U.S. patent application number 11/699047 was filed with the patent office on 2007-06-21 for method for use of igf-binding protein for selective sensitization of target cells in vivo.
This patent application is currently assigned to Insmed, Inc.. Invention is credited to Desmond Mascarenhas.
Application Number | 20070141069 11/699047 |
Document ID | / |
Family ID | 22878903 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141069 |
Kind Code |
A1 |
Mascarenhas; Desmond |
June 21, 2007 |
Method for use of IGF-binding protein for selective sensitization
of target cells in vivo
Abstract
New methods for the treatment of human disease are provided.
IGFBP-3 is administered together with a co-administered agent to
subjects having disease, thereby alleviating the symptoms of the
disease, under conditions where administration of IGFBP-3 alone at
the maximum practicable dose has no measurable beneficial effect on
the disease condition.
Inventors: |
Mascarenhas; Desmond; (Los
Altos Hills, CA) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Insmed, Inc.
|
Family ID: |
22878903 |
Appl. No.: |
11/699047 |
Filed: |
January 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09956508 |
Sep 18, 2001 |
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11699047 |
Jan 29, 2007 |
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60233840 |
Sep 19, 2000 |
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Current U.S.
Class: |
424/155.1 ;
424/185.1; 514/109; 514/251; 514/27; 514/283; 514/34; 514/449;
514/49; 514/651 |
Current CPC
Class: |
A61K 31/337 20130101;
A61P 35/00 20180101; A61K 31/704 20130101; A61K 38/30 20130101;
A61K 31/513 20130101; A61K 31/337 20130101; A61K 2300/00 20130101;
A61K 31/513 20130101; A61K 2300/00 20130101; A61K 31/704 20130101;
A61K 2300/00 20130101; A61K 38/30 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/155.1 ;
424/185.1; 514/034; 514/027; 514/109; 514/049; 514/449; 514/283;
514/251; 514/651 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61K 31/66 20060101
A61K031/66; A61K 31/525 20060101 A61K031/525; A61K 31/7048 20060101
A61K031/7048; A61K 31/704 20060101 A61K031/704; A61K 31/337
20060101 A61K031/337; A61K 31/7072 20060101 A61K031/7072; A61K
31/138 20060101 A61K031/138; A61K 31/4745 20060101
A61K031/4745 |
Claims
1. A method of treating a patient having a tumor that is
unresponsive to IGFBP-3 alone, comprising administering to said
patient a co-administered agent or treatment regimen together with
an effective amount of IGFBP-3.
2. The method of claim 1, wherein said co-administered agent is a
chemical agent selected from the group consisting of alkylating
agents, antimetabolites, Vinca alkaloids, podophyllotoxins,
antitumor antibiotics, nitrosoureas, metallic DNA modifying
compounds and microtubule stabilizers.
3. The method of claim 1, wherein said co-administered agent is a
biological agent selected from the group consisting of nutrient
limitation, antibodies, vaccines, peptides, cytokines, receptor
ligands and nucleic acids.
4. The method of claim 1, wherein said co-administered agent is a
physical agent selected from the group consisting of heat,
pressure, osmolarity, acidity and radiation.
5. The method of claim 2, wherein the cancer is selected from the
group consisting of breast, prostate, colon, ovarian, pancreatic,
gastric and lung cancer.
6. The method of claim 5, wherein the co-administered agent is a
chemical agent selected from the group consisting of doxorubicin,
paclitaxel, methotrexate, tamoxifen, cyclophosphamide, vincristine,
etoposide, streptozotocin and 5-fluorouracil.
7. The method of claim 6, wherein said cancer is breast cancer.
8. The method of claim 6, wherein said cancer is prostate
cancer.
9. The method of claim 6, wherein said cancer is colon cancer.
10. The method of claim 6, wherein said cancer is lung cancer.
11. The method of claim 6, wherein said co-administered agent is
paclitaxel.
12. The method of claim 6, wherein said co-administered agent is
5-fluorouracil.
13. The method of claim 6, wherein said co-administered agent is
doxorubicin.
14. The method of claim 7, wherein said co-administered agent is
paclitaxel.
15. The method of claim 7, wherein said co-administered agent is
5-fluorouracil.
16. The method of claim 7, wherein said co-administered agent is
doxorubicin.
17. The method of claim 8 wherein said co-administered agent is
paclitaxel.
18. The method of claim 8, wherein said co-administered agent is
5-fluorouracil.
19. The method of claim 8, wherein said co-administered agent is
doxorubicin.
20. The method of claim 1, wherein said IGFBP-3 is administered at
about 0.01 to about 50 milligrams per kilogram total body weight
per day (mg/kg/day).
21. A method for alleviating a symptom of cancer, comprising
administering a co-administered agent or treatment regimen together
with an effective amount of IGFBP-3 to a subject having cancer,
wherein the administration of IGFBP-3 alone has no measurable
effect on the symptom.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 USC .sctn. 119(e)
to U.S. Provisional Application Ser. No. 60/233,840, filed Sep. 19,
2000, which is incorporated by reference herein.
TECHNICAL FIELD
[0002] The invention relates to the field of treatment of disease,
and particularly to the use of insulin-like growth factor binding
protein for selective sensitization of target cells in vivo.
BACKGROUND ART
[0003] Growth factors are polypeptides which stimulate a wide
variety of biological responses (e.g. DNA synthesis, cell division,
expression of specific genes, etc.) in a defined population of
target cells. A variety of growth factors have been identified,
including the transforning growth factor beta family (TGF-.beta.s),
epidermal growth factor and transforming growth factor alpha (the
TGF-.alpha.s), the platelet-derived growth factors (PDGFs), the
fibroblast growth factor family (FGFs) and the insulin-like growth
factor family (IGFs), which includes IGF-I and IGF-II. Many growth
factors have been implicated in the pathogenesis of cancer.
[0004] IGF-I and IGF-II (the "IGFs") are related in amino acid
sequence and structure, with each polypeptide having a molecular
weight of approximately 7.5 kilodaltons (kDa). IGF-I mediates the
major effects of growth hormone, and is thus the primary mediator
of growth after birth. IGF-I has also been implicated in the
actions of various other growth factors, since the treatment of
cells with such growth factors leads to increased production of
IGF-I. In contrast, IGF-II is believed to have a major role in
fetal growth. Both IGF-I and IGF-II have insulin-like activities
(hence their names), and are mitogenic (stimulate cell
division).
[0005] IGF-I has been found to stimulate the growth of cells from a
number of different types of cancer (Butler et al., 1998 Cancer
Res. 58(14):3021-3027; Favoni R E, et al., 1998, Br. J. Cancer
77(12): 2138-2147). Additionally, IGF-I has additionally been found
to exert anti-apoptotic effects on a number of different cell
types, including tumor cells (Giuliano M, et al., 1998 Invest
Ophthalmol. Vis. Sci. 39(8): 1300-1311; Zawada W M, et al., 1998,
Brain Res. 786(1-2): 96-103; Kelley K W, et al., 1998, Ann. N.Y.
Acad. Sci. 840: 518-524; Toms S A, et al., 1998, J. Neurosurg.
88(5): 884-889; Xu F, et al., 1997, Br. J Haematol. 97(2):
429-440). Prospective studies have implicated IGF-I as a risk
factor for cancers of the prostate, breast, and colon, while
IGFBP-3, the major circulatory binding protein for IGFs, appears to
have a protective effect. A variety of other observations further
support the idea that the relative balance of IGFBP-3 to other
IGF-binding proteins (notably IGFBP-2) is somehow instrumental in
the control of tumor cell growth, both in vitro and in vivo. Recent
evidence also suggests that IGFBP-3 may play a central role in the
growth and apoptosis of tumor cells in an IGF-independent
manner.
[0006] Approximately half of the 1.3 million patients diagnosed
with cancer each year in the U.S. have (or will be at risk for)
systemic disease. Chemotherapy is the most common therapeutic
approach for these patients. Most chemotherapeutic agents are
effective primarily against dividing cells, and myelosuppression is
often the dose-limiting toxicity. Chemical agents fall into several
categories and have different mechanisms of action but, at
effective doses, most have side-effects which seriously impact the
patient's quality of life. doxorubicin (ADRLAMYCIN.RTM.),
irinotecan (CPT-11), paclitaxel (TAXOL.RTM.), cisplatin, tamoxifen,
methotrexate and 5-fluorouracil are popular agents used to treat a
variety of cancers, sometimes in combination. In addition to
myelosuppression, gastrointestinal effects, mucositis, alopecia,
and (in the case of doxorubicin) cardiac toxicities are also
observed with these agents.
[0007] Clearly, it would be of interest to find ways to make tumor
cells selectively sensitive to these chemical agents. One approach
might be to target the very properties that make cancer cells
unique. Cancer cells generally evolve strategies for circumventing
the normal cell cycle checkpoint controls that target cells for
self-destruction after sustaining the kind of DNA damage typically
inflicted by chemotherapeutic agents. If such functions could be
even partially restored in tumor cells by pretreatment with a
"sensitizing" agent, one would predict that such treatment would
exert a selective effect on such cells.
[0008] Almost all IGF circulates in a non-covalently associated
complex of IGF-I, insulin-like growth factor binding protein 3
(IGFBP-3) and a larger protein subunit termed the acid labile
subunit (ALS), such that very little free IGF-I is detectable. The
ternary complex is composed of equimolar amounts of each of the
three components. ALS has no direct IGF-binding activity and
appears to bind only to the IGF/IGFBP-3 complex (Baxter et al., J.
Biol. Chem. 264(20):11843-11848, 1989), although some reports
suggest that IGFBP-3 can bind to rat ALS in the absence of IGF (Lee
et al., Endocrinology 136:4982-4989, 1995). The ternary complex of
IGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa and
has a substantially increased half-life in circulation when
compared to binary IGF/IGFBP-3 complex or IGF alone (Adams et al.,
Prog. Growth Factor Res. 6(2-4):347-356; presented October 1995,
published 1996). This ternary complex is thought to act "as a
reservoir and a buffer for IGF-I and IGF-II preventing rapid
changes in the concentration of free IGF" (Blum et al. (1991),
"Plasma IGFBP-3 Levels as Clinical Indicators" in MODERN CONCEPTS
OF INSULIN-LIKE GROWTH FACTORS, pp. 381-393, E. M. Spencer, ed.,
Elsevier, New York). While there is essentially no excess (unbound)
IGFBP-3 in circulation, a substantial excess of free ALS does exist
(Baxter, J. Clin. Endocrinol. Metab. 67:265-272, 1988).
[0009] How IGFBP-3 mediates its cellular effects is not well
understood, although there is indirect evidence to suggest that it
mediates some of the effects of p53, a well-characterized tumor
suppressor (Ferry et al., (1999) Horm Metab Res 31(2-3):192-202).
IGFBP-3 is mobilized to the nucleus of rapidly growing cells
(Schedlich, et al., (1998) J. Biol. Chem. 273(29):18347-52; Jaques,
et al., (1997) Endocrinology 138(4):1767-70). A useful step toward
defining the functional interactions of IGFBP-3 would be to
identify protein domains involved in the ability of IGFBP-3 to
specifically bind a surprisingly large array of intracellular and
extracellular targets. Known targets include: IGF-I, IGF-II,
insulin (under some conditions), acid-labile subunit (ALS),
plasminogen, fibrinogen, transferrin, lactoferrin, collagen Type
Ia, prekallikrein, RXR-alpha, viral oncoproteins, heparin, specific
proteases, cellular receptors, a number of intracellular targets
identified in two-hybrid screens, and components of the nuclear
localization trasport machinery (Mohseni-Zadeh and Binoux (1997)
Endocrinology 138(12):5645-8; Collett-Solberg, et al. (1998) J.
Clin. Endocrinol Metab. 83(8):2843-8; Rajah, et al. (1995) Prog.
Growth Factor Res. 6(2-4):273-84; Fowlkes and Serra (1996) J. Biol.
Chem. 271:14676-14679; Campbell, et al. (1999) J. Biol Chem.
274(42):30215-21; Durham, et al. (1999) Horm Metab Res
31(2-3):216-25; Campbell, et al. (1998) Am J Physiol. 275(2Pt
1):E321-31). A better understanding of these binding interactions
might allow the generation of IGFBP-3 variants lacking one or more
of these functions. The activity of these and other variants in in
vivo models may suggest novel therapeutic strategies based either
on the variant proteins themselves, mimetics, or small organic
molecules selected from combinatorial chemistry libraries created
with the information gained from a study of these variants.
[0010] A recently described mutant in which residues 228-232 of
IGFBP-3 have been substituted with the corresponding residues from
IGFBP-1 (a closely related protein) shows impaired binding to ALS,
RXR-alpha, and plasminogen (Campbell, et al. (1998) Am. J. Physiol.
275(2 Pt 1):E321-31; Firth, et al. (1998) J. Biol. Chem.
273:2631-2638). Specific proteolysis of IGFBP-3 under certain
physiological conditions such as pregnancy and critical illness can
lead to altered binding and release of its IGF ligand. The binary
complex of IGFBP-3 with IGF-I or IGF-II (both growth factors bind
IGFBP-3, with similar affinities) can extravasate across
endothelial junctions to the intercellular milieu where IGFBP-3 can
interact specifically with glycosaminoglycans, specific proteases,
and cell-surface proteins.
[0011] It should be noted that, while IGFBP-3 is the most abundant
of the IGF binding proteins ("IGFBPs"), at least five other
distinct IGFBPs have been identified in various tissues and body
fluids. Although these proteins bind IGFs, they originate from
separate genes and have distinct amino acid sequences. Unlike
IGFBP-3, other circulating IGFBPs are not saturated with IGFs.
IGFBP-3 and IGFBP-5 are the only known IGFBPs which can form the
150 kDa ternary complex with IGF and ALS. The IGF and ALS binding
domains of IGFBP-3 are thought to be in the N-terminal portion of
the protein, as N-terminal fragments of the protein isolated from
serum retain these binding activities. However, some of the other
IGFBPs have also been suggested for use in combination with IGF-I
as therapeutics.
[0012] In addition to its role as the major carrier protein for IGF
in serum, IGFBP-3 has been recently shown to have a number of
different activities. IGFBP-3 can bind to an as-yet unidentified
molecule on the cell surface, where it can inhibit the activity of
exogenously-added IGF-I (Karas et al., 1997, J. Biol. Chem.
272(26):16514-16520). Although the binding of IGFBP-3 to cell
surfaces can be inhibited by heparin, the unidentified cell surface
binding molecule is unlikely to be a heparin-like cell surface
glycosaminoglycan, because enzymatic removal of heparin
glycosaminoglycans has no effect on IGFBP-3 cell surface binding
(Yang et al., 1996, Endocrinology 137(10):4363-4371). It is not
clear if the cell surface binding molecule is the same or different
than the IGFBP-3 receptor that was identified by Leal et al. (1997,
J. Biol. Chem. 272(33):20572-20576), which is identical to the type
V transforming growth factor-beta (TGF-.beta.) receptor.
[0013] IGFBP-3, when used alone in in vitro assays, has also been
reported to promote apoptosis. Interestingly, IGFBP-3 has been
shown to promote apoptosis in cells with and without functional
type 1 IGF receptors (Nickerson et al., 1997, Biochem. Biophys.
Res. Comm. 237(3):690-693; Rajah et al., 1997, J. Biol. Chem.
272(18):12181-12188). However, there are conflicting reports as to
whether apoptosis is induced by full length IGFBP-3 or a
proteolytic fragment of IGFBP-3 (Rajah et al., ibid; Zadeh et al.,
1997, Endocrinology 138(7):3069-3072). More recently, a wealth of
unpublished data gathered in a number of laboratories fails to
support some of the claims made in the above publications. In in
vivo models tested to date, infused IGFBP-3 protein alone has
showed mixed results in limiting tumor growth.
[0014] U.S. Pat. No. 5,681,818 claims the administration of IGFBP-3
for controlling the growth of somatomedin dependent tumors in the
treatment of cancer. U.S. Pat. No. 5,840,673 also describes the
indirect intracellular modulation of IGFBP-3 levels as a method for
controlling tumor growth. U.S. Pat. No. 6,015,786 discloses the use
of IGFBP-3 complexed with mutant IGF for the treatment of
IGF-dependent tumors. However, each of these patents discloses a
direct in vivo effect of administered IGFBP-3 protein on tumor
growth. None of these patents envisages a situation where IGFBP-3
has no effect on tumors on its own, yet sensitizes tumors to the
action of other agents. Numerous publications (Williams, et al.,
Cancer Res 60(1):22-7, 2000; Perks, et al., J Cell Biochem
75(4):652-64, 1999; Maile et al., Endocrinology 140(9):4040-5,
1999; Gill, et al., J Biol Chem 272(41):25602-7, 1997) further
demonstrate combined effects of IGF binding proteins, radiation and
ceramide on cultured cells. However, it is difficult or impossible
to extrapolate from tissue culture results to effectiveness in
vivo. In one report (Portera et al, Growth Hormone & IGF
Research 2000, Supplement A, S49-S50, 2000) IGFBP-3 combined with
CPT-11 showed additive effects in a colon cancer model both in vivo
and in vitro, but IGFBP-3 alone also showed effects on tumor growth
in this model. At the present time, a widely held belief among
skilled practitioners in the field is that IGFBP-3 alone may
sometimes control tumor growth directly. No one has shown that
systemically administered IGFBP-3 can sensitize tumor cells in
animals to the action of co-administered agents, without inhibiting
tumor growth when used on its own at similar doses.
[0015] Such a distinction is of considerable practical importance.
Among other things, it means that many types of tumors that may
appear to be recalcitrant to IGFBP-3 treatment when used singly, or
to some other agent used on its own, may in fact be quite
susceptible to the combination. Unless this fact is appreciated,
even the testing of certain combinations of substances which, on
their own, are known to have no effects on tumor growth, may never
be undertaken.
[0016] IGF-I and IGFBP-3 may be purified from natural sources or
produced by recombinant means. For instance, purification of IGF-I
from human serum is well known in the art (Rinderknecht et al.
(1976) Proc. Natl. Acad. Sci. USA 73:2365-2369). Production of
IGF-I by recombinant processes is shown in EP 0 128 733, published
in December of 1984. IGFBP-3 may be purified from natural sources
using a process such as that shown in Baxter et al. (1986, Biochem.
Biophys. Res. Comm. 139:1256-1261). Altematively, IGFBP-3 may be
synthesized by recombinantly as discussed in Sommer et al., pp.
715-728, MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS (E. M.
Spencer, ed., Elsevier, New York, 1991). Recombinant IGFBP-3 binds
IGF-I in a 1:1 molar ratio.
[0017] Topical administration of IGF-I/IGFBP-3 complex to rat and
pig wounds is significantly more effective than administration of
IGF-I alone (Id.). Subcutaneous administration of IGF-I/IGFBP-3
complex to hypophysectomized, ovariectomized, and normal rats, as
well as intravenous administration to cynomolgus monkeys,
"substantially prevents the hypoglycemic effects" of IGF-I
administered alone (Id.).
[0018] The use of IGF/IGFBP-3 complex has been suggested for the
treatment of a wide variety of disorders (see, for example, U.S.
Pat. Nos. 5,187,151, 5,527,776, 5,407,913, 5,643,867, 5,681,818 and
5,723,441, as well as International Patent Applications Nos. WO
95/03817, WO 95/13823, and WO 96/02565. IGF-I/IGFBP-3 complex is
also under development by Insmed Pharmaceuticals, Inc., as a
treatment for several indications, including diabetes and recovery
from hip fracture surgery.
[0019] For practitioners skilled in the art, the complex of IGF-I
and IGFBP-3 is generally considered to be a different compound, and
to have different biological effects, than IGFBP-3 alone.
[0020] While there are a large number of cytotoxic drugs available
for the treatment of cancer, these drugs are generally associated
with a variety of serious side effects, including alopecia,
leukopenia, mucositis. Accordingly, there is a need in the art for
cancer therapies that do not induce the serious side effects
associated with conventional cytotoxic chemotherapy. One method for
achieving this goal is to make target cells (such as tumor cells)
selectively sensitive to cytotoxic drugs, thereby permitting the
effective use of such drugs at lower doses not associated with
serious side effects.
[0021] A number of reports claim IGFBP-3 alone can cause apoptosis
in tumor cells in culture, and others have described additive
effects of combining IGFBP-3 treatment with various chemical agents
in tissue culture (cited above). However, it is unclear how these
effects relate, if at all, to in vivo models.
[0022] The key assumption in all of the above examples, is that the
efficacy of combination treatments involving administration of
IGFBP-3 and- other agents is investigated only after IGFBP-3 has
shown efficacy on its own. Given this state of thinking, which is
established in the field at this time, it is therefore unlikely
that effective combination regimens will be identified unless
IGFBP-3 alone is shown to have efficacy in the first place.
Synergistic effects with co-administered agents showing marginal
efficacy themselves would be even harder to identify.
[0023] Herceptin, a humanized antibody used in the treatment of
breast cancer, has exemplified the use of large proteinaceous
molecules to extend the therapeutic efficacy of chemical agents
(Pegram, et al. (1998) J. Clin. Oncol. 16(8):2659-71). However,
this molecule was approved for clinical use based on its own
efficacy on tumors and survival, when used alone. Additive effects
have been observed when this molecule is administered in
combination with chemical agents.
DISCLOSURE OF THE INVENTION
[0024] The inventor has surprisingly found that IGFBP-3 may not be
generally effective in controlling tumor growth when used alone, as
was previously postulated by several investigators. Nevertheless,
the inventor has found that IGFBP-3 is effective in synergistically
sensitizing tumor cells to the stressful effects of co-administered
agents such as adriamycin and taxol, when administered systemically
to animals in doses at which IGFBP-3 itself is ineffective in
controlling tumor growth when administered alone. This was true
even when the dose of IGFBP-3 used in the experiment approximated
the maximum practical dose of IGFBP-3 usable in a clinical scenario
for economic, technical, or other reasons.
[0025] This finding was unexpected because IGFBP-3 had previously
been thought to control tumor cell growth on its own (Sommer et
al., supra), based on the in vitro data. The synergistic
sensitization phenomenon disclosed herein may also explain why
IGFBP-3 has been sporadically associated with negative effects on
cell survival. The combination of stress or damage (whether
chemically induced, biologically induced, physically induced, or
otherwise effected by cell culture conditions) and IGFBP-3
administration may be the true cause of apoptosis in such cases.
One way to show this directly is to place cultured tumor cells
under nutritional stress by growing them at sub-optimal nutrient
concentrations. By the line of thinking posited above, the
subsequent addition of IGFBP-3 should have a dramatically greater
effect on cell death (apoptosis), than the same dose of IGFBP-3
added to the same cell line growing under normal nutrient
concentrations.
[0026] Disclosed herein are methods for alleviating the symptoms of
disease. In one embodiment, an effective amount of IGF-binding
protein or derivative thereof is systemically co-administered with
a chemotherapeutic agent to a subject having cancer, thereby
alleviating the symptoms of the cancer.
[0027] In another embodiment, IGF-binding protein or a derivative
thereof is systemically co-administered with other biological
modifiers such as ligands of retinoid or thyroid receptors, or
antibodies capable of binding target cell molecules, to the subject
with disease.
[0028] In yet another embodiment, IGF-binding protein or derivative
thereof is administered as described in the other embodiments, but
the administration occurs indirectly, using a gene sequence
delivered by a viral vector or other vehicle, or using an inducer
or antagonist.
[0029] In certain aspects, the invention provides methods for
alleviating the symptoms of cancer, by administering a
co-administered agent together with an effective amount of
insulin-like growth factor binding protein-3 (IGFBP-3) or
derivative thereof to a subject having cancer under conditions
wherein the administration of IGFBP-3 or derivative alone does not
alleviate said symptoms of cancer.
[0030] In some embodiments, the co-administered agent is a chemical
agent selected from the group consisting of alkylating agents,
antimetabolites, Vinca alkaloids, podophyllotoxins, antitumor
antibiotics, nitrosoureas, metallic DNA modifying compounds and
microtubule stabilizers, a biological agent selected from the group
consisting of nutrient limitation, antibodies, vaccines, peptides,
cytokines, receptor ligands and nucleic acids, or a a physical
agent selected from the group consisting of heat, pressure,
osmolarity, acidity and radiation. Preferred co-administered agents
include chemical agents selected from the group consisting of
doxorubicin, paclitaxel, methotrexate, tamoxifen, cyclophosphamide,
vincristine, etoposide, streptozotocin and 5-fluorouracil.
[0031] In certain embodiments, the cancer treated is breast,
prostate, colon, ovarian, pancreatic, gastric or lung cancer.
[0032] In some embodiments, the IGFBP-3 is administered at about
0.01 to about 50 milligrams per kilogram total body weight per day
(mg/kg/day).
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows sequences IGFBP-3 in single letter amino acid
code. FIG. 1A shows the amino acid sequences of native human
IGFBP-3 (Ala.sub.5 allelic variant). FIG. 1B shows [N109D]-hIGFBP-3
derivative (Ala.sub.5 allelic variant).
[0034] FIG. 2 depicts the results of the experiment described in
Example 1. The upper panel shows the effects of IGFBP-3 and
5-fluoruracil (5-FU) on 16C mammary adenocarcinoma cells implanted
in mice. At each time point the columns show, left to right,
control, IGFBP-3 alone, 5-FU alone, and IGFBP-3 plus 5-FU. The
lower panel shows the effects of IGFBP-3 and doxorubicin
(ADRIAMYCIN.RTM.) on 16C mammary adrenocarcinoma cells implanted in
mice. At each time point the columns show, left to right, control,
IGFBP-3 alone, doxorubicin alone, and IGFBP-3 plus doxorubicin.
[0035] FIG. 3 depicts the results of the experiment described in
Example 2. The upper panel shows the effects of IGFBP-3 and
paclitaxel (TAXOL.RTM.) on MDA-MB-231 cells implanted in mice. At
each time point, the columns show, left to right, control, IGFBP-3
alone, paclitaxel alone, and IGFBP-3 plus paclitaxel. The lower
panel shows the effects of IGFBP-3 and cisplatin on MDA-MB-23 1
tumors implanted in mice. At each time point, the columns show,
left to right, control, IGFBP-3 alone, cisplatin alone, and IGFBP-3
plus cisplatin.
BEST MODE FOR CARRYING OUT THE INVENTION
[0036] Disclosed herein are new methods for the treatment of
cancer. An effective amount of IGFBP-3 and a co-administered agent
are systemically administered to a subject suffering from cancer,
thereby alleviating the symptoms of the cancer. The combined
effects of the IGFBP-3 and the chemical insult synergistically slow
the growth rate of cancer, thereby alleviating the symptoms of, or
slowing the progression of the cancer. While not wishing to be
bound by any particular theory, the inventor believes that the
administration of IGFBP-3 restores a more "normal" response within
tumor cells to cell cycle checkpoints. When simultaneously stressed
by chemical or radiation damage, the tumor cells become more
responsive to apoptotic signals in the presence of IGFBP-3.
[0037] The inventor has surprisingly found that systemic
administration of IGFBP-3 alone at the substantial dose of 4 mg/kg
for 21 days was completely ineffective at slowing tumor growth in
more than one animal model of cancer. This was surprising because
it was believed that IGFBP-3 alone could cause the death of some
tumor cells. The dose chosen is likely to be close to the maximum
practicable dose that would be systemically delivered in a human
clinical setting. The maximum practicable dose is determined by the
cost, pharmacokinetics, toxicology, solubility of the formulation,
route of administration, and other practical considerations.
Definitions
[0038] As used herein, the terns "IGF-binding protein" and "IGFBP"
refer to natural and derivative molecules based on any of the six
human insulin-like growth factor binding proteins 1 through 6.
"Derivatives" refers to point mutants, deletion mutants, peptides,
peptidomimetics, small organic molecules, nucleic acids (such as
RNAS), or any other molecules which retain, modify or mimic those
structural properties of IGFBPs that are relevant to their ability
to sensitize target cells. Derivatives also include nucleic acid
molecules encoding IGF-binding proteins, such as DNA gene
sequences.
[0039] The term "co-administered agent", as used herein, refers to
a chemical agent; a biological agent such as an antibody, vaccine,
nutrient, cytokine, nucleic acid or receptor ligand such as growth
factor, retinoid or thyroid receptor ligand; and a physical agent,
such as radiation, acidity and heat. Co-administered agents
preferably have an anti-tumor activity when administered in the
absence of IGFBP.
[0040] "Chemical agents" include all common chemotherapeutic agents
such as alkylating agents (e.g. busulfan, cyclophosphamide,
ifosfamide), antimetabolites (e.g. Ara-C, 5-fluorouracil,
methotrexate), Vinca alkaloids (e.g. vinblastine, vincristine),
podophyllotoxins (e.g. VM-26, etoposide), antibiotics (e.g.
bleomycin, doxorubicin/ADRIAMYCIN.RTM.), nitrosoureas (e.g. BCNU,
streptozotocin), and metallic DNA modifying compounds (e.g.
carboplatin, cisplatin), and microtubule stabilizers (e.g.,
paclitaxel/TAXOL.RTM.). Chemical agents also include chemical
compounds that directly affect a targeted receptor by reducing
levels of the cognate ligand, by acting on the targeted receptor or
acting on the signalling pathway of the targeted receptor. For
example, the thyroid axis may be indirectly manipulated via
antagonists such as thyroid axis antagonists. As an example, the
term "thyroid axis antagonist" refers to a compound which acts to
decrease thyroid hormone activity in a subject. Thyroid axis
antagonists include 6-n-propyl-2-thiouracil (propylthiouracil or
PTU), methimazole, carbimazole, and other compounds known to the
art to reduce thyrotropic hormones, thyroid hormones, or thyroid
receptor signaling.
[0041] The term "treatment regimen" refers to a course of therapy.
Treatment regimens may utilize a single agent such as a single
chemical agent, but more typically involve two or more different
agents (e.g., combination therapy with multiple different cytotoxic
chemotherapry agents), and may involve two or more different types
of agents (e.g., administration of a chemical agent such as
paclitaxel in combination with a physical agent such as ionizing
radiation).
[0042] The term "alleviating", as used herein, refers to an
improvement, lessening, or diminution of a symptom of cancer.
"Alleviating" also includes slowing or halting progression of a
symptom.
[0043] The term "subject", as used herein, refers to a vertebrate
individual, including avian and mammalian individuals, and more
particularly to sport animals (e.g., dogs, cats, and the like),
agricultural animals (e.g., cows, horses, sheep, and the like), and
primates (e.g., humans).
[0044] As used herein, the term "comprising" and its cognates are
used in their inclusive sense; that is, equivalent to the term
"including" and its corresponding cognates.
[0045] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0046] IGF-binding protein, in combination with agents causing
cellular damage or stress, may be used to treat any cancer,
preferably carcinomas such as breast, prostate, colon and lung
cancers. Treatment with IGF-binding protein in combination with an
agetn which causes cellular damage or stress alleviates at least
one symptom of the cancer being treated. The particular symptom
alleviated will, as will be understood by one of skill in the art,
vary depending on the type of cancer, location of the primary tumor
and any local, regional, or distant spread, and the natural history
of the particular cancer. Alleviation of symptoms by administration
of an IGF-binding protein in combination with an agent may include,
but are not limited to, reduction in or elimination of tumor size,
reduction in or elimination of tumor-related pain, prevention of or
an increased time to disease progression, elimination or reduction
of symptoms secondary to the cancer (e.g., reduction or elimination
of a bowel obstruction due to a colon tumor), increased disease
free interval, and increased overall survival time.
[0047] IGF-binding protein for use in accordance with the instant
inventive methods may be derived from any species, although
species-matched IGF-binding protein (i.e., IGF-binding protein or
derivative based on the native sequence from the same species as
the subject to which the IGF-binding protein is to be administered)
is preferred. IGF-binding protein for use in the instant invention
is uncomplexed IGF-binding protein, that is, administered in the
absence of IGF (e.g., not administered as IGF-I/IGFBP-3 complex),
and is preferably administered without any IGF protein.
[0048] One of the naturally occurring protein sequences for IGFBP-3
is shown in FIG. 1. Human IGFBP-3 is found in two naturally
occuring allelic variants; alanine may be found at position 5 of
the mature protein (shown in FIG. 1a), or alternately glycine may
be found in this position. Additionally, other variants of IGFBP-3
may be created. For example, [N109D]-IGFBP-3 is a derivative of
IGFBP-3 that has an amino acid sequence alteration at position 109
of the mature sequence but behaves very similarly to wild type
IGFBP-3 in most assays tested to date. Point mutant derivatives
also include mutants selectively debilitated in their ability to
bind IGF-I, IGF-II, or any other known ligands of IGFBPs. For
example, it has been shown that point mutations at positions
corresponding to one or more of the conserved or semi-conserved
residues Val.sub.49, Tyr.sub.50, Pro.sub.62, Lys.sub.68,
Pro.sub.69, Leu.sub.70, Ala.sub.72, Leu.sub.73, and Leu.sub.74 of
IGFBP-5 may be debilitated in IGF-I binding. Many of these residues
are well-conserved in the other IGF-binding proteins as well.
Mutations at positions 228 and 230 the mature sequence of IGFBP-3
are believed to affect nuclear translocation and binding to
extracellular matrix proteins such as collagen.
[0049] Deletion mutants of IGFBP-3 or peptide derivatives based on
parts of the IGFBP-3 sequence, may also be used. The IGFBP-3
molecule consists of 264 amino acids and has three major structural
domains. The cysteine-rich amino terminal domain (roughly the first
100 amino acids of the mature sequence) is known to be essential
for high-affinity binding of IGFs. The middle domain (about 80
amino acids) has no cysteine residues, and is very susceptible to
proteases. It may also play a role in binding specific cellular
receptors. The carboxy-terminal domain (about 80 amino acids) is
also cysteine-rich and contains sequences essential for binding
extracellular matrix molecules such as heparin and collagen, serum
molecules such as ALS, plasminogen, and fibrinogen, nuclear
receptors such as RXR, and importin. Methods for nucleic acid
manipulation, protein expression and protein purification for
obtaining deletion or point mutants are known in the art.
[0050] Once a domain of IGFBP-3 has been defined by point mutation
or deletion analysis as necessary and sufficient for a particular
biological activity, such as the sensitization of target cells, it
is possible to design smaller molecules, such as peptides,
consisting of part of the IGFBP sequence. For example, one or both
of the sequences: TABLE-US-00001
(H2N)...DKKGFYKKKQCRPSKGRKRGFCW...(COOH); and
(H2N)...QCRPSKGRKRGFCW...(COOH)
may be sufficient to mimic some of the biological effects of
IGFBP-3.
[0051] Also disclosed herein is a methodology for creating and
recovering such derivative molecules. As disclosed in Example 5,
the inventor has discovered the presence of a metal-binding motif
in the IGFBP-3 molecule, allowing practical recovery of domains
containing this motif. Also disclosed are methods for generating
properly folded sub-domains of IGFBP-3, by engineering target sites
for a specific protease at strategic locations in the IGFBP-3
sequence, expressing the construct, and cleaving the expressed
protein with the cognate protease. The significance of this
approach in the case of IGFBP-3 is that numerous unsuccessful
attempts have already been made, in a number of laboratories, to
express truncated segments of IGFBP-3 in properly folded form. To
date, these have proved relatively unsuccessful in generating such
properly folded molecules as a major percentage of the total
expressed product. By generating the intact molecule and cleaving
it post facto, it is possible to generate folded domains with
substantially higher efficiencies.
[0052] Small organic molecules designed or selected based on the
IGFBP-3 sequence may also be created by computational and other
methods. Any of these derivative molecules may be assayed for the
desired biological activities, including the ability to sensitize
target cells to chemical treatments. Based on the results of these
assays, a small number of IGFBP-3 mutants or derivatives with
altered characteristics may be selected for clinical testing in the
context of human disease.
[0053] The IGF-binding protein or derivative is normally produced
by recombinant methods, which allow the production of all possible
variants in IGFBP sequence. Techniques for the manipulation of
recombinant DNA are well known in the art, as are techniques for
recombinant production of proteins (see, for example, in Sambrook
et al., MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-3 (Cold
Spring Harbor Laboratory Press, 2 ed., (1989); or F. Ausubel et
al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing and
Wiley-Interscience: New York, 1987) and periodic updates).
[0054] Preferably, the IGF-binding protein or derivative is
produced using a bacterial cell strain as the recombinant host
cell. An expression construct (i.e., a DNA sequence comprising a
sequence encoding the desired IGF-binding protein or derivative
operably linked to the necessary DNA sequences for proper
expression in the host cell, such as a promoter and/or enhancer
elements at the 5'end of the construct and terminator elements in
the 3'end of the construct) is introduced into the host cell. The
DNA sequence encoding the IGF-binding protein or derivative may
optionally linked to a sequence coding another protein (a "fusion
partner"), to form a fusion protein. Preferably, the DNA sequence
encoding the IGF-binding protein or derivative is linked to a
sequence encoding a fusion partner as described in U.S. Pat. No.
5,914,254. The expression construct may be an extrachromosomal
construct, such as a plasmid or cosmid, or it may be integrated
into the chromosome of the host cell, for example as described in
U.S. Pat. No. 5,861,273.
[0055] IGF-binding protein or derivative is preferably administered
by parenteral administration, including but not limited to
intravenous (IV), intraperitoneal (IP), intramuscular (IM),
subcutaneous (SC), intradermal (ID), transdermal, inhaled, and
intranasal routes. IV, IP, IM, and ID administration may be by
bolus or infusion administration. For SC administration,
administration may be by bolus, infusion, or by implantable device,
such as an implantable minipump (e.g., osmotic or mechanical
minipump) or slow release implant. The IGF-binding protein or
derivative may also be delivered in a slow release formulation
adapted for IV, IP, IM, ID or SC administration. Inhaled
IGF-binding protein or derivative is preferably delivered in
discrete doses (e.g., via a metered dose inhaler adapted for
protein delivery). Administration of IGF-binding protein or
derivative via the transdermal route may be continuous or
pulsatile. Administration of derivatives may also occur orally.
[0056] For parenteral administration, compositions of IGF-binding
protein or derivative may be in dry powder, semi-solid or liquid
formulations. For parenteral administration by routes other than
inhalation, the IGF-binding protein or derivative is preferably
administered in a liquid formulation. IGF-binding protein or
derivative formulations may contain additional components such as
salts, buffers, bulking agents, osmolytes, antioxidants,
detergents, surfactants, and other pharmaceutical excipients as are
known in the art.
[0057] IGF-binding protein or derivative is administered to
subjects having cancer at a dose of about 0.01 to about 40
mg/kg/day, more preferably about 0.1 to about 10 mg/kg/day, more
preferably 0.5 to about 4 mg/kg/day, even more preferably about 1
to about 2 mg/kg/day.
[0058] As an alternative to administration of IGFBP or a derivative
thereof, a nucleic acid construct encoding the IGFBP may be
administered. The construct contains a polynucleotide sequence
encoding the IGFBP, and normally contains sequences operably linked
to the IGFBP sequence which result in expression and translation of
the IGFBP sequence in the cells (e.g., a promoter/enhancer,
translation initiation site, polyadenylation signal, etc.),
although constructs which are designed to integrate into the cell
chromosome are also contemplated (e.g., where the construct
contains sequence which facilitates integration into the host
chromosome, such as sequences homologous to the recipient cells'
chromosome flanking the IGFBP sequence).
[0059] Methods of gene transfer are well known in the art, and
include in vitro methods (e.g., transformation of cultured cells,
preferably autologous cells, which are reintroduced into the
subject), ex vivo methods (e.g., transformation of cells which have
not been cultured in vivo, preferably autologous cells, with are
reintroduced into the subject), and in vivo methods (e.g.,
transformation of cells in situ by administration of a nucleic acid
construct to the subject). Methods for accomplishing such gene
transfer are well known in the art, and include standard
transformation methods including calcium phosphate transformation,
ballistic transformation, electroporation, lipid-mediated
transformation, naked DNA transfer, and viral-mediated
transfer.
[0060] The IGF-binding protein or derivative is administered to the
subject together with one or more of the following co-administered
agents: a chemotherapeutic agent; an antibody, physical stress,
such as radiation; or a ligand of a receptor present on the target
cells, such as retinoid receptors and thyroid receptors. The
administration of the two agents may be simultaneous, overlapping,
or separated in time, as long as the subject experiences exposure
to both agents at the same time. Where the two agents are
formulated for the same route and schedule of administration, the
administration is preferably simultaneous or nearly simultaneous
(e.g., concurrent or serial injections). However, in some
embodiments, the routes and schedules of administration for the two
agents will be different, making simultaneous administration
inconvenient. A subject will be considered to have been
administered both agents if the subject experiences simultaneous
systemic exposure to both compounds, regardless of when or how the
compounds were administered.
[0061] In methods requiring the administration of co-administered
agent with the IGF-binding protein or derivative, the dose of the
co-administered agent is normally titrated for the individual
subject, as is known in the art for that agent. Co-administered
agents may be produced in any formulation known to the art,
including parenteral and oral dosage forms. Oral formulations are
preferred, but parenteral formulations are also acceptable, and may
be more convenient in an in-patient setting. Formulations for
parenteral administration are generally formulated as liquids, but
may also be in gel or solid depot form. Formulations for oral
administration are generally in tablet or capsule form, although
syrups and liquids are also acceptable. Formulations of
co-administered agents generally include excipients, such as salts,
buffers, bulking agents, detergents, binding agents, surfactants,
stabilizers, preservatives, anti-oxidants, lubricants, coating
agents, and other pharmaceutically acceptable excipients as are
known in the art.
[0062] The dosage and mode of administration of the co-administered
agent should be adjusted according to the identity, formulation,
route of administration and other relevant characteristics
pertaining to the co-administered agent, as is known in the
art.
[0063] Inducers and antagonists would be administered in a similar
way. As an example: Where the antagonist is propylthiouracil, the
dose of propylthiouracil may be from 1 to 400 mg/day. A subject is
normally initiated with a dose of 50 to 400 mg/day, typically
divided into three equal doses, and maintained at 50 to 100 mg/day
divided into two or three equal doses. For methimazole and
carbimazole, the dose may be from 0.1 to 50 mg/day. Typically, a
subject is initiated with 5 to 50 mg/day, and maintained on 1 to 5
mg/day.
[0064] As will be understood by those of skill in the art, the
symptoms of cancer alleviated by the instant methods, as well as
the methods used to measure the symptom(s) will vary, depending on
the particular cancer and the individual patient. For solid tumors,
the most significant symptom is usually tumor size (either primary
tumor size or metastasis size). Tumor size may be measured by any
method known in the art, including radiological methods (e.g.,
x-ray, CT scan, MRI, PET scan, and the like), markers associated
with tumor size (e.g., serum prostate specific antigen, or PSA,
levels in prostate cancer, carcinoembryonic antigen, or CEA, levels
in colon and other cancers, and the like), direct physical
measurement, etc. Because many cancers, particularly advanced stage
cancers, are physically debilitating, performance-based
measurements such as activities of daily living (ADLs) or Karnofsky
performance score are useful in measuring a patient's response to
treatment. For tumors that secrete one or more hormones, serum
levels of the hormone secreted by the tumor may be used as a marker
of tumor size.
[0065] Many tumors induce physical symptoms due to their anatomic
location, and these secondary symptoms are considered symptoms of
the cancer. For example, bone tumors frequently cause significant
bone pain, and colorectal tumors can result in blockage of the
colon. These secondary symptoms can be measured using appropriate
methods known in the art (e.g., visual scales for measurement of
pain).
[0066] The invention also provides kits for use in the methods of
the invention. Kits of the invention comprise one or more
containers comprising IGFBP, and may optionally comprise one or
more containers of a co-administered agent and/or a set of
instructions, generally written instructions, relating to the use
of IGFBP with a co-administered agent for the treatment of
cancer.
[0067] The kits comprise IGFBP (and optional co-administered agent)
in any convenient, appropriate packaging. For example, if the IGFBP
or the co-administered agent is a dry formulation (e.g., freeze
dried or a dry powder), a vial with a resilient stopper is normally
used, so that the IGFBP or co-administered agent may be easily
resuspended by injecting fluid through the resilient stopper.
Ampoules with non-resilient, removable closures (e.g., sealed
glass) or resilient stoppers are most conveniently used for liquid
formulations of IGFBP or co-administered agent. Also contemplated
are packages for use in combination with a specific device, such as
an inhaler, nasal administration device (e.g., an atomizer) or an
infusion device such as a minipump. While it is contemplated that
the IGFBP and the co-administered agent can be supplied as a
mixture, it is generally preferred that the IGFBP and the
co-administered agent be supplied in separate containers.
[0068] The instructions relating to the use of IGFBP and
co-administered agent for the treatment of cancer generally include
information as to dosage, dosing schedule, and route of
administration for the treatment. The containers of IGFBP (and
optional co-administered agent) may be unit doses, bulk packages
(e.g., multi-dose packages) or sub-unit doses. Instructions
supplied in the kits of the invention are typically written
instructions on a label or package insert (e.g., a paper sheet
included in the kit), but machine-readable instructions (e.g.,
instructions carried on a magnetic or optical storage disk) are
also acceptable.
[0069] The patents, patent applications, and publications cited
throughout the disclosure are incorporated herein by reference in
their entirety.
EXAMPLES
Example 1
Treatment of Nutritionally Stressed A293 Kidney Cells with
IGFBP-3
[0070] Human embryonal kidney A293 cells were grown in Dulbecco's
Modified Eagle Medium (D-MEM) supplemented with fetal calf serum at
2%, 4%, 6%, or 8%. When the cells reached 80-85% confluency (cell
titer approximately 2.1.times.10.sup.6 cells per plate), 5 ug of
IGFBP-3 or buffer control was added to each plate. The cells were
incubated at 37 C overnight. The next day the medium was removed
and the cells were rinsed with trypsin-EDTA (0.25% trypsin, 1 mM
EDTA) plus 1.times. phosphate buffered saline. The cells were
cetrifuged and the supernatant was removed. ApoAlert caspase-3
assay kit from Clontech Inc (Palo Alto, Calif.) was used to measure
apoptosis. The cells were resuspended in 50 ul of chilled cell
lysis buffer and incubated on ice for 10 minutes. The resulting
cell lysates were centrifuged at 14000 rpm in a Beckman
microcentrifuge for 3 minutes at 4 C. The supernatant was
transferred to new tubes and 50 ul of 2.times. reaction buffer/DTT
plus 5 ul of 1 mM caspase-3 substrate was added to each tube. After
incubating at 37 C for 1 hour in a water bath, the samples were
read at 405 nm in a microplate reader. The results of this
experiment are shown in FIG. 2.
Example 2
Treatment of 16C Mammary Tumors with IGFBP-3 and Co-Administered
Agents
[0071] Female C3H mice (8-11 animals per group) received early SC
implants of 16C mammary adenocarcinoma tumor fragments (near 100%
"take") and were treated with vehicle, [N109D]-IGFBP-3 (4 mg/kg/day
SC .times.21), 5-FU (10 mg/kg/day IP .times.5), adriamycin (2
mg/kg/dose IV on day 1 and day 8 post-implant), or the indicated
combinations. Tumors were measured twice weekly. Animals were
sacrificed at Day 21. The doses of doxorubicin and 5-fluorouracil
used in this experiment were chosen to be marginally effective,
based on previous experience with these chemical agents in this
model. [N109D]-IGFBP-3 significantly (p<0.01) potentiated the
effects of both agents, but had no measurable effects on its own.
The results are shown graphically in FIG. 2 Table 1 below shows the
delay (in days) in tumor growth (endpoint of 1500 mg tumor weight).
TABLE-US-00002 TABLE 1 Treatment Group Days Delay CONTROL 0
[N109D]-IGFBP-3 ALONE -0.2 ADRIAMYCIN .RTM. +2.5* ADRIAMYCIN .RTM.
+ [N109D]-IGFBP-3 +4.4** 5-FLUOROURACIL +1.9 5-FLUOROURACIL +
[N109D]-IGFBP-3 +3.1** *p < 0.05; **p < 0.001
[0072] In order to gain further insights into the mechanism of
action of exogenously added IGFBP-3 in these mouse models, tumor
tissues from the groups treated with vehicle, doxorubicin, or
doxorubicin plus IGFBP-3, were further analyzed using gene array
technology. RNA was extracted from tumor tissue. cDNA was prepared,
labelled and used to probe Atlas 1.2k-I mouse gene arrays (Clontech
Laboratories Inc., Palo Alto, Calif.). These arrays contain 1,176
mouse gene sequences. The results indicated that adriamycin alone
produced differential expression of 26 genes (2%), 16 of which were
down-regulated. Interestingly, some of the latter included genes
whose products are known agents in cell cycle checkpoint control,
growth-related responses, as well as a number of
cytoskeletal/extracellular matrix proteins. In general, adriamycin
appears to blunt some of the very mechanisms that could hasten the
demise of treated tumor cells, but these were normalized by IGFBP-3
co-treatment. Interestingly, RXR-alpha levels are 50% inhibited in
tumors from animals treated with a combination of doxorubicin and
IGFBP-3 (but not when treated with doxorubicin alone).
Example 3
Treatment of MDA-MB-231 Mammary Tumors with IGFBP-3 and
Co-Administered Agents
[0073] MDA-MB-231 tumors were implanted in nude mice. Ten animals
(NCr-nu) were allocated to each group. The tumors were implanted SC
as trocar fragments and allowed to increase in size to
approximately 170 mg before treatment began on Day 15 post-implant.
The treatment regimen for [N109D]-IGFBP-3 was 4.0 mg/kg/dose, daily
for 21 days, SC beginning on Day 15. The treatment regimen for
Taxol was 5.0 mg/kg/day, daily for five days, IV. The treatment
regimen for Cisplatin was 4.0 mg/kg/dose, administered every fourth
day for three treatments, IP. Treatment regimens for TAXOL.RTM. and
Cisplatin began on Day 18. Tumor measurements were performed daily.
TABLE-US-00003 TABLE 2 MEDIAN TUMOR VOLUME IN MILLIGRAMS ON DAYS
INDICATED 15 19 22 26 29 33 CONTROL 171 .+-. 26 464 .+-. 244 694
.+-. 311 877 .+-. 664 1116 .+-. 858 1965 .+-. 1384 IGFBP-3 161 .+-.
35 367.5 .+-. 244 565 .+-. 510 818.5 .+-. 750 1089 .+-. 851 1936
.+-. 1825 CISPLATIN 171 .+-. 33 368 .+-. 124 446 .+-. 175 563 .+-.
214 675 .+-. 220 922 .+-. 299 CIS + BP3 162 .+-. 37 384 .+-. 117
432 .+-. 173 608 .+-. 316 649 .+-. 347 908 .+-. 516 TAXOL .RTM. 153
.+-. 32 327 .+-. 131 384 .+-. 337 512 .+-. 489 648 .+-. 538 900
.+-. 1067 TAXOL .RTM. + BP3 162 .+-. 44 302 .+-. 192 416 .+-. 241
416 .+-. 262 432 .+-. 351 513 .+-. 757
[0074] Cisplatin and TAXOL.RTM.groups and combination groups were
significantly different from control. [N109D]-IGFBP-3 group was not
significantly different from control. Strong synergistic effects
were seen in the TAXOL.RTM.+IGFBP-3 combination treatment compared
to TAXOL.RTM. alone, but not in the cisplatin+IGFBP-3 combination
compared to cisplatin alone. The results are shown in FIG. 3. Table
3 below shows the delay (in days) in tumor growth (endpoint of
3.times. doubling of tumor weight). TABLE-US-00004 TABLE 3
Treatment Group Days Delay CONTROL 0 [N109D]-IGFBP-3 ALONE -2.2
TAXOL +3.5 TAXOL + [N109D]-IGFBP-3 >+8.9* *p < 0.05;
Example 4
Treatment of LAPC-4 Prostate Tumor Cells with IGFBP-3 and
Co-Administered Agents
[0075] A study was performed to analyze the effects of IGFBP-3 in
combination with Taxol on the growth and death of prostate cancer
cells utilizing the LAPC-4 xenograft model. One million cells (in
100 mcl) were injected SQ into SCID mice. After 4 weeks palpable
tumors were observed. 4 groups were treated (6 mice per group): 1)
saline control; 2) IGFBP-3 (4 mg/kg/day intra-peritoneally); 3)
taxol (2 mg/kg/day intraperitoneally on days 5 through 8); 4) taxol
and IGFBP-3 combination. Tumors were analyzed for size by palpation
weekly and serum collected. Animals were sacrificed at day 21 and
tumor weight assessed. The results of this experiment demonstrated
a trend for reduced tumor size (40%) with combination therapy.
Example 5
Generation of Defined Sub-Domains of IGFBP-3 by Engineering 3C
Protease Target Sites Into the Primary Sequence of the Protein
[0076] Defined IGFBP-3 sub-domains were generated from constructs
expressed as soluble fusion proteins in an E. coli expression
system. The general structures of the fusions are: [0077] IVS-1:
DsbA(mut) . . . [3C] . . . domain 1 . . . [3C] . . . domain 2/3
[0078] IVS-2: DsbA(mut) . . . [3C] . . . domain 1/2 . . . [3C] . .
. domain 3 where [3C] is the peptide sequence recognized by HRV 3C
proteinase. Yields are comparable to wild type, and a substantial
fraction is believed to be correctly folded, based on the
demonstrated ability of the protein to bind IGF-I. After cleavage,
the sub-domains of IGFBP-3 generated from the IVS-1 construct
(domains 1, 2/3) are captured on hydrophobic interaction resins
such as Phenyl-Sepharose or (less desirably) on cation exchange
resins such as SP-Sepharose. Other resins, such as immobilized
heparin can also be used. Efficient on-column cleavage of IVS-1
fusion with 3C proteinase has been demonstrated using 1:10
(protease to substrate) ratios at 4 degrees Celsius or room
temperature. Complete cleavage has been seen in less than 20
minutes. In the past, amino acid sequencing of cleavage products
has shown that the enzyme cleaves in an unusually clean manner
(<5% "ragged" ends). From 2 grams of wet cell paste, a few
milligrams of purified IGFBP-3 2/3 domain can be captured on
Phenyl-Sepharose. Further purification to near homogeneity can be
achieved on nickel- or zinc-affinity chromatography, as this
invention further demonstrates, for the first time, the metal
binding properties of IGFBP-3 and, furthermore, that the
determinants of this characteristic of the protein are located
primarily in the C-terminal portion of the molecule (i.e. the 2/3
domain). Apparently, metal-binding does not require the
amino-terminal .about.100 amino acids of the protein. The
amino-terminal .about.100 amino acids are deemed to constitute the
primary domain for IGF-I binding in the IGFBP-3 molecule.
[0079] The present invention has been detailed both by direct
description and by example. Equivalents and modifications of the
present invention will be apparent to those skilled in the art, and
are encompassed within the scope of the invention.
Sequence CWU 1
1
4 1 23 PRT Homo sapien 1 Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln
Cys Arg Pro Ser Lys Gly 1 5 10 15 Arg Lys Arg Gly Phe Cys Trp 20 2
14 PRT Homo sapien 2 Gln Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly
Phe Cys Trp 1 5 10 3 264 PRT Homo sapien 3 Gly Ala Ser Ser Ala Gly
Leu Gly Pro Val Val Arg Cys Glu Pro Cys 1 5 10 15 Asp Ala Arg Ala
Leu Ala Gln Cys Ala Pro Pro Pro Ala Val Cys Ala 20 25 30 Glu Leu
Val Arg Glu Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu 35 40 45
Ser Glu Gly Gln Pro Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly 50
55 60 Leu Arg Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala
Leu 65 70 75 80 Leu Asp Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val
Ser Arg Leu 85 90 95 Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro
Gly Asn Ala Ser Glu 100 105 110 Ser Glu Glu Asp Arg Ser Ala Gly Ser
Val Glu Ser Pro Ser Val Ser 115 120 125 Ser Thr His Arg Val Ser Asp
Pro Lys Phe His Pro Leu His Ser Lys 130 135 140 Ile Ile Ile Ile Lys
Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys 145 150 155 160 Val Asp
Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu 165 170 175
Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp 180
185 190 Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly
Val 195 200 205 His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys
Lys Gln Cys 210 215 220 Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys
Trp Cys Val Asp Lys 225 230 235 240 Tyr Gly Gln Pro Leu Pro Gly Tyr
Thr Thr Lys Gly Lys Glu Asp Val 245 250 255 His Cys Tyr Ser Met Gln
Ser Lys 260 4 264 PRT Homo sapien VARIANT (1)...(264)
[N109D]-hIGFBP-3 derivative. A non-naturally occurring derivative.
4 Gly Ala Ser Ser Ala Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys 1
5 10 15 Asp Ala Arg Ala Leu Ala Gln Cys Ala Pro Pro Pro Ala Val Cys
Ala 20 25 30 Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Leu Thr
Cys Ala Leu 35 40 45 Ser Glu Gly Gln Pro Cys Gly Ile Tyr Thr Glu
Arg Cys Gly Ser Gly 50 55 60 Leu Arg Cys Gln Pro Ser Pro Asp Glu
Ala Arg Pro Leu Gln Ala Leu 65 70 75 80 Leu Asp Gly Arg Gly Leu Cys
Val Asn Ala Ser Ala Val Ser Arg Leu 85 90 95 Arg Ala Tyr Leu Leu
Pro Ala Pro Pro Ala Pro Gly Asp Ala Ser Glu 100 105 110 Ser Glu Glu
Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser 115 120 125 Ser
Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser Lys 130 135
140 Ile Ile Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys
145 150 155 160 Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe
Ser Ser Glu 165 170 175 Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg
Arg Glu Met Glu Asp 180 185 190 Thr Leu Asn His Leu Lys Phe Leu Asn
Val Leu Ser Pro Arg Gly Val 195 200 205 His Ile Pro Asn Cys Asp Lys
Lys Gly Phe Tyr Lys Lys Lys Gln Cys 210 215 220 Arg Pro Ser Lys Gly
Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys 225 230 235 240 Tyr Gly
Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val 245 250 255
His Cys Tyr Ser Met Gln Ser Lys 260
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