U.S. patent application number 09/929552 was filed with the patent office on 2002-09-05 for inhibiting proliferation of cancer cells.
This patent application is currently assigned to Tufts University. Invention is credited to Sonnenschein, Carlos, Soto, Ana M..
Application Number | 20020123080 09/929552 |
Document ID | / |
Family ID | 25086386 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020123080 |
Kind Code |
A1 |
Sonnenschein, Carlos ; et
al. |
September 5, 2002 |
Inhibiting proliferation of cancer cells
Abstract
A method of testing cancer cells is described. Assays are
provided for determining the potential for inhibiting cancer cells
proliferation using albumin-derived peptides. The methods of the
present invention allow for drug screening as well as for
evaluation of biopsied tumors.
Inventors: |
Sonnenschein, Carlos;
(Boston, MA) ; Soto, Ana M.; (Boston, MA) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
101 HOWARD STREET
SUITE 350
SAN FRANCISCO
CA
94105
US
|
Assignee: |
Tufts University
|
Family ID: |
25086386 |
Appl. No.: |
09/929552 |
Filed: |
August 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09929552 |
Aug 14, 2001 |
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08769746 |
Dec 19, 1996 |
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6274305 |
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/5091
20130101 |
Class at
Publication: |
435/7.23 |
International
Class: |
G01N 033/574 |
Claims
1. A method of testing human cancer cells, comprising: a)
providing: i) a human cancer patient, ii) an antibody to the
receptor for human albumin; b) obtaining cancer cells from said
patient; and c) contacting said cells ex vivo with said
antibody.
2. The method of claim 1, wherein said cancer cells are obtained
from a biopsy.
3. The method of claim 1, wherein said cancer cells are selected
from the group consisting of breast cancer cells and prostate
cancer cells.
4. The method of claim 1, further comprising after step (b) and
prior to step (c) the step of culturing said cancer cells.
5. The method of claim 4, wherein said culturing is performed in
serum-free media.
6. The method of claim 1, further comprising after step (c) the
step of detecting said antibody bound to said cells.
7. A method of testing human cancer cells, comprising: a)
providing: i) human cancer cells obtained from a biopsy; ii) an
antibody specific for the human albumin receptor; and b) contacting
said cells ex vivo with said antibody.
8. The method of claim 7, wherein said cancer cells are selected
from the group consisting of breast cancer cells and prostate
cancer cells.
9. The method of claim 7, further comprising prior to step (b) the
step of culturing said cancer cells.
10. The method of claim 9, wherein said culturing is performed in
serum-free media.
11. The method of claim 7, further comprising after step (b) the
step of detecting said antibody bound to said cells.
Description
FIELD OF THE INVENTION
[0001] The invention generally relates to the inhibition of cancer
cell proliferation, and, more specifically, to the testing of
cancer cells for their ability to be inhibited, and to the
identification and use of drugs to inhibit cancer cell
proliferation.
BACKGROUND
[0002] The term "chemotherapy" simply means the treatment of
disease with chemical substances. The father of chemotherapy, Paul
Ehrlich, imagined the perfect chemotherapeutic as a "magic bullet;"
such a compound would kill invading organisms without harming the
host. This target specificity is sought in all types of
chemotherapeutics, including anticancer agents.
[0003] However, specificity has been the major problem with
anticancer agents. In the case of anticancer agents, the drug needs
to distinguish between host cells that are cancerous and host cells
that are not cancerous. The vast bulk of anticancer drugs are
indiscriminate at this level. Typically anticancer agents have
negative hematological effects (e.g., cessation of mitosis and
disintegration of formed elements in marrow and lymphoid tissues),
and immunosuppressive action (e.g., depressed cell counts), as well
as a severe impact on epithelial tissues (e.g., intestinal mucosa),
reproductive tissues (e.g., impairment of spermatogenesis), and the
nervous system. P. Calabresi and B.A. Chabner, In: Goodman and
Gilman The Pharmacological Basis of Therapeutics (Pergamon Press,
8th Edition) (pp. 1209-1216).
[0004] What is needed is a specific anticancer approach that is
particularly suitable for specific cancer cells. Importantly, the
treatment must be effective with minimal host toxicity.
SUMMARY OF THE INVENTION
[0005] The invention generally relates to the inhibition of cancer
cell proliferation, and, more specifically, to the testing of
cancer cells for their ability to be inhibited, and to the
identification and use of drugs to inhibit cancer cell
proliferation. The present invention provides A) an in vitro model
for testing cancer cells and evaluating their potential for being
inhibited, B) a screening assay for identifying drugs that inhibit
cancer cell proliferation, and C) chemotherapeutics for inhibiting
cancer cell proliferation in vivo.
[0006] A variety of assay formats are contemplated for testing the
potential for inhibiting cancer cells. In one embodiment, a portion
of a patient's tumor is obtained (e.g., by biopsy) and placed in
tissue culture. Thereafter, the response of the cancer cells to a
albumin-derived peptide is assessed. Where the albumin-derived
peptide inhibits proliferation, the tumor can be considered to be
expressing the corresponding plasma membrane receptor and such a
tumor may be suitable for chemotherapeutics that target this
receptor. The potential for reversing or overcoming this inhibition
with hormone (e.g. estradiol) can also be assessed by adding the
hormone to the culture. Where the inhibition with the
albumin-derived peptide is cancelled by the presence of the
hormone, the tumor can be considered to be expressing the
corresponding plasma membrane hormone-binding receptor and such a
tumor may be suitable for chemotherapeutics that target this
particular receptor. Moreover, other therapies may be adopted that
those skilled in the art recognize to be appropriate for "hormone
sensitive" tumors.
[0007] In one embodiment, the present invention contemplates a
method of evaluating human cancer comprising: a) providing i) a
human cancer patient, and ii) an albumin-derived peptide; b)
obtaining cancer cells from said patient; c) contacting said cells
ex vivo with said albumin-derived peptide; and d) measuring cancer
cell proliferation. Preferably the cancer cells are cultured in
serun-free culture media so as to essentially avoid introducing
complicating factors. In another embodiment, the present invention
provides a method of testing human cancer cells comprising: a)
providing i) a human cancer patient, ii) an albumin-derived
peptide, and iii) one or more hormones or hormone analogues; b)
obtaining cancer cells from said patient; c) culturing said cells
in serum-free culture media in the presence of said albumin-derived
peptide and said one or more hormones or hormone analogues; and d)
measuring cancer cell proliferation.
[0008] As noted above, the present invention also contemplates a
screening assay for identifying drugs that inhibit tumor invasion.
The present invention contemplates a screening assay utilizing the
binding activity of albumin-derived peptides. In one embodiment,
cancer cells (whether obtained from a primary tumor or grown as an
established cell line) are placed in tissue culture in the presence
of an albumin-derived peptide. It is contemplated that an
inhibitable tumor cells cultured in the presence of the
albumin-derived peptide will not proliferate. In the drug screening
assay, candidate drug inhibitors are added to a second tissue
culture containing the inhibitable tumor cells (this can be done
individually or in mixtures). Where the inhibitable cells are found
to be similarly inhibited by the candidate drug, a drug inhibitor
is indicated (hereinafter a "type 1 drug inhibitor"). It is also
contemplated that the drug screening be done in the presence of a
blocking hormone, i.e. a hormone that overcomes or cancels the
inhibition caused by the albumin-derived peptide. For example,
where the inhibition by the albumin-derived peptide is cancelled by
the addition of estradiol, a candidate drug can be added in an
attempt to interfere with the action of the hormone. Where the
presence of estradiol does not result in proliferation, a second
type of drug inhibitor is indicated (hereinafter a "type 2 drug
inhibitor"). It is not intended that the present invention be
limited by the nature of the drugs screened in the screening assay
of the present invention. A variety of drugs, including peptides,
are contemplated. Antibodies to albumin, the hormone or the
corresponding receptors are contemplated as convenient positive
controls.
[0009] In one embodiment, the present invention contemplates a
method of screening drugs comprising: a) providing: i)
albumin-inhibitable tumor cells, ii)an inhibitor selected from the
group consisting of albumin, an albumin-derived peptide, and an
albumin-derived peptide analogue, iii) one or more steroid
hormones, and iv) a candidate drug; b) contacting said cells in
vitro with said inhibitor, said one or more steroid hormones and
said candidate drug; and c) measuring the extent of tumor cell
proliferation.
[0010] It is not intended that the present invention be limited by
the means by which the extent of proliferation is measured. A
variety of quantitative and qualitative means is known in the art,
including (but not limited to): 1) the uptake of radiolabelled
nucleic acid precursors (e.g. tritiated thymidine), 2) microscopic
examination, and 3) automated cell counting (including lysing cells
and counting nuclei).
[0011] It is not intended that the present invention be limited by
the nature of the cancer cells used for drug screening. Both i)
cancer cells from established cancer cell lines and ii) cancer
cells obtained from patients (e.g. from a biopsy) are contemplated.
A variety of tumor types are contemplated as well, including, but
not limited to, breast cancer cells and prostate cancer cells.
[0012] Finally, the present invention contemplates
chemotherapeutics for treating cancer in vivo. In one embodiment,
the present invention contemplates chemotherapeutics to inhibit
cancer cell proliferation. Both type 1 and type 2 drug inhibitors
identified through the above-described screening assay are
contemplated. Moreover, albumin-derived peptides and peptide
analogues are specifically contemplated for in vivo use. In one
embodiment, the method comprises administering an albumin-derived
peptide or peptide analogue as adjunct therapy with additional
chemotherapeutics.
DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows an amino acid sequence representative of human
serum albumin.
[0014] FIG. 2 depicts the DNA sequence coding for mature HSA.
[0015] FIG. 3 shows the SDS-PAGE results for albumin and
albumin-derived peptides.
[0016] FIG. 4 is a graph showing the dose response of cancer cells
to albumin in the presence and absence of estradiol.
DEFINITIONS
[0017] The term "drug" as used herein, refers to any medicinal
substance used in humans or other animals. Encompassed within this
definition are naturally occurring and synthetic organic compounds,
as well as naturally occurring and synthetic recombinant
pharmaceuticals (whether hormones, peptides or peptide
analogues).
[0018] The term "hormone" refers to trace substances produced by
various endocrine glands which serve as chemical messengers carried
by the blood to various target organs, where they regulate a
variety of physiological and metabolic activities in vertebrates.
The steroid hormones include the estrogens, or female sex hormones,
the androgens, or male hormones (testosterone,
dihydrotestosterone); the progestational hormone progesterone; and
the steroid hormones of the adrenal cortex (major forms, cortisol,
aldosterone, and corticosterone).
[0019] The term "albumin-derived peptide" refers to a peptide
having a sequence that is identical to a portion of the amino acid
sequence of albumin. The present invention also contemplates
analogues. In one embodiment, an "albumin-derived peptide analogue"
comprises a peptide having a sequence that is similar (but not
identical) to a portion of the amino acid sequence of albumin. In
another embodiment, an "albumin-derived peptide analogue" is a
"mimetic." Mimetics are compounds mimicking the necessary
conformation for recognition and docking to the receptor binding to
the albumin-derived peptide.
[0020] The term "receptors" refers to structures expressed by cells
and which recognize binding molecules (e.g. ligands).
[0021] The term "antagonist" refers to molecules or compounds which
inhibit the action of a "native" or "natural" compound (such as
albumin). Antagonists may or may not be homologous to these natural
compounds in respect to conformation, charge or other
characteristics. Thus, antagonists may be recognized by the same or
different receptors that are recognized by the natural
compound.
[0022] The term "host cell" refers to any cell which is used in any
of the screening assays of the present invention. "Host cell" also
refers to any cell which either naturally expresses particular
receptors of interest or is genetically altered so as to produce
these normal or mutated receptors.
DESCRIPTION OF THE INVENTION
[0023] As noted above, chemotherapeutic agents are currently
employed to reduce the unrestricted growth of cancer cells.
However, better agents are needed that more specific and less
toxic. The invention generally relates to the inhibition of cancer
cell proliferation, and, more specifically, to the testing of
cancer cells for their ability to be inhibited, and to the
identification and use of drugs to inhibit cancer cell
proliferation.
[0024] A. Assays For Inhibiting Cancer Cell Proliferation
[0025] Discovering how to inhibit the proliferation of tumor cells
first requires the development of assays with which to test the
potential for tumor cells to be inhibited. The present invention
contemplates a variety of in vitro assays involving the use of
albumin and/or albumin-derived peptides.
[0026] Albumin and Making Albumin-Derived Peptides
[0027] In one assay system, the present invention contemplates
using albumin and/or albumin-derived peptides. Human serum albumin
(HSA) is the most abundant plasma protein. A molecule of HSA
consists of a single non-glycosylated polypeptide chain of 585
amino acids of formula molecular weight 66,500. A representative
amino acid sequence of HSA is shown in FIG. 1. Variations in the
sequence are known. As used herein, "variants of human serum
albumin" are those sequence showing greater than 80% homology, and
preferably greater than 90% homology, and most preferably greater
than 95% homology, to the sequence set forth in FIG. 1.
[0028] Albumin-derived peptides are those peptides having a
sequence that is identical to a portion of the amino acid sequence
of albumin as set forth in FIG. 1. In one embodiment, the present
invention contemplates an albumin-derived peptide comprising the
N-terminal portion of human serum albumin up to amino acid residue
n, where n is between 360 and 430, are more preferably between 369
and 419.
[0029] In one embodiment, an "albumin-derived peptide analogue"
comprises a peptide having a sequence that is similar (but not
identical) to a portion of the amino acid sequence of albumin as
set forth in FIG. 1. Such similar sequences are contemplated to
have conservative substitutions and/or deletions and/or
additions.
[0030] Conservative substitutions are those where one or more amino
acids are substituted for others having similar properties in the
understanding of one skilled in the art. Typical substitutions
include, but are not limited by, substitutions of alanine or valine
for glycine, arginine or asparagine for glutamine, serine for
threonine and histidine for lysine.
[0031] Analogues having deletions are those having up to ten (and
preferably only one or two) amino acid residues lacking (in
comparison to the sequence set forth in FIG. 1). Preferably, such
deletions occur in the portion between 1 and 370, and more
preferably between 100 and 369.
[0032] Analogues having additions are those peptides that encompass
additional amino acid residues, including whole sequences which are
not native to HSA. In one embodiment, the peptide analogue having
additions comprises a peptide between one hundred and five hundred
amino acids in length. In one embodiment, the peptide analogue
having additions comprises additional amino acids added to the
amino terminus of an albumin-derived sequence. In one embodiment,
the peptide analogue having additions comprises additional amino
acids added to the carboxy terminus of an albumin-derived sequence.
In another embodiment, the peptide analogue having additions
comprises additional amino acids added to both the amino and
carboxy termini.
[0033] One common methodology for evaluating sequence homology, and
more importantly statistically significant similarities, is to use
a Monte Carlo analysis using an algorithm written by Lipman and
Pearson to obtain a Z value. According to this analysis, a Z value
greater than 6 indicates probable significance, and a Z value
greater than 10 is considered to be statistically significant. W.
R. Pearson and D. J. Lipman, Proc. Natl. Acad. Sci. (USA),
85:2444-2448 (1988); D. J. Lipman and W. R. Pearson, Science,
227:1435-1441 (1985). In the present invention, synthetic
albumin-derived peptide analogues are those peptides with
statistically significant sequence homology and similarity (Z value
of Lipman and Pearson algorithm in Monte Carlo analysis exceeding
6).
[0034] Preferred albumin-derived peptides include, but are limited
to, HSA (1-373) (i.e. where the C-terminal amino acid is Val); HSA
(1-387) (i.e. where the C-terminal amino acid is Leu); HSA (1-388)
(i.e. where the C-terminal amino acid is I1e); HSA (1-379) (i.e.
where the C-terminal amino acid is Lys); HSA (1-390) (i.e. where
the C-terminal amino acid is Gln); and HSA (1-407) (i.e. where the
C-terminal amino acid is Leu).
[0035] It is not intended that the present invention be limited by
the manner in which the albumin-derived peptide is made. In one
embodiment, the peptide is made by enzymatic digestion. For
example, a trypsin-like enzyme will cleave HSA between Lys (389)
and Gln (390), as well as at other sites. In another embodiment,
the peptide is made by peptide synthesis. In still another
embodiment, Albumin-derived peptides are conveniently made by
recombinant techniques. See U.S. Pat. No. 5,380,712, hereby
incorporated by reference. FIG. 2 depicts the DNA sequence coding
for mature HSA. This sequence can be used together with standard
recombinant DNA procedures to construct expression vectors for the
expression of albumin-derived peptides.
[0036] It is not intended that the present invention be limited by
the precise amount of albumin or albumin-derived peptide used in
the assays of the present invention. When albumin is used, the
inhibiting concentration of albumin is typically between one
microgram and two milligram per milliliter of culture fluid, and
more preferably greater than one hundred micrograms per milliliter
and less than one milligram per milliliter.
[0037] B. Drug Screening Assays
[0038] As noted above, the present invention also contemplates a
screening assay for identifying drugs that inhibit cancer cell
proliferation. The present invention contemplates a screening assay
utilizing the binding activity of albumin and/or albumin-derived
peptides, including but not limited to the above-described
peptides. In one embodiment, an inhibitable tumor cell line is
placed in tissue culture. The tumor cells (under ordinary
conditions) will proliferate; however, in the presence of albumin
and/or albumin-derived peptides, the cell proliferation will be
inhibited.
[0039] In the drug screening assay of the present invention,
candidate drug inhibitors are added to the tissue culture (this can
be done individually or in mixtures). Where the cancer cells are
found to be inhibited, a drug inhibitor is indicated.
[0040] It is not intended that the present invention be limited by
the nature of the drugs screened in the screening assay of the
present invention. A variety of drugs, including peptides, are
contemplated.
[0041] It is also not intended that the present invention be
limited by the particular tumor cells used for drug testing. A
variety of tumor cells (for both positive and negative controls)
are contemplated (including but not limited to the cells set forth
in Table 1 below). In addition, primary tumor cells from patients
are contemplated.
[0042] Where the inhibitable cells are found to be similarly
inhibited by the candidate drug, a drug inhibitor is indicated
(hereinafter a "type 1 drug inhibitor"). It is also contemplated
that the drug screening be done in the presence of a blocking
hormone, Lie. a hormone that overcomes or cancels the inhibition
caused by the albumin-derived peptide. Where the presence of a
hormone does not result in proliferation, a second type of drug
inhibitor is indicated (hereinafter a "type 2 drug inhibitor"). It
is not intended that the present invention be limited by the nature
of the hormone used in conjunction with the drug screening assay.
As discussed below, the present invention contemplates hormones
mediating cell proliferation.
[0043] Hormones Mediating Proliferation of Cancer Cells
[0044] While an understanding of the mechanisms involved in cancer
is not necessary to the successful practice of the present
invention, it is believed that hormones can mediate the
proliferation of cancer cells. The present invention contemplates
the use of steroid sex hormones in conjunction with the assays for
testing cancer cells.
1TABLE 1 Designation And Origin Of Human Cell Lines And
Strains.sup.1 ORIGIN CELL LINES OR STRAINS Colonic carcinoma
SW1116, HCT116, SKCO-1, HT-29, KM12C, KM12SM, KM12L4, SW480
Pancreatic carcinoma BxPC-3, AsPC-1, Capan-2, MIA PaCa-2, Hs766T
Colon adenoma VaCo 235 Lung carcinoma A549 Prostate carcinoma PC-3,
DU-145 Breast cancer 009P, 013T, MCF-7, MDA-MB231 Lymphoma Daudi,
Raji Breast epithelium 006FA Diploid fibroblast HCS (human corneal
stroma), MRC-5 .sup.1The SW1116, HT-29, SW480, Raji lymphoblastoid
cells, and the pancreatic lines are obtained from the American Type
Culture Collection.
[0045] Naturally occurring or endogenous estrogens constitute one
class of steroid sex hormones which are produced in the ovaries and
other tissues in the body. The naturally occurring estrogens are
estrone (also known as E.sub.1), estradiol-17B (also known as
E.sub.2), and estriol (also known as E.sub.3). Synthetic compounds
having estrogenic properties include ethinyl estradiol (Estinyl);
3-methyl-ethinyl estradiol (Mestranol); and diethylstilbestrol
(DES); methallenestril (Vallestril); and doisynoestrol
(Fenocylin).
[0046] The present invention contemplates testing the response of
the cancer cells to albumin and albumin-derived peptides. Where the
albumin-derived peptide inhibits proliferation, the tumor can be
considered to be expressing the corresponding plasma membrane
receptor and such a tumor may be suitable for chemotherapeutics
that target this receptor. The potential for reversing or
overcoming this inhibition with hormone (e.g. estradiol) can also
be assessed by adding the hormone to the culture. Where the
inhibition with the albumin-derived peptide is cancelled by the
presence of the hormone, the tumor can be considered to be
expressing the corresponding plasma membrane hormone-binding
receptor and such a tumor may be suitable for chemotherapeutics
that target this particular receptor. Moreover, other therapies may
be adopted that those skilled in the art recognize to be
appropriate for "hormone sensitive" tumors.
[0047] A variety of formats and protocols for testing hormones is
contemplated. Illustrative formats and protocols are described in
U.S. Pat. Nos. 4,859,585 and 5,135,849, both of which are hereby
incorporated by reference.
[0048] C. Administering Chemotherapeutics
[0049] It is contemplated that albumin, albumin-derived peptides,
and albumin-derived peptide analogues, as well as the type 1 and
type 2 drugs discussed above, can be administered systemically or
locally to inhibit tumor cell proliferation in cancer patients.
They can be administered intravenously, intrathecally,
intraperitoneally as well as orally. Moreover, they can be
administered alone or in combination with anti-proliferative
drugs.
[0050] Where combinations are contemplated, it is not intended that
the present invention be limited by the particular nature of the
combination. The present invention contemplates combinations as
simple mixtures as well as chemical hybrids. An example of the
latter is where the peptide or drug is covalently linked to a
targeting carrier or to an active pharmaceutical. Covalent binding
can be accomplished by any one of many commercially available
crosslinking compounds.
[0051] It is not intended that the present invention be limited by
the particular nature of the therapeutic preparation. For example,
such compositions can be provided together with physiologically
tolerable liquid, gel or solid carriers, diluents, adjuvants and
excipients.
[0052] These therapeutic preparations can be administered to
mammals for veterinary use, such as with domestic animals, and
clinical use in humans in a manner similar to other therapeutic
agents. In general, the dosage required for therapeutic efficacy
will vary according to the type of use and mode of administration,
as well as the particularized requirements of individual hosts.
[0053] Such compositions are typically prepared as liquid solutions
or suspensions, or in solid forms. Oral formulations for cancer
usually will include such normally employed additives such as
binders, fillers, carriers, preservatives, stabilizing agents,
emulsifiers, buffers and excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, cellulose, magnesium carbonate, and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations, or powders, and
typically contain 1%-95% of active ingredient, preferably
2%-70%.
[0054] The compositions are also prepared as injectables, either as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid prior to injection may also be
prepared.
[0055] The antagonists of the present invention are often mixed
with diluents or excipients which are physiological tolerable and
compatible. Suitable diluents and excipients are, for example,
water, saline, dextrose, glycerol, or the like, and combinations
thereof. In addition, if desired the compositions may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, stabilizing or pH buffering agents.
[0056] Additional formulations which are suitable for other modes
of administration, such as topical administration, include salves,
tinctures, creams, lotions, and, in some cases, suppositories. For
salves and creams, traditional binders, carriers and excipients may
include, for example, polyalkylene glycols or triglycerides.
[0057] Designing Mimetics
[0058] It may be desirable to administer an analogue of an
albumin-derived peptide. As mentioned previously, the present
invention contemplates compounds mimicking the necessary
conformation for recognition and docking to the albumin receptor. A
variety of designs for such mimetics are possible. For example,
cyclic peptides, in which the necessary conformation for binding is
stabilized by nonpeptides, are specifically contemplated. U.S. Pat.
No. 5,192,746 to Lobl et al., U.S. Pat. No. 5,169,862 to Burke, Jr.
et al., U.S. Patent No. 5,539,085 to Bischoff et al., U.S. Patent
No. 5,576,423 to Aversa et al., U.S. Pat. No. 5,051,448 to
Shashoua, and U.S. Patent No. 5,559,103 to Gaeta et al., all hereby
incorporated by reference, describe multiple methods for creating
such compounds.
[0059] Synthesis of nonpeptide compounds that mimic peptide
sequences is also known in the art. Eldred et al., (J. Med. Chem.
37:3882 (1994)) describe nonpeptide antagonists that mimic the
Arg-Gly-Asp sequence. Likewise, Ku et al., (J. Med. Chem. 38:9
(1995)) give further elucidation of the synthesis of a series of
such compounds. Such nonpeptide compounds are specifically
contemplated by the present invention.
[0060] The present invention also contemplates synthetic mimicking
compounds that are multimeric compounds that repeat the relevant
peptide sequence. As is known in the art, peptides can be
synthesized by linking an amino group to a carboxyl group that has
been activated by reaction with a coupling agent, such as
dicyclohexyl-carbodiimide (DCC). The attack of a free amino group
on the activated carboxyl leads to the formation of a peptide bond
and the release of dicyclohexylurea. It can be necessary to protect
potentially reactive groups other than the amino and carboxyl
groups intended to react. For example, the (x-amino group of the
component containing the activated carboxyl group can be blocked
with a tertbutyloxycarbonyl group. This protecting group can be
subsequently removed by exposing the peptide to dilute acid, which
leaves peptide bonds intact.
[0061] With this method, peptides can be readily synthesized by a
solid phase method by adding amino acids stepwise to a growing
peptide chain that is linked to an insoluble matrix, such as
polystyrene beads. The carboxyl-terminal amino acid (with an amino
protecting group) of the desired peptide sequence is first anchored
to the polystyrene beads. The protecting group of the amino acid is
then removed. The next amino acid (with the protecting group) is
added with the coupling agent. This is followed by a washing cycle.
The cycle is repeated as necessary.
[0062] Experimental
[0063] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0064] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); mM (millimolar); N (Normal); mol (moles); mmol
(millimoles); .mu.mol (micromoles); nmol (nanomoles); g (grams); mg
(milligrams); .mu.g (micrograms); L (liters); ml (milliliters);
.mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers);.degree. C. (degrees Centigrade);
mAb (monoclonal antibody); MW (molecular weight); PBS (phosphate
buffered saline); U (units); d(days).
[0065] A clonal population of the human breast cancer MCF-7 cell
line was used in some of the experiments described below. These
cells were routinely grown in 5% fetal bovine serum (FBS)
supplemented-Dulbecco's Modified Eagle's Medium (DME).
Charcoal-dextran (CD) stripped serum-supplemented medium inhibits
their proliferation and estradiol cancels this effect. Estrogen
non-target, serum-insensitive human breast cancer MDA-MB231 cells
(ATCC, Rockville, Md.) were used as controls; they were grown under
the same conditions as MCF-7 cells.
[0066] Recombinant human albumin (rHA), recombinant Domain I (aa
1-194, rDI) and Domain I+II (aa 1-387, rDI+II) were obtained from
Delta Biotechnology Ltd., Nottingham, U. K. rHA and truncated HA
transcripts were produced in yeast grown in synthetic medium
comprising sucrose, vitamins and inorganic salts.
[0067] At chosen intervals after exposure to 2 mg/ml rHA with or
without 100 pM estradiol, cells were trypsinized, pelleted by
centrifugation at 100 g for 3 min, resuspended in 10% DMSO-10%
CDHuS and snap frozen. Cells were kept at -20.degree. C. for up to
7 days. Cells were quickly thawed at 37.degree. C., centrifuged and
resuspended at a density of 10.sup.6 cells/ml in a solution
containing 0.1% Triton X-100, 0.1 mg/ml propidium iodide (PI)
(Sigma) in DME. Total DNA was quantified by propidium iodide
binding. The RNAse treatment used in the original method to
hydrolyze double stranded RNA did not significantly affect the DNA
fluorescence and was omitted. Cells were analyzed in a
Becton-Dickinson FACSCAN flow cytometer. Ten thousand cells were
collected for each point. Data were collected and compiled with
Becton-Dickinson Lysis II and Cell Fit software.
[0068] HA-free serum was obtained by Cibracron Blue and by hexyl-S
agarose chromatography. Ten millilitres of CD stripped serum were
dialyzed against start buffer [500 mM NaCl, 500 mM K.sub.2SO.sub.4,
50 mM sodium phosphate buffer (SPB) pH 7.6 containing 10 uM
butylated hydroxytoluene (BHT)] and chromatographed through a
1.6.times.12 cm hexyl-S agarose column. After extensive elution of
the breakthrough proteins, the retained fraction (HA) was eluted
first with 5 mM SPB, pH 7.6, containing 10 uM BHT, and finally with
40% ethylene glycol in 50 mM SPB, pH 7.6, containing 10 uM BHT.
Removal of HA was monitored electrophoretically and by immunoblots.
The three hexyl-S agarose fractions were dialyzed against a buffer
suitable for tissue culture (100 mM NaCl, 25 mM Hepes, pH 7.4,
containing 1 uM BHT) and their effect on cell proliferation tested
at concentrations ranging from 0.25 to mg/ml of protein in
ITDME.
[0069] In some experiments, serum and recombinant proteins were
analyses by ID-sodium dodecylsulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) using 12.5% homogeneous and 10-15%
gradient polyacrylamide gels and SDS buffer strips in the
electrophoresis Phast System (Pharmacia, Piscataway, N.J.). Gels
were stained with Coomassie blue. Gel images were digitized and
analyses using the BioImage, whole band software package (Millipore
Corp., Bedford, Mass.).
[0070] A polyclonal antibody was obtained in rabbits using as
antigen serum albumin (Sigma; cat. no. A1887); it was purified by
chromatography through tandem columns of butyl-S and hexyl-S
agarose as described by Porath. The immunoglobulin fraction was
precipitated with 2.2 M (NH.sub.4).sub.2SO.sub.4, dialyzed and
purified by affinity chromatography using as immunoadsorbant rHA
coupled to CNBr-activated Sepharose-4B. A monoclonal mouse anti-HA
IgG (Sigma; cat. no. A6684) and a polyclonal anti-HA serum (cat.
no. 126582, lot 703293) supplied by Calbiochem (Richmond, Calif.)
gave similar results. However, the monoclonal antibody was the most
sensitive one; as little as 0.5 mg HA/lane were clearly resolved as
a band by the Biolmage software. Serum proteins were resolved by
SDS-PAGE, transferred electrophoretically onto Immobilon-P
membranes using the Phast System (ID-PAGE). Membranes were blocked
with 60 mg/l teleostean gelatin, reacted with the monoclonal
antibody (1:20000), rinsed, and processed with a VEctastain ABC-Ap
kit (Vector, Inc., Burlingame, Calif.) for reaction with the second
antibody and alkaline phosphatase following the protocol, described
by the manufacturer. Immunoreactive bands were visualized after
incubation with the substrate [5-bromo-1-chloro-3-indolyl
phosphate/nitroblue tetrazolium BCIP/NBT substrate kit
(Vector)].
EXAMPLE 1
Inhibitory Effects of Albumin
[0071] To assess the inhibitory effect of serum or purified protein
preparations, 4.times.10.sup.4 cells/well were seeded in 12-well
Falcon Multiplates in 5% FBS; they were allowed to attach for 24 h
before exposing them to test medium. Cell proliferation yields were
measured after 4 days in DME plus 25 ug transferrin (T)/ml and 100
ng insulin (I)/ml (TTDME) alone, or ITDME plus CD human serum
(CDHuS), or purified protein preparations. Each sample was tested
in duplicate in the presence or absence of 100 pM estradiol
(E.sub.2) and each experiment was repeated at least three times.
Phenol red-free media were used in all experiments involving cell
proliferation rates or yields. Cells were lysed and nuclei counted
on a Coulter Counter Model ZM. Estrogens were removed from serum,
HA (Sigma Chemical Co.; cat. no. A1887) and from rHA preparations
by CD stripping.
[0072] As seen in FIG. 4, CDHuS (open diamonds) and CDHA (open
circles) inhibited the proliferation of MCF-7 cells in a
dose-dependent manner; similarly, rHA (closed diamonds) inhibited
the proliferation of these cells. Estradiol (closed squares)
reversed the inhibitory effect. Progesterone,
5.alpha.-dihydrotestosterone, the synthetic androgen R1881,
hydrocortisone, and thyroxine failed to reverse the effect of rHA
(data not shown).
EXAMPLE 2
Inhibitory Activity of Truncated Forms of rHA
[0073] To assess whether the inhibitory activity of albumin was
encoded in a specific sequence within the albumin molecule, the
inhibitory effect of truncated peptides encoding the first domain
alone and the first and second domains (see FIG. 3) was tested in
the manner that rHA was tested in Example 1.
[0074] The inhibitory activity of dDI+II was comparable to that of
rHA; rDI also showed inhibitory activity, albeit quantitatively
lower than that of rHA or rDI+II, both when expressed as a molar
concentration and as mg/ml (data not shown).
EXAMPLE 3
Conjugation of Albumin-derived Peptides
[0075] In this example, the preparation of a peptide conjugate is
described. As noted above, the albumin-derived peptides of the
present invention can be made synthetically or recombinantly. A
cysteine can be added to facilitate conjugation to other
proteins.
[0076] In order to prepare a protein for conjugation, it is
dissolved in buffer (e.g., 0.01 M NaPO.sub.4, pH 7.0) to a final
concentration of approximately 20 mg/ml. At the same time
n-maleimidobenzoyl-N-hydroxysucc- inimide ester ("MBS" available
from Pierce) is dissolved in N,N-dimethyl formamide to a
concentration of 5 mg/ml. The MBS solution, 0.51 ml, is added to
3.25 ml of the protein solution and incubated for 30 minutes at
room temperature with stirring every 5 minutes. The MBS-activated
protein is then purified by chromatography on a Bio-Gel P-10 column
(Bio-Rad; 40 ml bed volume) equilibrated with 50 mM NaPO.sub.4, pH
7.0 buffer. Peak fractions are pooled (6.0 ml).
[0077] The above-described cysteine-modified peptide (20 mg) is
added to the activated protein mixture, stirred until the peptide
is dissolved and incubated 3 hours at room temperature. Within 20
minutes, the reaction mixture becomes cloudy and precipitates form.
After 3 hours, the reaction mixture is centrifuged at-
10,000.times.g for 10 min and the supernatant analyzed for protein
content. The conjugate precipitate is washed three times with PBS
and stored at 4.degree. C.
[0078] From the above, it should be clear that the present
invention provides a method of testing of cancer cells,and in
particular identifying cancer cells that are inhibitable as well as
hormone sensitive. With regard to the later, distinguishing such
tumors allows the physician to change and/or optimize therapy.
Importantly, the albumin-derived peptides of the present invention
(and other drugs developed by use of the screening assay of the
present invention) will provide treatment associated with minimal
host toxicity.
* * * * *