heavy
chains, three in light chains) that are the regions of the mouse
antibody which bind to a specific antigen. Transplantation of CDRs
is achieved by genetic engineering whereby CDR DNA sequences are
determined by cloning of murine heavy and light chain variable (V)
region gene segments, and are then transferred to corresponding
human V regions by site directed mutagenesis. In the final stage of
the process, human constant region gene segments of the desired
isotype (usually gamma I for CH and kappa for CL) are added and the
humanized heavy and light chain genes are co-expressed in mammalian
cells to produce soluble humanized antibody.
[0135] The transfer of these CDRs to a human antibody confers on
this antibody the antigen binding properties of the original murine
antibody. The six CDRs in the murine antibody are mounted
structurally on a V region "framework" region. The reason that
CDR-grafting is successful is that framework regions between mouse
and human antibodies may have very similar 3-D structures with
similar points of attachment for CDRs, such that CDRs can be
interchanged. Such humanized antibody homologs may be prepared, as
exemplified in Jones et al., 1986, Nature 321, 522-525; Riechmann,
1988, Nature 332, 323-327; Queen et al., 1989, Proc. Nat. Acad.
Sci. USA 86, 10029; and Orlandi et al., 1989, Proc. Nat. Acad. Sci.
USA 86, 3833.
[0136] Nonetheless, certain amino acids within framework regions
are thought to interact with CDRs and to influence overall antigen
binding affinity. The direct transfer of CDRs from a murine
antibody to produce a recombinant humanized antibody without any
modifications of the human V region frameworks often results in a
partial or complete loss of binding affinity. In a number of cases,
it appears to be critical to alter residues in the framework
regions of the acceptor antibody in order to obtain binding
activity.
[0137] Queen et al., 1989 (supra) and WO 90/07861 (Protein resign
Labs) have described the preparation of a humanized antibody that
contains modified residues in the framework regions of the acceptor
antibody by combining the CDRs of a murine mAb (anti-Tac) with
human immunoglobulin framework and constant regions. They have
demonstrated one solution to the problem of the loss of binding
affinity that often results from direct CDR transfer without any
modifications of the human V region framework residues; their
solution involves two key steps. First, the human V framework
regions are chosen by computer analysts for optimal protein
sequence homology to the V region framework of the original murine
antibody, in this case, the anti-Tac mAb. In the second step, the
tertiary structure of the murine V region is modeled by computer in
order to visualize framework amino acid residues which are likely
to interact with the murine CDRs and these murine amino acid
residues are then superimposed on the homologous human framework.
See also Protein Design Labs--U.S. Pat. No. 5,693,762.
[0138] One may use a different approach (Tempest et al., 1991,
Biotechnology 9, 266-271) and utilize, as standard, the V region
frameworks derived from NEWM and REI heavy and light chains
respectively for CDR-grafting without radical introduction of mouse
residues. An advantage of using the Tempest et al., approach to
construct NEWM and REI based humanized antibodies is that the
3-dimensional structures of NEWM and REI variable regions are known
from x-ray crystallography and thus specific interactions between
CDRs and V region framework residues can be modeled.
[0139] Regardless of the approach taken, the examples of the
initial humanized antibody homologs prepared to date have shown
that it is not a straightforward process. However, even
acknowledging that such framework changes may be necessary, it is
not possible to predict, on the basis of the available prior art,
which, if any, framework residues will need to be altered to obtain
functional humanized recombinant antibodies of the desired
specificity. Results thus far indicate that changes necessary to
preserve specificity and/or affinity are for the most part unique
to a given antibody and cannot be predicted based on the
humanization of a different antibody.
[0140] Preferred antagonists useful in the present invention
include chimeric recombinant and humanized recombinant antibody
homologs (i.e., intact immunoglobulins and portions thereof) with B
epitope specificity that have been prepared and are described in
co-pending U.S. patent application Ser. No. 08/004,798, filed Jan.
12, 1993, PCT Publication US94/00266, filed Jan. 7, 1994. The
starting material for the preparation of chimeric (mouse V-human C)
and humanized anti-VLA-4 antibody homologs may be a murine
monoclonal anti-VLA-4 antibody as previously described, a
monoclonal anti-VLA-4 antibody commercially available (e.g., HP2/1,
Amae International, Inc., Westbrook, Me.), or a monoclonal
anti-VLA-4 antibody prepared in accordance with the teaching
herein. For example, the variable regions of the heavy and light
chains of the anti-VLA-4 antibody HP 1/2 have been cloned,
sequenced and expressed in combination with constant regions of
human immunoglobulin heavy and light chains. Such HP 1/2 antibody
is similar in specificity and potency to the murine HP 1/2
antibody, and may be useful in methods of treatment according to
the present invention.
[0141] Other preferred humanized anti-VLA4 antibody homologs are
described by Athena Neurosciences, Inc. in PCT/US95/01219 (27 Jul.
1995). These humanized anti-VLA-4 antibodies comprise a humanized
light chain and a humanized heavy chain. The humanized light chain
comprises three complementarity determining regions (CDR1, CDR2 and
CDR3) having amino acid sequences from the corresponding
complementarity determining regions of a mouse 21-6 immunoglobulin
light chain, and a variable region framework from a human kappa
light chain variable region framework sequence except in at least
position the amino acid position is occupied by the same amino acid
present in the equivalent position of the mouse 21.6 immunoglobulin
light chain variable region framework. The humanized heavy chain
comprises three complementarity determining regions (CDR1, CDR2 and
CDR3) having amino acid sequences from the corresponding
complementarity determining regions of a mouse 21-6 immunoglobulin
heavy chain, and a variable region framework from a human heavy
chain variable region framework sequence except in at least one
position the amino acid position is occupied by the same amino acid
present in the equivalent position of the mouse 21-6 immunoglobulin
heavy chain variable region framework.
Therapeutic Applications
[0142] In this method according to the first aspect of the
invention, VLA-4 binding agents, in particular, VCAM fusions and
anti-VLA-4 antibody homologs are preferably administered
parenterally. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion
techniques.
[0143] The VLA-4 binding agents are preferably administered as a
sterile pharmaceutical composition containing a pharmaceutically
acceptable carrier, which may be any of the numerous well known
carriers, such as water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol, and the like, or combinations thereof.
The compounds of the present invention may be used in the form of
pharmaceutically acceptable salts derived from inorganic or organic
acids and bases. Included among such acid salts are the following:
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate
and undecanoate. Base salts include ammonium salts, alkali metal
salts, such as sodium and potassium salts, alkaline earth metal
salts, such as calcium and magnesium salts, salts with organic
bases, such as dicyclohexylamine salts, N-methyl-D-glucamine,
tris(hydroxymethyl)methylamine and salts with amino acids such as
arginine, lysine, and so forth. Also, the basic nitrogen-containing
groups can be quaternized with such agents as lower alkyl halides,
such as methyl, ethyl, propyl, and butyl chloride, bromides and
iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and
diamyl sulfates, long chain halides such as decyl, lauryl, myristyl
and stearyl chlorides, bromides and iodides, aralkyl halides, such
as benzyl and phenethyl bromides and others. Water or oil soluble
or dispersible products are then obtained.
[0144] The pharmaceutical compositions of this invention comprise
any of the compounds of the present invention, or pharmaceutically
acceptable derivatives thereof, together with any pharmaceutically
acceptable carrier. The term "carrier" as used herein includes
acceptable adjuvants and vehicles. Pharmaceutically acceptable
carriers that may be used in the pharmaceutical compositions of
this invention include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, serum proteins, such as human
serum albumin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty acids, water, salts or electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0145] According to this invention, the pharmaceutical compositions
may be in the form of a sterile injectable preparation, for example
a sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to techniques-known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as do natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as Ph. Helv or
similar alcohol.
[0146] The pharmaceutical compositions of this invention, in
particular small molecule antagonists of the VLA-4/VCAM-1
interaction, may be given parenterally or orally. If given orally,
they can be administered in any orally acceptable dosage form
including, but not limited to, capsules, tablets, aqueous
suspensions or solutions. In the case of tablets for oral use,
carriers which are commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried corn starch. When aqueous suspensions are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
Topically-transdermal patches may also be used. The pharmaceutical
compositions of this invention may also be administered by nasal
aerosol or inhalation through the use of a nebulizer, a dry powder
inhaler or a metered dose inhaler. Such compositions are prepared
according to techniques well-known in the art of pharmaceutical
formulation and may be prepared as solutions in saline, employing
benzyl alcohol or other suitable preservatives, absorption
promoters to enhance bioavailability, fluorocarbons, and/or other
conventional solubilizing or dispersing agents.
[0147] According to another embodiment compositions containing a
compound of this invention may also comprise an additional agent
selected from the group consisting of corticosteroids,
anti-inflammatories, immunosuppressants, antimetabolites, and
immunomodulators. Specific compounds within each of these classes
may be selected from any of those listed under the appropriate
group headings in "Comprehensive Medicinal Chemistry", Pergamon
Press, Oxford, England, pp. 970-986 (1990), the disclosure of which
is herein incorporated by reference. Also included within this
group are compounds such as theophylline, sulfasalazine and
aminosalicylates (anti-inflammatories); cyclosporin, FK-506, and
rapamycin (immunosuppressants); cyclophosphamide and methotrexate
(antimetabolites); steroids (inhaled, oral or topical) and
interferons (immunomodulators).
[0148] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated, and the particular mode of
administration. It should be understood, however, that a specific
dosage and treatment regimen for any particular patient will depend
upon a variety of factors, including the activity of the specific
compound employed, the age, body weight, general health, sex, diet,
time of administration, rate of excretion, drug combination, and
the judgment of the treating physician and the severity of the
particular disease being treated. The amount of active ingredient
may also depend upon the therapeutic or prophylactic agent, if any,
with which the ingredient is co-administered.
[0149] The dosage and dose rate of the compounds of this invention
effective to prevent, suppress or inhibit cell adhesion will depend
on a variety of factors, such as the nature of the inhibitor, the
size of the patient, the goal of the treatment, the nature of the
pathology to be treated, the specific pharmaceutical composition
used, and the judgment of the treating physician. Dosage levels of
between about 0.001 and about 100 mg/kg body weight per day,
preferably between about 0.1 and about 50 mg/kg body weight per day
of the active ingredient compound are useful. Most preferably, the
VLA-4 binding agent, if an antibody or antibody derivative, will be
administered at a dose ranging between about 0.1 mg/kg body
weight/day and about 20 mg/kg body weight/day, preferably ranging
between about 0.1 mg/kg body weight/day and about 10 mg/kg body
weight/day and at intervals of every 1-14 days. For non-antibody or
small molecule binding agents, the dose range should preferably be
between molar equivalent amounts to these amounts of antibody.
Preferably, an antibody composition is administered in an amount
effective to provide a plasma level of antibody of at least 1
mg/ml. Optimization of dosages can be determined by administration
of the binding agents, followed by assessment of the coating of
VLA-4-positive cells by the agent over time after administered at a
given dose in vivo.
[0150] Myeloma cells contained in a sample of the individual's
peripheral blood (or bone marrow cells) should be probed for the
presence of the agent in vitro (or ex vivo) using a second reagent
to detect the administered agent. For example, this may be a
fluorochrome labeled antibody specific for the administered agent
which is then measured by standard FACS (fluorescence activated
cell sorter) analysis. Alternatively, presence of the administered
agent may be detected in vitro (or ex vivo) by the inability or
decreased ability of the individual's cells to bind the same agent
which has been itself labeled (e.g., by a fluorochrome). The
preferred dosage should produce detectable coating of the vast
majority of VLA-4-positive cells. Preferably, coating is sustained
in the case of an antibody homolog for a 1-14 day period.
Combination Therapy
[0151] In some embodiments of this invention, one or more
antagonists of an interaction between an integrin with an .alpha.4
subunit and a ligand for this integrin could be administered in
combination with one or more compounds that may not be (preferably
are not) an antagonist of an interaction between an integrin with
an .alpha.4 subunit and a ligand for this integrin. Such compound
could be a chemotherapeutic agent or another agent, including
without limitation: melphalan, bisphosphonates (examples of which
are ibandronate and pamidronate), thalidomide, erythropoietin, and
antagonists, such as mAb blockers, of IL6 and IL15.
[0152] Multiple myeloma is currently treated inefficiently with
standard chemotherapeutic regimens. In some embodiments of this
invention, an antagonist of an interaction between an integrin with
an .alpha.4 subunit and a ligand for this integrin could be
administered in combination with one or more standard agents for
treatment of multiple myeloma, such as chemotherapeutic agents.
Hence, the two compounds (an antagonist of an interaction between
an integrin with an .alpha.4 subunit and a ligand for this integrin
and a compound that may not be (preferably is not) an antagonist of
an interaction between an integrin with an .alpha.4 subunit and a
ligand for this integrin; said compound that may not be (preferably
is not) an antagonist of an interaction between an integrin with an
.alpha.4 subunit and a ligand for this integrin could be a
chemotherapeutic agent or another agent for treatment or prevention
of multiple myeloma, including, inter alia: melphalan,
bisphosphonates (examples of which are ibandronate and
pamidronate), thalidomide, erythropoietin, and antagonists, such as
mAb blockers, of IL6 and IL15) could act to prevent or treat
multiple myeloma synergistically; or lower dosage of either or both
compounds (an antagonist of an interaction between an integrin with
an .alpha.4 subunit and a ligand for this integrin and a compound
that may not be (preferably is not) an antagonist of an interaction
between an integrin with an .alpha.4 subunit and a ligand for this
integrin, such as one or more standard agents for treatment of
multiple myeloma) needed to provide the same effect as a higher
dosage of either compound alone.
[0153] In some embodiments, this invention provides methods for
treating or preventing multiple myeloma comprising administering to
an individual a therapeutically or prophylactically effective
amount of a first composition comprising an antagonist of an
interaction between an .alpha.4 subunit-bearing integrin and a
ligand for an .alpha.4 subunit-bearing integrin, wherein said first
composition is administered in combination with a therapeutically
or prophylactically effective amount of a second composition
comprising a compound that may not be (preferably is not) an
antagonist of an interaction between an .alpha.4 subunit-bearing
integrin and a ligand for an .alpha.4 subunit-bearing integrin. In
certain embodiments, this invention provides methods for treating
or preventing multiple myeloma comprising administering to an
individual a therapeutically or prophylactically effective amount
of a first composition comprising an antagonist of an interaction
between an .alpha.4 subunit-bearing integrin and a ligand for an
.alpha.4 subunit-bearing integrin, wherein said first composition
is administered in combination with a therapeutically or
prophylactically effective amount of a second composition
comprising a compound that may not be (preferably is not) an
antagonist of an interaction between an .alpha.4 subunit-bearing
integrin and a ligand for an .alpha.4 subunit-bearing integrin,
wherein, to be therapeutically or prophylactically effective, a
dosage of said antagonist is lower when administered in combination
with said compound than not administered in combination with said
compound; or
a dosage of said compound is lower when administered in combination
with said antagonist than not administered in combination with said
antagonist, or both. Such compound could be a chemotherapeutic
agent or another agent for treating or preventing multiple myeloma,
including, for example: melphalan, bisphosphonates (examples of
which are ibandronate and pamidronate), thalidomide,
erythropoietin, and antagonists, such as mAb blockers, of IL6 and
IL15.
[0154] In some embodiments, this invention provides methods for
inhibiting bone resorption associated with tumors of bone marrow,
the methods comprising administering to a mammal with said tumors
an antagonist of an interaction between an .alpha.4 subunit-bearing
integrin and a ligand for an .alpha.4 subunit-bearing integrin, in
an amount effective to provide inhibition of said bone resorption,
wherein said antagonist is administered in combination with a
compound, in an amount effective to provide inhibition of said bone
resorption, that may not be (preferably is not) an antagonist of an
interaction between an .alpha.4 subunit-bearing integrin and a
ligand for an .alpha.4 subunit-bearing integrin. In certain
embodiments, this invention provides methods for inhibiting bone
resorption associated with tumors of bone marrow, the methods
comprising administering to a mammal with said tumors an antagonist
of an interaction between an .alpha.4 subunit-bearing integrin and
a ligand for an .alpha.4 subunit-bearing integrin, in an amount
effective to provide inhibition of said bone resorption, wherein
said antagonist is administered in combination with a compound, in
an amount effective to provide inhibition of said bone resorption,
that may not be (preferably is not) an antagonist of an interaction
between an .alpha.4 subunit-bearing integrin and a ligand for an
.alpha.4 subunit-bearing integrin, wherein, to be therapeutically
or prophylactically effective,
a dosage of said antagonist is lower when administered in
combination with said compound than not administered in combination
with said compound; or a dosage of said compound is lower when
administered in combination with said antagonist than not
administered in combination with said antagonist, or both. Such
compound could be a chemotherapeutic agent or another agent for
inhibiting bone resorption associated with tumors of bone marrow,
including, for example: melphalan, bisphosphonates (examples of
which are ibandronate and pamidronate), thalidomide,
erythropoietin, and antagonists, such as mAb blockers, of IL6 and
IL15.
[0155] In some embodiments, this invention provides methods of
treating or preventing a subject having a disorder characterized by
the presence of osteoclastogenesis, the methods comprising
administering to the subject an antagonist of an interaction
between an .alpha.4 subunit-bearing integrin and a ligand for an
.alpha.4 subunit bearing integrin, in an amount sufficient to
suppress or prevent the osteoclastogenesis, wherein said antagonist
is administered in combination with a compound, in an amount
sufficient to suppress or prevent the osteoclastogenesis, that may
not be (preferably is not) an antagonist of an interaction between
an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4
subunit-bearing integrin. In certain embodiments, this invention
provides methods of treating or preventing a subject having a
disorder characterized by the presence of osteoclastogenesis, the
methods comprising administering to the subject an antagonist of an
interaction between an .alpha.4 subunit-bearing integrin and a
ligand for an .alpha.4 subunit bearing integrin, in an amount
sufficient to suppress or prevent the osteoclastogenesis, wherein
said antagonist is administered in combination with a compound, in
an amount sufficient to suppress or prevent the osteoclastogenesis,
that may not be (preferably is not) an antagonist of an interaction
between an .alpha.4 subunit-bearing integrin and a ligand for an
.alpha.4 subunit-bearing integrin, wherein, to be therapeutically
or prophylactically effective,
a dosage of said antagonist is lower when administered in
combination with said compound than not administered in combination
with said compound; or a dosage of said compound is lower when
administered in combination with said antagonist than not
administered in combination with said antagonist, or both. Such
compound could be a chemotherapeutic agent or another agent for
treating or preventing a subject having a disorder characterized by
the presence of osteoclastogenesis, including, for example:
melphalan, bisphosphonates (examples of which are ibandronate and
pamidronate), thalidomide, erythropoietin, and antagonists, such as
mAb blockers, of IL6 and IL15.
[0156] In some embodiments, the pharmaceutical or prophylactic
composition of this invention can also include a pharmaceutically
or prophylactically effective amount of a chemotherapeutic agent or
another agent, including without limitation: melphalan,
bisphosphonates (examples of which are ibandronate and
pamidronate), thalidomide, erythropoietin, and antagonists, such as
mAb blockers, of IL6 and IL15. Said chemotherapeutic agent or
another agent could be included in a second composition.
Animal Models
[0157] The animal model has been described in detail (Garrett
1997). Briefly, Radl et al. (1988) had described a murine model of
myeloma which arose spontaneously in aged C57BL/KaLwRij mice. This
condition occurred in approximately 1 in 200 animals as they aged,
and led to a monoclonal gammopathy with some of the features of
human disease (Radl 1988). To develop a better and more
reproducible animal model we have established and subcloned a cell
line from this murine myeloma called 5TGM1, and found that it
causes lesions in mice characteristic of human myeloma, such as
severe osteolysis and the involvement of non-bone organs including
liver and kidney (Garrett 1997). Mice inoculated with cultured
cells develop disease in a highly predictable and reproducible
manner, which includes formation of a monoclonal gammopathy and
radiologic bone lesions. Furthermore, some of the mice become
hypercalcemic, and the bone lesions are characterized by increased
osteoclast activity. Thus, based on histological examination of
affected organs in 5TGM1-bearing mice and increased serum levels of
IgG2b, 5TGM1 is defined as a murine myeloma which recapitulates
accurately the hallmarks of human disease.
[0158] The following examples are intended to further illustrate
certain preferred embodiments of the invention and are not intended
to be limiting in nature. In the following examples, the necessary
restriction enzymes, plasmids, and other reagents and materials may
be obtained from commercial sources and cloning, ligation and other
recombinant DNA methodology may be performed by procedures
well-known in the art.
Example 1
Materials and Methods
5TGM1 Myeloma Cells
[0159] 5TGM1 myeloma cells were initially derived from a myeloma
which arose spontaneously in aged C57BL/KaLwRij mice (Garrett 1997,
Vanderkerken 1997). Cells were grown in Isocove's Modified
Dulbecco's Medium (IMDM, Life Technologies Inc., Gaithersburg, Md.)
supplemented with 10% fetal bovine serum (FBS, Summit, Fort
Collins, Colo.) and 1% penicillin-streptomycin solution (GIBCO,
Grand Island, N.Y.) at 37 C in 5% CO.sub.2 atmosphere. For in vitro
experimentation described below, 5TGM1 cells between passage 25 and
30 were used.
Antibodies, Soluble VCAM-1
[0160] Neutralizing antibodies against murine VCAM-1 (M/K-2.7),
integrin VLA-4 (PS/2 mAb), and Intercellular Adhesion Molecule-1
(ICAM-1, YN1/1.7), were kindly gifted by Dr. Kensuke Miyake (Saga
Medical University, Saga, Japan). Recombinant soluble VCAM-1 (Lobb
et al., 1991), containing the 7 extracellular domains of human
VCAM-1, was the gift of Dr. Roy Lobb, Biogen Inc., Cambridge,
Mass.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
[0161] Using RT-PCR, we confirmed expression of VCAM-1 and integrin
.alpha.4 in bone marrow stromal cells and 5TGM1, respectively.
Total RNA was prepared from 5TGM1, a primary culture of bone marrow
stromal cells and an ST2 marrow stromal cell line (RIKEN Cell Bank,
Tsukuba, Japan) by the single-step RNA isolation method using
TRIzol reagent (GIBCO). Three .mu.g of RNA was incubated with 50 ng
of random hexamer at 70.degree. C. for 10 min and chilled on ice,
then converted to first strand cDNA using reverse transcriptase
(Perkin-Elmer, Branchburg, N.J.) according to the manufacturers
instruction. The primers used for PCR were as follows: murine
VCAM-1 5'-primer; 5'-OH-GCTGCGCGTCACCATTGTTCTC-3'-OH (SEQ ID NO:
1]; murine VCAM-1 3'-primer, 5'-OH-ACCACCCTCTTGAAGCCTTGTG-3'-OH
[SEQ NO: 2]; murine integrin .alpha.4 5'-primer,
5'-OH-CCCCTCAACACGAACAGATAGG-3'-OH [SEQ ID NO: 3]; murine integrin
.alpha.4 3'-primer; 5'-OH-GCCTTGTCCTTAGCAACACTGC-3'-OH [SEQ ID
NO:4].
[0162] PCR was performed for 30 cycles consisting of 1 min at
94.degree. C., 1 min at 55.degree. C. and 2 min at 72.degree. C.
PCR reaction mixture (total 50 .mu.l) contained 10 microliters.
First strand cDNA, 50 mM KCL, 10 mM Tris-HCL (pH 8.3), 2 mM
MgCl.sub.2, deoxy-NTP mix (0.2 mM each), the pair of primers (0.15
micromolar each) and 2 U Taq DNA polymerase (Perkin-Elmer,
Branchburg, N.J.). The PCR products were separated on 2.5% agarose
gels containing ethidium bromide and visualized under ultraviolet
light. The size of the fragments were confirmed by reference to
molecular weight markers.
Attachment of 5TGM1 Cells onto Bone Marrow Stromal Cells
[0163] For heterotypic cell-cell adhesion assays, ST2 cells (5 e
4/well) were seeded in 48-well culture plates (Costar, Cambridge,
Mass.) and cultured 48 h in alphaMEM supplemented with 10% FBS
until confluency. 5TGM1 cells (5 e 6) were labeled by incubation
with 10 microCi [methyl-3H] thymidine (New England Nuclear) for 24
h at 37.degree. C. in the culture medium. After the ST2 monolayer
was formed, it was incubated with 1% bovine serum albumin (BSA,
Sigma, St Louis, Mo.) in serum-free alphaMEM for 1 hour and
tritium-labeled 5TGM1 cells were plated onto the monolayer. The
system was incubated in the absence or presence of antibodies to
VCAM-1 or .alpha.4.beta.1 integrin at 37.degree. C. for 1 h.
Non-adherent cells were removed by washing with 5% trichloroacetic
acid twice and PBS twice, and adherent cells were solubilized in
300 microliters of 0.25 mM NaOH, neutralized with the same volume
of 0.25 mm HCl and the radioactivity was determined in a liquid
scintillation counter.
Osteoclast Formation Assay in the Co-Culture of 5TGM1 and Mouse
Bone Marrow Cells
[0164] Mouse bone marrow cells were obtained from 5-week-old male
C57BL mice as described previously (Yoneda 1993). Femurs and tibiae
were dissected aseptically and both ends cut off. Bone marrow cells
were flushed out, collected and incubated in alphaMEM supplemented
with 10% FBS (Hyclone, Logan, Utah) and 1% penicillin-streptomycin
in 100 mm-culture dishes (Becton Dickinson Labware, Bedford, Mass.)
at 37.degree. C. for 2 h. Non-adherent cells containing hemopoietic
osteoclast precursors and stromal cells were harvested. Bone marrow
cells (1 e 6) and 5TGM1 cells (1 e 3) in 300 microliters of the
culture medium were plated onto 48-well culture plates (day 0). On
day 2, 300 microliters of fresh culture medium was gently added to
each well, and on day 4, 300 microliters of spent medium was
replaced with the same volume of fresh medium. On day 6, the
cultures were fixed and stained for tartrate-resistant acid
phosphatase (TRAP) using commercial kits (Sigma). TRAP-positive
multinucleated cells with more than 3 nuclei were defined as
osteoclast-like (OC-like) cells, and manually counted under
microscope. To confirm that these OC-like cells have the capability
to resorb bone, 5TGM1 cells and marrow cells were co-cultured on
5.times.5 mm whale dentine slices in the same condition, and
resorption pits formed on these dentine slices were examined by
scanning electron microscopy as described (Yoneda 1992).
[0165] In some experiments, co-cultures of 5TGM1 myeloma cells and
marrow cells were performed using transwell inserts (Becton
Dickinson Ubware) to prevent direct contact between these two types
of cells. (2 e 6, 24-well plates, Costar). Marrow cells were plated
in the lower chambers and 5TGM1 myeloma cells (2 e 3) were then
plated in either lower (direct contact) or upper (no contact)
chambers.
Organ Cultures of 45Ca-labeled Fetal Rat Long Bones
[0166] Conditioned media harvested from 5TGM1 cultures were assayed
for bone-resorbing activity by organ cultures of 45Ca-labeled fetal
rat long bones as described previously (Mbalaviele 1995). Pregnant
rats were injected with 250 uCi of 45Ca (New England Nuclear) on
the 18th day of gestation. Radius and ulna bone shafts were
obtained from 19-day fetuses by microdissection, and precultured
for 24 h in BGJ medium (Sigma) supplemented with 0.1% BSA between
air and liquid-phase on stainless mesh grids. Bones were then
cultured in the presence of conditioned media (50% v/v) or in
control medium for 120 hours. The media were changed once at 48
hours. At the end of the culture, bones were incubated in ice-cold
5% trichloroacetic acid for 2 h, and 45Ca radioactivity in bones
and media determined in a liquid scintillation counter. Bone
resorption was quantitated as the percentage of 45Ca released into
the medium from bones as calculated by: (45Ca count in
medium)/(45Ca count in medium and bone).times.100.
Co-culture of 5TGM1 Myeloma Cells with Mouse Stromal Cell Line ST2
Cells
[0167] ST2 cells (0.5 e 6) and 5TGM1 (4 e 6) cells were plated
together onto 60-mm culture dishes (Becton Dickinson) in 10%
FBS-supplemented IMDM and cultured overnight, washed with
serum-free IMDM twice, and incubated in 5 ml of serum-free IMDM.
After 48 h, conditioned media were harvested and stored at
-70.degree. C. until use.
Effect of mAb PS2 to VLA-4 on serum IgG2b elevation in
5TGM1-bearing mice
[0168] Mice were injected with 1e 5 5TGM1 cells, which were allowed
to colonize the bone marrow. Mice were split into two groups of
three, one serving as a control group, and the second treated
biweekly beginning on day 8 with 80 .mu.g mAb PS/2 (4 mg/kg).
Levels of IgG2b, the antibody isotype produced by 5TGM1 myeloma
cells, were measured weekly from weeks 1 to 6.
Results
[0169] Expression of VCAM-1, VLA-4, and effect of Antibodies
Against VCAM-1 and VLA-4 on 5TGM1 Attachment to ST2 Monolayers
[0170] Using RT-PCR, we confirmed the expression of VCAM-1 and
integrin VLA-4 in bone marrow stromal cells and myeloma cells,
respectively. As expected, both the ST2 stromal cell line and
primary bone marrow stromal cells expressed VCAM-1, while 5TGM1 did
not. In contrast, the 5TGM1 myeloma cells expressed integrin VLA-4,
whereas stromal cells did not (data not shown). In addition, both
anti-VCAM-1 antibody (10 .mu.g/ml) and VLA-4 antibody (10 .mu.g/ml)
partially (50-80%) inhibited the attachment of 5TGM1 cells to ST2
monolayers, showing that VCAM-1 and the VLA-4 integrin expressed on
these cells are biologically functional and that these antibodies
have neutralizing activity (data not shown).
[0171] OC-like Cell Formation in the Coculture of 5TGM1 Myeloma
Cells with Mouse Bone Marrow Cells
[0172] On day 6 of the coculture of 5TGM1 cells and mouse marrow
cells, numerous TRAP-positive multinucleated osteoclast-like
(OC-like) cells were formed. These OC-like cells exhibited
resorption pit formation on dentine slices, demonstrating that
these cells were capable of resorbing bone, and possess an
osteoclastic phenotype. In experiments using transwell inserts,
formation of OC-like cells was observed when 5TGM1 cells were
cultured in direct contact with bone marrow cells. In contrast,
there was only a marginal number of OC-like cells formed when 5TGM1
cells were separated from marrow cells by the transwell membrane.
Thus 5TGM1 cells induce osteoclast formation in mixed marrow
cultures, and this induction requires direct cell-cell contact.
Effect of Antibodies Against VCAM-1 and Integrin VLA4 on OC-like
Cell Formation in the Co-culture of 5TGM1 and Marrow Cells
[0173] Both anti-VCAM-1 antibody (VCAM-1 Ab, 10 .mu.g/ml) and anti
VLA-4-integrin antibody (.alpha.4.beta.1 Ab, 10 .mu.g/ml)
dramatically inhibited OC-like cell formation. In contrast, mAb
against ICAM-1, another adhesion molecule on marrow stromal cells
implicated in stromal/myeloma interactions, had no effect on
OC-like cell formation (FIG. 1).
[0174] To determine whether this inhibition by VCAM-1 and VLA-4
mAbs was specific for 5TGM1-induced OC-like cell formation and was
not due to cytotoxicity, the effects of these antibodies were
examined on OC-like cell formation induced by 1.25
(OH).sub.2D.sub.3, a widely-used stimulator of osteoclastogenesis
in mouse bone marrow cell cultures (Takahashi 1988). Neither VCAM-1
Ab nor VLA-4 mAb inhibited OC-like cell formation induced by
vitamin D3, which itself had no effect on VCAM-1 expression in
stromal cells (data not shown).
Effect of conditioned Medium Harvested from the Co-culture of 5TGM1
and ST2 on Bone Resorption
[0175] Conditioned medium from the co-culture of 5TGM1 cells and
ST2 cells showed a marked increase in bone resorption in the fetal
rat long bone assay (FIG. 2), while conditioned medium of 5TGM1
caused only a marginal increase, as compared to control medium.
Conditioned medium from ST2 cells showed no increase in bone
resorption. Thus direct cell-cell contact via VCAM-1 and VLA-4 both
induces osteoclast-like cells and production of bone-resorbing
factors in vitro.
Effect of Recombinant Soluble VCAM-1 (sVCAM-1) on the Production of
Bone-resorbing and Osteoclastogenic Activity by 5TGM1 Cells
[0176] Conditioned medium of 5TGM1 treated with a soluble
recombinant form of VCAM-1 (sVCAM-1) increased bone resorption in
fetal rat long bones in a dose-dependent manner, while conditioned
medium obtained from untreated 5TGM1 only marginally increased bone
resorption. Soluble VCAM-1 itself had no effects on bone resorption
(data not shown). In the mouse marrow culture system, conditioned
medium harvested from 5TGM1 cells treated with sVCAM-1 showed
increased activity of OC-like cell formation, while conditioned
medium of untreated 5TGM1 exhibited only marginal activity of
OC-like cell formation (FIG. 3).
Expression of Rank Ligand mRNA in Marrow Stromal Cells (ST2)
Cultured in the Presence and Absence of Murine Myeloma Cells
[0177] Because Rank ligand appears to be an important mediator of
OCL formation and may be the final common pathway for the effects
of osteoclastogenic cytokines on OCL formation, we have examined
the expression of Rank ligand in 5TGM1 and ST2 cells both
individually and when cocultured. We find that coculture of 5TGM1
and ST2 cells induces Rank ligand mRNA in the ST2 cells.
Furthermore, while 5TGM1 cells do not express Rank ligand, they do
so when treated with sVCAM-1 (not shown). Finally, the conditioned
medium from 5TGM1 cells treated with sVCAM-1 induced Rank ligand
mRNA in ST2 cells, suggesting that the VCAM-1/VLA-4 pathway
produces a cytokine in myeloma cells that enhances Rank ligand
expression by marrow stromal cells (data not shown).
[0178] In summary, we show that 5TGM1 cells alone produce marginal
amount of activity that stimulates OC-like cell formation and bone
resorption. However, when 5TGM1 myeloma cells were co-cultured with
bone marrow cells containing hemopoietic osteoclast precursors and
stromal cells, they strongly adhered to the stromal cells and
increased OC-like cell formation. There were no OC-like cells
formed in the co-cultures in which 5TGM1 cells were prevented from
contacting stromal cells. Furthermore, in organ cultures of fetal
rat long bones the conditioned medium harvested from the cocultures
of 5TGM1 myeloma cells and ST2 bone marrow stromal cells had
increased bone resorbing activity compared with conditioned medium
of either ST2 or 5TGM1 alone. These data are consistent with the
notion that direct cell-cell contact of 5TGM1 cells with bone
marrow stromal cells is required for the production of
osteoclast-stimulating and bone-resorbing activity. We then
determined what cell adhesion molecules were involved in the direct
cell-cell interaction between 5TGM1 cells and marrow stromal cells
that is necessary for the production of osteoclastogenic activity.
Our data indicate that VCAM-1 and VLA-4 integrin play a role in
this cell-cell interaction, since neutralizing antibodies to these
two adhesion molecules profoundly decreased OC-like cell formation
in the co-cultures. The VCAM-1/VLA-4 integrin interaction is
responsible for the cell-cell communication between marrow stromal
cells and 5TGM1 myeloma cells leading to increased production of a
osteoclastogenic and bone-resorbing activity. Finally, this bone
resorbing activity in part is due to induction of Rank ligand.
Example 2
In Vivo Experiments
[0179] Our in vitro studies suggest that the interaction between
VLA-4 on myeloma cells with VCAM-1 on marrow stromal cells may play
a key role in the induction of bone resorbing activity by myeloma.
We have taken the key step of testing this hypothesis in vivo in an
animal model which accurately reflects human disease.
[0180] A. In this experiment, mice were injected with 1 e 5 5TGM1
myeloma cells, which were allowed to colonize the bone marrow. Mice
were split into two groups of three, one serving as a control
group, and the second treated biweekly beginning on day 8 with mAb
PS/2. Levels of IgG2b, the antibody isotype produced by 5TGM1
myeloma cells, were measured weekly from weeks 1 to 6. Treatment
with mAb at a dose of 80 .mu.g per injection (.about.4 mg/kg)
biweekly strongly inhibited IgG2b production, indicative of
significant inhibition of myeloma cell survival and growth in vivo
(FIG. 4). Further, the treated mice showed reduced incidence of
paraplegia (all 3 untreated animals showed paraplegia on day 42,
while only one of the treated animals showed paraplegia). The two
treated animals with no paraplegia also showed a reduction in
spleen and liver weights, which also correlate with tumor burden.
Finally, the treated animals showed a reduction in tumor area by
histology (from 6.71+/-1.74 to 0.05+/-0.08 square millimeters) in
the tibia and femurs. There was no effect of treatment on serum
calcium levels (data not shown)
[0181] B. In a parallel experiment, treatment with 40 .mu.g PS/2
mAb biweekly had no effect on IgG2b levels (not shown). These data
show that mAb PS/2 to VLA-4 strongly inhibits the growth of
established myeloma cells in a dose-dependent fashion.
[0182] C. In another in vivo experiment, 18 SCID mice were injected
with 5TGM1 myeloma cells at day 0. Four mice were treated with PBS;
4 mice were treated in a prophylactic protocol with mAb M/K 2.7
reactive against to mouse VCAM1 at a dosage of 80 .mu.g (-4 mg/kg)
every 3 days starting at day -1 (i.e. days -1, 2, 5, 8, and 11). In
a parallel experiment using the same protocol, five mice were
treated with 160 .mu.g mAb M/K 2.7. In addition, five mice were
treated with 160 .mu.g mAb M/K 2.7 starting at day 8 (i.e. days 8,
11, 14, 17, and 20) in a therapeutic protocol. Serum was taken from
all mice on days 21, 28, and 35, and animals were X-rayed then
sacrificed for histology on day 35. All three treatment groups
showed a reduction in serum IgG2b levels, indicative of reduced
myeloma cell burden (FIG. 5). A significant effect was also
observed on spleen weights at the low dose prophylactic protocol
relative to control (0.23+/-0.14 g for control versus 0.08+/-0.04
for treated). In the prophylactic high dose group 4 of 5 animals
showed a clear reduction in spleen weight, but the overall value
was not significant because of one animal with a large spleen
weight (data not presented).
[0183] D. One can investigate whether an initial high bolus dose of
.alpha.4 integrin antagonist, followed by a maintenance dose,
improves efficacy. The myeloma cells are already established in the
marrow compartment, and their tight VLA-4-dependent interaction
with VCAM-1 needs to be inhibited. Furthermore, presumably the
greater the number of established myeloma cells, the higher the
initial dose required to flush cells out into the peripheral
circulation.
[0184] A larger study with the anti-VLA-4 antibody PS/2 (PS/2=PS2)
was therefore performed. Twenty eight SCID mice were injected with
5TGM1 myeloma cells at day 0. Nine mice received no treatment; 9
mice received an isotype-matched control IgG mAb; 10 mice were
treated with mAb PS/2 to alpha 4 integrin. A different therapeutic
regimen was given, in which mice were given a high dose of mAb (200
.mu.g) on days 4, 5, and 6, then a maintenance dose of 80 .mu.g (-4
mg/kg) every 3 days starting at day 8.
[0185] There was a statistically significant reduction in serum
IgG2b when the treated group was compared to either the untreated
or control IgG-treated group at weeks 3 and 4 (data not presented).
Importantly, when the treated group was compared to either the
untreated or control IgG-treated group there was a clear effect on
survival (FIG. 6).
Example 3
Other In Vivo Experiments
[0186] Based on the information presented herein for the first
time, persons having ordinary skill in the art can readily confirm
and extend the importance of the .alpha.4 integrins and their
ligands in multiple myeloma using the murine animal model
described.
[0187] The following series of experiments are well within the
level of skill in the art based upon the present disclosure but
serve merely to exemplify, and not limit, the types of work. [0188]
1) Dose response to mAb PS/2 to determine the optimal biweekly
maintenance dose. 80 .mu.g shows good efficacy, but 40 .mu.g was
without effect. One examines higher doses up to 20 mg/kg two or
three times weekly to determine optimal dosing. [0189] 2) Patients
present with disease at different stages of severity, linked to
increased tumor burden. One examines the efficacy of mAb PS/2 given
at different times after establishment of disease, i.e., one
compares treatment initiation at 8 days (see for example FIG. 4) to
initiation after two, three, four and five weeks post inoculation
to see how late mAb can be given to provide sonic relief of
symptoms. [0190] 3) The effects of mAb MK-2 to murine VCAM-1 are
examined, following the same parameters outlined above (dosing,
timing of dosing) for mAb to VLA-4. It is anticipated that similar
dosing levels will be required to see efficacy. [0191] 4) Further
markers of myeloma progression are examined, including tumor burden
in both marrow and extramedullary sites, quantification of bone
lesions by radiographic analysis of the skeleton by
histomorphometry; measurement of rates of bone reportion by
evaluation of collagen crosslinks in plasma; measurement of
monoclonal protein production in plasma; hypercalcemia where
present; and mortality. [0192] 5) Multiple myeloma is currently
treated inefficiently with standard chemotherapeutic regimens. The
additive or synergistic effects of mAbs at optimal dosing in
conjunction with, or either before or after, dosing with
appropriate chemotherapeutic regimens is examined. [0193] 6) The
ability of a small molecule .alpha.4 integrin inhibitor that is
selective for one particular .alpha.4 integrin or is selective for
several .alpha.4 integrins at once or the ability of combinations
of such inhibitors, to mimic the effects of mAbs and block myeloma
progression is examined using the protocols and outcomes described
above. Small molecule inhibitors are delivered parenterally or
orally, in the dosing range of 0.1 to 30 mg/kg, once or twice
daily, or twice or three times weekly.
Example 4
Anti-.alpha.4 Integrin Antibody Enhanced Sensitivity of Myeloma
Cells to Melphalan (Chemotherapy Combination Experiments)
[0194] Cell-cell contact of myeloma cells with stromal cells via
.alpha.4.beta.1 integrin and VCAM-1 apparently facilitate their
arrest, proliferation, survival and production of osteoclast
activating factors in bone marrow cavity. Therefore, interference
with stromal cell/myeloma cell interactions is a potential adjuvant
intervention to enhance the efficacy of anti-cancer agents in
myeloma bone disease.
[0195] Here, we studied the effects of a neutralizing antibody to
.alpha.4 integrin (.alpha.4Ab), which disrupts stromal
cells/myeloma cell interactions, on the sensitivity of myeloma to
melphalan, a most widely-used chemotherapeutic agent for myeloma,
using the 5TGM1 mouse myeloma cells that reproducibly cause
extensive osteolysis in tumor-bearing animals. The .alpha.4Ab
(100-200 .mu.g/mouse, i.p., 2 or 3 times a week) and melphalan (50,
100, 200 .mu.g/mouse, i.p., once a week) were administered
following inoculation of 5TGM1 cells in the tail vein in male
xid-nu-bg mice. Melphalan alone at doses of 50 and 100 .mu.g/mouse
failed to suppress serum IgG2 levels, a systemic indicator of
myeloma tumor burden, whereas serum IgG2 levels were significantly
suppressed by 200 .mu.g/mouse melphalan. Combined treatment with
melphalan (50 or 100 .mu.g/mouse) and .alpha.4Ab suppressed serum
IgG2 levels to a greater extent than melphalan (200 .mu.g/mouse)
alone. Moreover, histomorphometric examination revealed that
melphalan (50 .mu.g/mouse) combined with .alpha.4Ab significantly
decreased 5TGM1 tumor volume in bone compared with melphalan (200
.mu.g/mouse) alone.
[0196] To study the role of cell-cell contact of 5TGM1 cells with
marrow stromal cells in the sensitivity to melphalan, the effects
of melphalan on 5TGM1 cells cultured in contact with the ST2 mouse
marrow stromal cells were examined. We found that 5TGM1 cells
cultured on ST2 cells exhibited increased survival and reduced
apoptosis in the presence of melphalan compared with 5TGM1 cells
cultured on tissue culture plates. Furthermore, .alpha.4Ab reduced
survival and increased apoptosis in 5TGM1 cells in these cultures.
In summary, our data show that .alpha.4Ab enhanced the sensitivity
of 5TGM1 cells to melphalan and suggested that disruption of
stromal cell/myeloma cell interactions using .alpha.4Ab is an
effective adjuvant therapy, allowing dosage reduction of
chemotherapeutic agents and thereby lowering the risk of adverse
effects.
[0197] In one particular set of experiments on the combined effects
of melphalan and anti-alpha 4 integrin Ab in 5TGM1 model, as shown
in an experimental protocol in FIG. 7, an initial high dose of 200
.mu.g mAb was given during the first week, followed by a
maintenance dose of 100 .mu.g. Melphalan was given three times.
Animals (scid/nu/bg mice) were sacrificed at day 28 and serum IgG2b
levels (a surrogate marker of tumor burden) and tumor burden in
bone were measured (histomorphometric analysis of bones). The serum
IgG2b was only modestly affected by either melphalan or
anti-.alpha.4 mAb, while the combination produced a significant
drop in IgG2b levels (FIG. 8). The tumor volume in bone marrow,
measured as a ratio of treated to untreated (so that a reduction
results in <100%), was also reduced (FIG. 9). The data were a
summary of two experiments combined.
[0198] We also examined the effects of the combined administration
of melphalan (100 .mu.g/mouse) and PS/2 mAb on survival of
5TGM1-bearing mice (scid/nu/bg mice). An experimental protocol of
these experiments is shown in FIG. 35. We evaluated survival by the
onset of hindleg paralysis (i.e., the onset of hindleg paralysis
was the end-point in these experiments). As shown in FIG. 36,
melphalan (100 .mu.g/mouse) alone had no effect on the onset of
hindleg paralysis and anti-.alpha.4Ab slightly but significantly
delayed the onset of hindleg paralysis. Combined administration of
melphalan and anti-.alpha.4Ab significantly delayed the onset of
hind-leg paralysis. Kaplan-Meier analysis of these data
demonstrated that the effects of anti-.alpha.4Ab and the
combination of anti-.alpha.4Ab and melphalan were significantly
different from the untreated. Melphalan alone, at a dose of 200
.mu.g/mouse, significantly delayed the onset. Thus, we can reduce
the dose of Melphalan to a quarter in the presence of the
anti-.alpha.4Ab, as compared to melphalan alone. These data
suggested that combined use of the anti-.alpha.4Ab enables the
reduction of the dose of melphalan, thereby decreasing adverse
effects.
[0199] We also wish to study the dose-response of melphalan with a
fixed dose of an anti-.alpha.4 mAb.
Example 5
In Vivo Experiments Conducted in Immuno-Competent C57BL/KaLwRij
Mice
[0200] Results reported below indicated a beneficial therapeutic
effect of treatment with the PS/2 mAb in a mouse model of myeloma
when initiated on day four after infusion of myeloma cells. Mice
were treated with PS/2 mAb periodically and were sacrificed for
analysis on day 20 and 21.
[0201] These experiments showed an effect of the PS/2 mAb on a
number of disease parameters treatment relative to untreated
controls: [0202] 1. Splenomegaly was reduced by 24% and tumor
burden in the spleen was reduced by 75%. A disease dependent
increase in non-myeloma cells was not affected by PS/2 mAb
treatment. [0203] 2. Tumor burden in the bone marrow was reduced by
22%. [0204] 3. Myeloma cells in the peripheral blood was reduced by
57%. [0205] 4. Increased LDH levels were reduced by 34% and AST
levels were reduced by 42%. Phosphorus levels, which decrease
slightly with disease, improved by 13%. [0206] 5. Circulating
mIgG2b levels were reduced by 50%.
[0207] At the conclusion of the experiment, circulating levels of
PS/2 mAb were undetectable in most mice. This indicated a possible
reduction in the effectiveness of mAb treatment, probably either
due to an immune response to the rat IgG or the antibody were bound
up by the expanding myeloma population.
Methods
[0208] C57BL/KaLwRij mice were inoculated, via the tail vein, with
1e6 5TGM1 myeloma cells. The mice were randomly divided into three
groups of ten animals each. One group was left untreated, one group
received PS/2 mAb treatment and the third group received rat IgG2b
isotype control mAb. The antibody treated groups received 200
micrograms of antibody on days 4, 5, and 6. After that the mice
were injected twice weekly with 200 micrograms of antibody. The
mice were sacrificed for analysis on days 20 and 21. Blood was
removed via cardiac puncture for FACS analysis of cell populations.
The plasma was used for quantification of circulating mIgG2b, PS/2
mAb and numerous blood chemistry parameters. The spleen was removed
and the wet weight recorded. A portion of the spleen was fixed in
4% paraformaldehyde for histological evaluation while the remainder
of the spleen was used for FACS analysis of cell populations. The
liver was also removed and fixed for histological evaluation. Both
femur/tibia pairs were removed. One set was fixed in 4%
paraformaldehyde for histological evaluation. The bone marrow was
flushed from the other pair, the cells were counted and the cell
populations were analyzed by FACS.
[0209] For FACS analysis of cell populations, the cells were
surface stained with a cocktail of lineage markers, which included
CD4, CD8, CD19, B220, MAC1, GR1 and NK1.1. The cells were then
double stained for cytoplasmic mIgG2b. Another set was surface
stained for B220 alone and double stained for cytoplasmic
mIgG2b.
[0210] Tumor burden was calculated by quantifying the total number
of cells in either the spleen or the bone marrow (one femur/tibia
pair) of each mouse. That number is then multiplied by the
percentage of myeloma cells, measured as mIgG2b positive cells. The
percentage of myeloma cells could also be measured as mIgG2b
positive cells and/or GFP+ cells if the 5TGM1 cells are transfected
with an expression vector containing the GFP gene and therefore the
cells express GFP (green fluorescent protein).
[0211] The levels of mIgG2b were determined by ELISA. Plasma was
incubated on plates coated with an anti mouse IgG2b antibody. The
trapped mIgG2b (mouse IgG2b) was then detected with labeled anti
mouse Fc.
[0212] Circulating PS/2 mAb levels were determined by a FACS
binding assay. 5TGM1 cells, grown in vitro, were incubated with
plasma from treated animals. The bound antibody was then detected
with labeled anti rat Fc antibody.
Results
[0213] FIG. 10 shows the spleen weights of each group. Spleen
weight increased from an average of 89 milligrams in the disease
free group to 298 milligrams in the untreated group. In the group
that received PS/2 mAb treatment, the spleens were 24% smaller than
the untreated group, while the control rat IgG2b isotype mAb
control group was only 6% smaller. Statistical analysis using the
Student t test showed that the effect of PS/2 mAb treatment was
significant (p<0.05) when compared to either the untreated or
the control rat IgG2b (rIgG2b) isotype mAb control treated group.
The same analysis showed that the small reduction in the control
rIgG2b isotype mAb control group was not significant as compared to
the untreated group.
[0214] FIG. 11 shows that PS/2 mAb treatment reduced the percentage
of myeloma cells in the spleen by 68%, as defined by the presence
of cytoplasmic mIgG2b positive cells. This reduction seen in the
PS/2 mAb treatment group was significant (p<0.001) when compared
to either the untreated or the rat IgG2b isotype mAb control
groups. FIG. 11 also shows that the mIgG2b positive cells were
almost equally split between two populations. One population was
lineage positive and the other was lineage negative. The effect of
PS/2 mAb treatment is equally effective in either the lineage
positive mIgG2b positive or lineage negative mIgG2b positive
populations. The graph also shows that the lineage positive
population was positive for the B cell marker B220. As shown in
FIG. 11, there was a disease dependent increase in the percentage
of non myeloma cells that was not affected by PS/2 mAb treatment.
These cells were most likely an expanding extramedullary
hematopoietic cell population due to the physiological stress of
bone marrow neoplasia.
[0215] FIG. 12 shows representative FACS plots of the staining for
lineage markers and cytoplasmic mIgG2b in splenocytes. The
reduction in mIgG2b positive cells in the PS/2 treated group was
clearly shown.
[0216] By using the tumor burden calculation to assess the effect
of PS/2 mAb treatment in the spleen, the reduction was 75%. The
effect of PS/2 mAb treatment was significant (p<0.001) when
compared to either control group. These results are shown in FIG.
13.
[0217] FIG. 14 shows that, at this point in disease development,
77% of bone marrow cells were now mIgG2b positive myeloma cells.
PS/2 mAb treatment resulted in a modest 15% reduction in the
percentage of cytoplasmic mIgG2b positive cells in the bone marrow
while there was a 6% reduction in the rat IgG2b isotype mAb control
group. Analysis by Student t test showed that the reduction of
mIgG2b positive cells in the PS/2 mAb treatment group was
significant (p<0.001) when compared to untreated group or the
rat IgG2b isotype mAb control group (p<0.05). As in the spleen,
the mIgG2b positive population was split between a lineage positive
and a lineage negative population and the effect of PS/2 mAb was on
both populations. FIG. 14 also shows a disease dependent depletion
in the percentage of non-myeloma cells (defined as mIgG2b negative)
in the bone marrow of all treatment groups. A higher percentage of
bone marrow cells were retained in PS/2 mAb groups as compared to
the untreated or rat IgG2b isotype mAb control groups. The PS/2 mAb
treatment was significant when compared to the untreated control
group (p<0.001) and the rat IgG2b control group (p<0.05).
[0218] FIG. 15 shows the results of the tumor burden calculations
for the bone marrow. These results showed that there was a 22%
reduction in the absolute number of mIgG2b positive cells in the
PS/2 treated group and no effect of the rat IgG2b mAb isotype
control. The effect of PS/2 mAb was significant (p<0.05) when
compared to either the untreated or rat IgG2b control groups.
[0219] The Experimental Pathology Report of the H+E stained
sections indicated that there were distinctly fewer neoplastic
infiltrates within the liver and spleen of PS/2 mAb treated animals
as compared to either the untreated or rat IgG2b control groups. In
the bone marrow, no distinctly apparent differences were noted
among the groups.
[0220] FIG. 16 shows that mIgG2b positive myeloma cells were
detected in the peripheral blood and PS/2 mAb treatment reduces
their numbers. The 57% reduction in mIgG2b positive cells with PS/2
mAb treatment was significant (p<0.001) when compared to either
the untreated or the rat IgG2b control groups.
[0221] FIGS. 17A, 17B and 17C show the results of the blood
chemistry study. As previously observed, there was an increase in
LDH (FIG. 17A) and AST (FIG. 17B) levels associated with myeloma.
PS/2 mAb treatment significantly reduced the levels of both enzymes
as shown by a Student t test p value <0.001. There was no effect
of the isotype control rat IgG2b antibody on these enzymes. Mice
with myeloma also had a slight decrease in phosphorus levels (FIG.
17C). PS/2 mAb treatment but not isotype control rat IgG2b mAb
reduced this decrease (FIG. 17C). The PS/2 mAb effect on the
phosphorus levels was significant with a Student t test p value of
<0.05
[0222] Circulating mIgG2b levels were measured by ELISA. FIG. 18
shows that at the conclusion of the experiment the untreated group
had an average of 2.4 mg/ml of circulating antibody while the PS/2
mAb group had a 50% reduction to 1.2 mg/ml. There was a small
reduction in the control rat IgG2b group to 2.0 mg/ml. The
reduction in the PS/2 mAb group was significant with a Student t
test p value of <0.05 as compared to either the untreated or rat
IgG2b control groups. The reduction in the rIgG2b control group was
not significant.
[0223] Results of the FACS binding assay, to determine circulating
PS/2 mAb levels, showed that only two of the ten animals that
received PS/2 mAb treatment had detectable levels of PS/2 mAb in
the plasma. This result indicated that the amount of circulating
PS/2 mAb was limited. This suggested that the PS/2 mAb was either
fully bound to the myeloma cells or that the antibody was cleared
by an immune response.
[0224] In summary, administration of PS/2 mAb in this mouse model
system of multiple myeloma had the following effects: splenomegaly
was reduced 24%, tumor burden in spleen by 75%, tumor burden in
marrow by 22% (FIG. 15), circulating myeloma cells by 57% (FIG. 16,
left hand panel [mIgG2b (pos) cells are the myeloma cells]),
reduced liver enzyme levels were normalized, suggesting a reduction
in liver tumor burden, and circulating plasma levels of IgG2b were
reduced 50%.
[0225] To evaluate the effects of mAb treatment at later times,
three `acute` treatment protocols were tried (FIG. 19). Mice were
injected with 5TGM1 cells, and then given mAb PS/2 on day 16 (1 day
treatment), days 16 and 17 (2 day treatment), or days 14-19 (6 day
treatment).
[0226] Remarkably, as shown in FIGS. 20A and 20B, the percentage of
IgG2b-positive (i.e. myeloma) cells was dramatically reduced in
spleen and blood with either 1 (FIG. 20A) or 2 day (FIG. 20B)
treatments. The tumor burden (i.e. the absolute number of myeloma
cells, rather than the % of total cells) was also reduced by both
treatments (FIG. 21). The 1 or 2 day treatments had no effect on
cells in bone marrow, however, whether expressed as % (FIGS. 20A
and 20B) or tumor burden (not shown). The six day protocol reduced
cells in both blood and spleen, as expected (FIG. 22). The
percentage of cells in bone marrow is unaffected, but there was a
clear effect when expressed as tumor burden (FIG. 23) because the
total cell number and myeloma cell numbers were reduced in
proportion.
[0227] These experiments are key because they suggest that: 1) very
late therapeutic intervention can be successful; 2) the therapeutic
effect of the anti-.alpha.4 mAb in the longer term experiments may
be lost over time, due to the intact immune response to the rat
antibody in the C57BL/KaLwRij mice; 3) the rapid drop in myeloma
cell numbers in blood and spleen is likely not due to decreased
growth but through effects on other parameters, such as
apoptosis.
Chemotherapy Combination Experiments
[0228] Additive/synergistic effects of the combination of PS/2 mAb
and melphalan were seen under most conditions. In these
experiments, the 5TGM1 cells injected into the animals were
transfected with an expression vector containing the GFP gene.
These myeloma cells expressed the GFP protein, allowing the myeloma
cells to be tracked directly as GFP+ myeloma cells. These myeloma
cells could also be tracked as mIgG2b positive cells. The data were
only useful on day 27. FIG. 24 shows a general experimental
protocol. FIG. 25 shows that mIgG2b levels were unaffected by PS/2
mAb or melphalan alone, but reduced (p<0.05) by the combination.
FIGS. 26A and 26B show that the percentages of GFP+ myeloma cells
in blood (FIG. 26A) and spleen (FIG. 268) were unaffected by
melphalan but reduced by PS/2 mAb. The combination further reduced
levels in spleen (FIG. 26B), but was not statistically significant
in blood (FIG. 26A) because PS/2 mAb alone was so effective.
Neither melphalan nor PS/2 mAb alone affected percentage of GFP+
myeloma cells in bone marrow (FIG. 26C), but the combination was
effective (p<0.05). If expressed as tumor burden, the
combination was once again statistically significantly better than
monotherapy in both spleen (FIG. 27A) and bone marrow (FIG. 27B).
FIG. 28 shows in tabular forms that PS/2 mAb plasma levels are
undetectable in all but one assay.
Example 6
In Vivo Experiments Conducted in Immuno-Competent C57BL/KaLwRij
Mice: VLA4 Small Molecule Experiments
[0229] To evaluate the efficacy of VLA4 binding small molecules,
BIO8809, and a related but non-VLA4 binding chemical control,
BIO9257, were used. These are PEGylated small molecules with
extended half lives. BIO8809 was described in PCT publication WO
01/12186 A1, published Feb. 22, 2001, the disclosure of which is
hereby incorporated by reference. The chemical syntheses and the
structures of BIO8809 and BIO9257 are shown in Example 7.
[0230] In these experiments, the 5TGM1 cells injected into the
animals were transfected with an expression vector containing the
GFP gene. These myeloma cells expressed the GFP protein, allowing
the myeloma cells to be tracked directly as GFP+ myeloma cells.
These myeloma cells could also be tracked as mIgG2b positive
cells.
[0231] Tumor burden was calculated by quantifying the total number
of cells in blood, the spleen or the bone marrow (one femur/tibia
pair) of each mouse. That number is then multiplied by the
percentage of myeloma cells, measured as GFP+ myeloma cells, to
yield the tumor burden.
[0232] In two experiments, BIO8809 showed statistically significant
inhibition of myeloma cell numbers in both blood and spleen, but
not in bone marrow.
[0233] In a long-term treatment experiment (the protocol is
summarized in FIG. 29), PS/2 mAb had the expected effect on the
percentage of myeloma cells in blood (FIG. 30A) and spleen (FIG.
30B), and surprisingly, in this experiment, on bone marrow as well
(FIG. 30C). The small molecule had statistically significant
effects in spleen (at all doses) (FIG. 30B), and in blood (0.03
mg/kg dose only) (FIG. 30A), but not in bone marrow (FIG. 30C) at
any dose. Replotting as tumor burden (i.e. the absolute number of
myeloma cells) did not affect the data (FIGS. 31A (blood), 31B
(spleen) and 31C (bone marrow)).
[0234] In an acute 6 day treatment experiment (see protocol
summarized in FIG. 32; 3 day treatment for small molecules), PS/2
mAb had the expected effect on the percentage of myeloma cells in
blood (FIG. 33A) and spleen (FIG. 33B). In this experiment, the
untreated control in bone marrow was very low, making these data
uninterpretable (FIG. 33C). The small molecule had statistically
significant effects in spleen (at all doses, with nice dose
response) (FIG. 33B), and in blood (all doses versus BIO9257
control; 3 mg/kg dose only versus untreated control) (FIG. 33A). In
bone marrow no dose showed significant inhibition, even versus
BIO9257 control (FIG. 33C). Replotting as tumor burden did not
affect the data (FIGS. 34A (spleen) and 34B (bone marrow)).
[0235] The small numbers (N=2 per dose) in these preliminary
experiments precluded more robust analysis or interpretation.
Nevertheless, these data already show that the small molecule
inhibitors can work as effectively as the mAb in reducing blood and
spleen myeloma burden.
Example 7
BIO-8809 and BIO-9257
[0236]
(2S,4R)-4-amino-1-[benzyloxycarbonyl]pyrrolidine-2-methylcarboxylat-
e hydrochloride (I) could be converted to the 4-tert-butoxycarbonyl
derivative II by protection of the 4-amino group with
di-tert-butyldicarbonate and subsequent removal of the
1-benzyloxycarbonyl group using hydrogenation over Pd/C catalyst.
Reaction with benzenesulfonyl chloride gave sulfonamide III.
Hydrolysis of the methyl ester with lithium hydroxide gave the
acid, which was coupled to amine salt IV using
[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate] (HATU) to give V. This methyl ester was
hydrolyzed with lithium hydroxide, followed by the deprotection of
the tert-butoxycarbonyl protected 4-amino group using acidic
conditions to give VI. This amino acid was then coupled to
methoxy-poly(ethylene glycol)-succinimidyl propionate (MW: 20,000)
to give BIO-8809 (VII).
##STR00002## ##STR00003##
[0237]
(2S,4R)-4-amino-1-[benzyloxycarbonyl]pyrrolidine-2-methylcarboxylat-
e hydrochloride (I) was coupled to
6-(tert-butoxycarbonylamino)caproic acid using HATU to give VIII.
The benzyloxycarbonyl protecting group was hydrogenated over Pd/C
catalyst, and this free amine was then coupled to benzenesulfonyl
chloride to give IX. The methyl ester was hydrolyzed with lithium
hydroxide, followed by the deprotection of the tert-butoxycarbonyl
protected 6-amino group using acidic conditions to give X. This
amino acid was then coupled to methoxy-poly(ethylene
glycol)-succinimidyl propionate (MW: 20,000) to give BIO-9257
(XI).
##STR00004##
Example 8
Other Compounds
[0238] Bisphosphonates are now standard of care in myeloma
treatment (see, e.g., Berenson et al., J Clin Oncol 16: 593-602
(1998)), and are effective at reducing bone lesions, but not tumor
burden, in a particular model (see, e.g., Dallas et al., Blood 93:
1697-1706 (1999)). Since all myeloma patients are now on
bisphosphonates, the efficacy of an VLA4 mAb or a small molecule,
such as BIO8809, in combination with bisphosphonates will be
tried.
[0239] Thalidomide is the latest drug with reported efficacy in
myeloma patients (Barlogie et al., Blood 98: 492-4 (2001)). Other
drug candidates include erythropoietin, and mAb blockers (or other
antagonists) of IL6 and IL15 (Mittelman et al., Proc Natl Acad Sci
USA, 98: 5181-5186 (2001); Tinhofer et al., Blood 95: 610-618
(2000); Bataille et al., 1989). The efficacy of an anti-VLA4 mAb or
a small molecule, such as BIO8809, in combination with any one of
these compounds, as well as with melphalan, could be tried.
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Sequence CWU 1
1
8122DNAArtificial SequenceDescription of Artificial Sequence Primer
1gctgcgcgtc accattgttc tc 22222DNAArtificial SequenceDescription of
Artificial Sequence Primer 2accaccctct tgaagccttg tg
22322DNAArtificial SequenceDescription of Artificial Sequence
Primer 3cccctcaaca cgaacagata gg 22422DNAArtificial
SequenceDescription of Artificial Sequence Primer 4gccttgtcct
tagcaacact gc 2258PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Glu Ile Leu Asp Val Pro Ser Thr 1
565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Glu Ile Leu Asp Val 1 575PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Leu
Asp Val Pro Ser 1 585PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Arg Cys Asp Xaa Cys 1 5
* * * * *