U.S. patent application number 17/151898 was filed with the patent office on 2021-06-17 for bi-functional allosteric protein-drug molecules for targeted therapy.
The applicant listed for this patent is University of Utah Research Foundation. Invention is credited to David A. Bull, Kwangsuk Lim, Youngwook Won.
Application Number | 20210177986 17/151898 |
Document ID | / |
Family ID | 1000005418115 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210177986 |
Kind Code |
A1 |
Won; Youngwook ; et
al. |
June 17, 2021 |
BI-FUNCTIONAL ALLOSTERIC PROTEIN-DRUG MOLECULES FOR TARGETED
THERAPY
Abstract
Disclosed herein, is a bi-functional allosteric protein-drug
molecule comprising a targeting moiety, one or more biological
binding domains, and one or more therapeutic agents, wherein the
therapeutic agent is allosterically bound to the biological binding
domain. Also described herein, are methods of incorporating a
bi-functional allosteric protein-drug molecule comprising a
targeting moiety, one or more biological binding domains that
captures the therapeutic agent without the formation of a chemical
bond, and one or more therapeutic agents physiologically acceptable
compositions including them; and methods of administering the
bi-functional allosteric protein-drug molecule to patients for the
treatment of cancer.
Inventors: |
Won; Youngwook; (Salt Lake
City, UT) ; Bull; David A.; (Salt Lake City, UT)
; Lim; Kwangsuk; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Utah Research Foundation |
Salt Lake City |
UT |
US |
|
|
Family ID: |
1000005418115 |
Appl. No.: |
17/151898 |
Filed: |
January 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15534948 |
Jun 9, 2017 |
10898582 |
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PCT/US15/65308 |
Dec 11, 2015 |
|
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17151898 |
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62090760 |
Dec 11, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6849 20170801;
A61K 47/6801 20170801; A61K 47/6803 20170801; A61K 47/6889
20170801; C07K 2319/33 20130101; A61K 9/0019 20130101; C07K 16/32
20130101; C07K 2317/622 20130101; A61K 31/27 20130101; A61K 31/337
20130101; A61K 47/6855 20170801; A61K 47/6879 20170801 |
International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/32 20060101 C07K016/32; A61K 9/00 20060101
A61K009/00; A61K 31/27 20060101 A61K031/27; A61K 31/337 20060101
A61K031/337 |
Claims
1. A bi-functional allosteric protein-drug molecule comprising a
targeting moiety, one or more biological binding domains, and one
or more therapeutic agents, wherein the therapeutic agent is
allosterically bound to the biological binding domain.
2. The bi-functional allosteric protein-drug molecule of claim 1,
wherein the targeting moiety is a peptide or protein.
3. The bi-functional allosteric protein-drug molecule of claim 1,
wherein the targeting moiety is an antibody.
4. The bi-functional allosteric protein-drug molecule of claim 3,
wherein the antibody is a single chain antibody (scFv) or a Fab
fragment.
5. The bi-functional allosteric protein-drug molecule of claim 3,
wherein the antibody is human, chimeric or humanized or a
biologically active variant thereof.
6. The bi-functional allosteric protein-drug molecule of claim 3,
wherein the antibody is a monoclonal antibody or a polyclonal
antibody.
7. The bi-functional allosteric protein-drug molecule of claim 4,
wherein the scFv or Fab fragment specifically binds a growth factor
receptor.
8. The bi-functional allosteric protein-drug molecule of claim 7,
wherein the growth factor receptor is a member of the epidermal
growth factor receptor (EGFR) family.
9. The bi-functional allosteric protein-drug molecule of claim 3,
wherein the antibody is trastuzumab, panitumumab or cetuximab, or a
biologically active variant thereof.
10. The bi-functional allosteric protein-drug molecule of claim 1,
wherein the biological binding domain and the therapeutic agent are
present in a ratio of 1:1 or 1:5 (binding domain:therapeutic).
11. The bi-functional allosteric protein-drug molecule of claim 1,
wherein the biological binding domain comprises an ATP binding
domain and/or a taxane binding domain.
12. The bi-functional allosteric protein-drug molecule of claim 11,
wherein the biological binding domains are two or more.
13. The bi-functional allosteric protein-drug molecule of claim 1,
wherein the therapeutic agent is an anti-cancer agent.
14. The bi-functional allosteric protein-drug molecule of claim 13,
wherein the anti-cancer agent is a derivate of geldanamycin, a
taxane or a HSP90 inhibitor.
15. The bi-functional allosteric protein-drug molecule of claim 14,
wherein the geldanamycin derivative is 17-AAG or 17-DMAG.
16. The bi-functional allosteric protein-drug molecule of claim 14,
wherein the taxane is paclitaxel or docetaxel.
17. The bi-functional allosteric protein-drug molecule of claim 14,
wherein the HSP90 inhibitor is 17-AAG, geldanamycin, 17-DMAG,
IPI-504, BIIB021, SNX-5422, STA-9090 or NVP-AUY922.
18. A pharmaceutical composition comprising the bi-functional
allosteric protein-drug molecule of claim 1 and a pharmaceutically
acceptable carrier.
19. (canceled)
20. The pharmaceutical composition of claim 18, wherein the
therapeutic agent is an anti-cancer agent and wherein the
pharmaceutical composition is formulated for intravenous
administration.
21. A method of treating a patient with cancer, the method
comprising: (a) identifying a patient in need of treatment; and (b)
administering to the patient a therapeutically effective amount of
the pharmaceutical composition of claim 20.
22.-28. (canceled)
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/534,948, filed Jun. 9, 2017, which claims
the benefit of priority under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2015/065308, filed on Dec. 11, 2015, which
claims priority to U.S. Provisional Application No. 62/090,760,
filed on Dec. 11, 2014. The content of these earlier filed
applications is hereby incorporated by reference in their
entirety.
BACKGROUND
[0002] Cancer is one of the leading causes of death in the world.
Despite improvements in prevention, early detection, treatment and
survival, the American Cancer Society states that breast cancer is
the second most common newly diagnosed cancer and second leading
cause of death among women in the United States. Targeted therapy
is one treatment option available to patients involving the
administration of drugs such as antibodies that are selective for
cancer cells leaving normal cells relatively unharmed. Conventional
antibody-drug conjugate technology for breast cancer is limited in
large part because the low concentration of antibody present in
antibody-drug molecules are conjugated through a synthetic linker.
Further limitations include one or more of the following: high
production cost, inherent immunogenicity, multiple steps required
for conjugation; and/or safety issues related to the synthetic
linker. Alternative approaches are needed for improving the
construction of antibody-drug complexes for targeted disease
therapy.
SUMMARY
[0003] Disclosed herein, are bi-functional allosteric protein-drug
molecules comprising a targeting moiety, one or more biological
binding domains, and one or more therapeutic agents, wherein the
therapeutic agent is allosterically bound to the biological binding
domain.
[0004] Disclosed herein, are pharmaceutical compositions comprising
bi-functional allosteric protein-drug molecules comprising a
targeting moiety, one or more biological binding domains, and one
or more therapeutic agents, wherein the therapeutic agent is
allosterically bound to the biological binding domain, and a
pharmaceutically acceptable carrier.
[0005] Disclosed herein, are methods of treating a patient with
cancer, the method comprising (a) identifying a patient in need of
treatment; and (b) administering to the patient a therapeutically
effective amount of a pharmaceutical composition comprising a
bi-functional allosteric protein-drug molecule comprising a
targeting moiety, one or more biological binding domains, and one
or more therapeutic agents, wherein the therapeutic agent is
allosterically bound to the biological binding domain, and a
pharmaceutically acceptable carrier. Any of the methods of
treatment can be configured as methods of "use."
[0006] Other features and advantages of the present compositions
and methods are illustrated in the description below, the drawings,
and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a picture of an SDS-PAGE of the eluted samples:
the protein transduction domain-ATP binding domain (PTD-ABD) (left
lane); the PTD-ABD captured by geldanamycin (GM) beads (middle
lane); and the addition of cell lysate to the PTD-ABD+GM beads
(right lane).
[0008] FIG. 2 shows the viability of MCF-7 breast cancer cells
after delivery of either protein transduction domain-ATP binding
domain (PTD)-ATP binding domain (ABD)-17-AAG, 17-AAG or
PTD-ABD.
[0009] FIGS. 3A-C show the uptake kinetics of geldanamycin into
MCF-7 cells. Time kinetics of GM internalization (A), mean
fluorescence intensity (MFI) values of the GM-positive cells (B),
and kinetics of GM internalization (C), in a dose-dependent
manner.
[0010] FIG. 4 is a graph demonstrating the effects of short-term
treatment with PTD-ABD-17-AAG.
[0011] FIG. 5 shows the results of live/dead assay determined by
fluorescence-activated cell sorting (FACS). Left: control; right:
PTD-ABD.
[0012] FIG. 6 is a picture of an SDS-PAGE showing the control and
ABD-geldanamycin (ATP binding domain-GM) (lane 1), the ABD-GM
incubated in the presence of serum for 2 hours at 37.degree. C.
(lane 2) and the ABD-GM incubated in the presence of serum for 4
hours at 37.degree. C. (lane 3).
[0013] FIGS. 7A-B illustrate the dissociation of
biotin-geldanamycin (BGM) in BT-474 (A) and MCF-7 (B) cancer cell
lines.
[0014] FIGS. 8A-B illustrate the dissociation of
biotin-geldanamycin (BGM) in MDA-MB-231 (A) and SK-BR-3 (B) cancer
cell lines.
[0015] FIGS. 9A-B are pictures depicting the binding of
geldanamycin (GM) to the HER2 scFv-ABD fusion protein in trial 1
(A) and trial 2 (B).
[0016] FIG. 10 illustrates a scheme showing the formation of the
scFv-ATP binding domain (ABD)-17-AAG and the mechanism of action in
HER2+ breast cancer.
[0017] FIG. 11 illustrates a scheme showing the scFv-taxane-binding
domain (TBD)-taxane molecule. A plasmid DNA encoding two protein
domains: a HER2-scFv domain and the taxane-binding pocket of
.beta.-tubulin. Expression of this plasmid in CHO or HEK293 cells
produces a recombinant fusion protein comprising the scFv domain
and the taxane-binding domain. The high binding affinity of taxane
to the luminal site drives the incorporation of PTX into the
scFv-TBD fusion protein, which in turn leads to the formation of
the scFv-TBD-PTX complex. In this formulation, the TBD not only
solubilizes taxane in physiological buffers but also serves to
prevent the exposure of taxane to aqueous environments. The shaded
solvent accessible surface of the taxane-binding pocket of
.beta.-tubulin, colored according to degrees of hydrophobicity,
appears on the right with the color key on the left (maximum, red;
minimum, dark blue). The empty PTX-binding pocket (burnt orange) is
highly hydrophobic (A). The binding center occupied by PTX, showing
excellent shape complementarity (B). Surface recoloring illustrates
the conversion of the hydrophobic cavity to a hydrophilic surface
following the binding of PTX (C; Snyder J P et al. PNAS 2001;
98:5312-5316).
[0018] FIG. 12A-B show SDS-PAGE (A) and Western blot (B) of the
proteins and the proteins bound to GM-biotin. Lane 1: Protein
marker; Lanes 2-4: Purified proteins; Lanes 5-7: GM-bound proteins;
Lanes 2 and 5: HER2 scFv-ABD; Lanes 3 and 6: HER2 scFv-MD; Lanes 4
and 7: the HER2 scFv-ABD.times.3. Arrows indicate the location of
the proteins.
[0019] FIG. 13A-E show that TAT-ABD facilitates the internalization
of geldanamycin (GA) into BT-474 cells. A. Control, non-treated
cells. B. FITC-GA, FITC-labeled GA internalized into the cells. C.
Rhodamine-TAT-ABD, Rhodamine-labeled TAT-ABD without GA resulting
in the internalization of the TAT-ABD protein into the cells. D.
Rhodamine-TAT-ABD+FITC-GA:, combination of B. plus C., resulting in
the internalization of both TAT-ABD and GA into the cells. E. A bar
graph showing the percent intracellular uptake of A.-D.
[0020] FIG. 14A-E show that TAT-ABD facilitates the internalization
of geldanamycin (GA) into MCF-7 cells. A. Control, non-treated
cells. B. FITC-GA, FITC-labeled GA internalized into the cells. C.
Rhodamine-TAT-ABD, Rhodamine-labeled TAT-ABD without GA resulting
in the internalization of the TAT-ABD protein into the cells. D.
Rhodamine-TAT-ABD+FITC-GA:, combination of B. plus C., resulting in
the internalization of both TAT-ABD and GA into the cells. E. A bar
graph showing the percent intracellular uptake of A.-D.
[0021] FIG. 15A-E show that TAT-ABD facilitates the internalization
of geldanamycin (GA) into MCF-7 cells. A. Control, non-treated
cells. B. FITC-GA, FITC-labeled GA internalized into the cells. C.
Rhodamine-TAT-ABD, Rhodamine-labeled TAT-ABD without GA resulting
in the internalization of the TAT-ABD protein into the cells. D.
Rhodamine-TAT-ABD+FITC-GA:, combination of B. plus C., resulting in
the internalization of both TAT-ABD and GA into the cells. E. A bar
graph showing the percent intracellular uptake of A.-D.
[0022] FIG. 16A-E show that TAT-ABD facilitates the internalization
of geldanamycin (GA) into MCF-7 cells. A. Control, non-treated
cells. B. FITC-GA, FITC-labeled GA internalized into the cells. C.
Rhodamine-TAT-ABD, Rhodamine-labeled TAT-ABD without GA resulting
in the internalization of the TAT-ABD protein into the cells. D.
Rhodamine-TAT-ABD+FITC-GA:, combination of B. plus C., resulting in
the internalization of both TAT-ABD and GA into the cells. E. A bar
graph showing the percent intracellular uptake of A.-D.
[0023] FIG. 17 shows that HER2-ABD/17-AAG (HA+17A) can improve the
anti-cancer activity of 17-AAG in HER2-positive cancer cells
(SKBr-3) compared to HER2-negative cancer cells (MDA-MB-231). Cells
were exposed to free-17-AAG (17A), free HER2-scFv-ABD (HA), or
HER2-scFv-ABD-17-AAG (HA+17A).
DETAILED DESCRIPTION
[0024] The present disclosure can be understood more readily by
reference to the following detailed description of the invention,
the figures and the examples included herein.
[0025] Before the present compositions and methods are disclosed
and described, it is to be understood that they are not limited to
specific synthetic methods unless otherwise specified, or to
particular reagents unless otherwise specified, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular aspects only and
is not intended to be limiting. Although any methods and materials
similar or equivalent to those described herein can be used in the
practice or testing of the present invention, example methods and
materials are now described.
[0026] Moreover, it is to be understood that unless otherwise
expressly stated, it is in no way intended that any method set
forth herein be construed as requiring that its steps be performed
in a specific order. Accordingly, where a method claim does not
actually recite an order to be followed by its steps or it is not
otherwise specifically stated in the claims or descriptions that
the steps are to be limited to a specific order, it is in no way
intended that an order be inferred, in any respect. This holds for
any possible non-express basis for interpretation, including
matters of logic with respect to arrangement of steps or
operational flow, plain meaning derived from grammatical
organization or punctuation, and the number or type of aspects
described in the specification.
[0027] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. The publications
discussed herein are provided solely for their disclosure prior to
the filing date of the present application. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided herein can be different
from the actual publication dates, which can require independent
confirmation.
Definitions
[0028] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise.
[0029] The word "or" as used herein means any one member of a
particular list and also includes any combination of members of
that list.
[0030] Ranges can be expressed herein as from "about" or
"approximately" one particular value, and/or to "about" or
"approximately" another particular value. When such a range is
expressed, a further aspect includes from the one particular value
and/or to the other particular value. Similarly, when values are
expressed as approximations, by use of the antecedent "about," or
"approximately," it will be understood that the particular value
forms a further aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units is
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0031] As used herein, the terms "optional" or "optionally" mean
that the subsequently described event or circumstance may or may
not occur and that the description includes instances where said
event or circumstance occurs and instances where it does not.
[0032] As used herein, the term "sample" is meant a tissue or organ
from a subject; a cell (either within a subject, taken directly
from a subject, or a cell maintained in culture or from a cultured
cell line); a cell lysate (or lysate fraction) or cell extract; or
a solution containing one or more molecules derived from a cell or
cellular material (e.g. a polypeptide or nucleic acid), which is
assayed as described herein. A sample may also be any body fluid or
excretion (for example, but not limited to, blood, urine, stool,
saliva, tears, bile) that contains cells or cell components.
[0033] As used herein, the term "subject" refers to the target of
administration, e.g., a human. Thus the subject of the disclosed
methods can be a vertebrate, such as a mammal, a fish, a bird, a
reptile, or an amphibian. The term "subject" also includes
domesticated animals (e.g., cats, dogs, etc.), livestock (e.g.,
cattle, horses, pigs, sheep, goats, etc.), and laboratory animals
(e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In one
aspect, a subject is a mammal. In another aspect, a subject is a
human. The term does not denote a particular age or sex. Thus,
adult, child, adolescent and newborn subjects, as well as fetuses,
whether male or female, are intended to be covered.
[0034] As used herein, the term "patient" refers to a subject
afflicted with a disease or disorder. The term "patient" includes
human and veterinary subjects. In some aspects of the disclosed
methods, the "patient" has been diagnosed with a need for treatment
for cancer, such as, for example, prior to the administering
step.
[0035] As used herein, the term "bi-functional allosteric
protein-drug molecule" refers to a composition comprising a
bi-functional allosteric protein and one or more therapeutic
agents, wherein the therapeutic agent is allosterically bound to
the biological binding domain of the bi-functional allosteric
protein. An example of a bi-functional allosteric protein-drug
molecule can be, for instance, "scFv-TBD-PTX" indicating a single
chain antibody bound to a taxane binding domain which is then
allosterically bound to paclitaxel (a drug molecule).
[0036] The bi-functional allosteric protein-drug molecules
described below have the following advantages over other known
antibody-drug conjugates: 1) one or more biological binding domains
that capture one or more therapeutic agents without the formation
of a chemical bond, thus avoiding over-conjugation of the
antibody-drug conjugate; 2) one or more therapeutic agents that
minimize the presence of free antibody; and 3) can enhance
half-life in the circulation while retaining affinity to an
antigen.
[0037] As used herein, the term "bi-functional allosteric protein"
refers to a protein comprising a targeting moiety and one or more
biological binding domains. Examples of bi-functional allosteric
proteins include, but are not limited to, bi-functional recombinant
scFv-ABD (ATP binding domain) fusion proteins, recombinant scFv-ABD
and scFv fusion proteins. In some aspects, a bi-functional
allosteric protein can comprise one or more biological binding
domains. For example, disclosed herein are bi-functional allosteric
proteins comprising an anti-HER2 antibody targeting moiety and one
or more biological binding domains, such as ATP, that then can
allosterically bind or capture one or more therapeutic agents, such
as 17-allyaminogeldanamycin (17-AGG).
[0038] As used herein, the term "targeting moiety" refers to the
portion of the bi-functional allosteric protein that specifically
binds a selected target. The targeting moiety can be, for example,
an antibody, peptide, polypeptide, growth factor or any ligand
protein. The target can be, for example, a receptor or an antigen
(e.g., HER2 (also called ErbB-2, ERBB2) or EGFR).
[0039] As used herein, the term "biological binding domain" or
"binding domain" refers to the portion of the bi-functional
allosteric protein that can allosterically bind to or capture a
therapeutic agent without the creation of chemical bonds. "Binding
domain" or "BD" can be an "ATP Binding Domain" or "ABD",
respectively.
Bi-Functional Allosteric Protein-Drug Molecules
[0040] Targeting moiety. In some aspects, the targeting moiety of
the bi-functional allosteric protein-drug molecule can be a
protein, including but not limited to a peptide or polypeptide, an
antibody or biologically active variant thereof, growth factor or
other ligand protein. For example, if the targeting moiety is an
antibody, the antibody can be a single chain antibody (scFv) or a
Fab fragment; a human, chimeric or humanized antibody or a
biologically active variant thereof and/or can be (or can be
derived from) a monoclonal or polyclonal antibody. The antibody can
be a naturally expressed antibody (e.g., a tetrameric antibody) or
a biologically variant thereof.
[0041] In some aspects, the targeting moiety of the bi-functional
allosteric protein-drug molecule can be a non-naturally occurring
antibody (e.g., a single chain antibody or diabody) or a
biologically active variant thereof. As noted above, the variants
include, without limitation, a fragment of a naturally occurring
antibody (e.g., an Fab fragment), a fragment of a scFv or diabody,
or a variant of a tetrameric antibody, an scFv, a diabody, or
fragments thereof that differ by an addition and/or substitution of
one or more amino acid residues. The antibody can also be further
engineered.
[0042] In other aspects, bi-functional allosteric protein-drug
molecules, described herein, comprise a targeting moiety, wherein
the targeting moiety is a scFv or Fab fragment that binds to a
growth factor receptor. The growth factor receptor can be a
receptor bound by a member of the epidermal growth factor (EGF)
family. Examples of receptors for proteins in the EGF family
include an EGF receptor (EGFR), a heparin-binding EGF-like growth
factor receptor (HB-EGFR), an amphiregulin receptor (AR), an
epiregulin receptor (EPR), a betacellulin receptor, and a receptor
for neuregulin (e.g., a receptor for neuregulin-1, neuregulin-2,
neuregulin-3, or neuregulin-4). Accordingly, in some embodiments,
the growth factor receptor is a member of the EGFR family (e.g.,
HER2 (human epidermal growth factor receptor 2), sometimes called
ERBB2, HER2/neu) and the targeting moiety is an antibody,
including, but not limited to, trastuzumab, cetuximab, or
panitumumab, or a biologically active variant thereof. In some
aspects, the scFv or Fab fragment can bind a cell surface receptor,
or a cell membrane protein (e.g., transport proteins, membrane
enzymes, and cell adhesion molecules). Other suitable targets to
which a "targeting moiety" can bind, include without limitation,
hormone receptors (e.g., estrogen, progesterone), cytokine
receptors (i.e., type I, such as growth hormone receptor,
prolactin, erythropoietin; type II; members of the immunoglobulin
superfamily, such as interleukin-1; tumor necrosis factor receptor
family, such as CD27, CD30, CD40; chemokine receptors, such as
interleukin-8, CCRI, CXCR4; transforming growth factor (TGF) beta
receptors); cell adhesion molecules (e.g., integrin); and vascular
endothelial growth factor (VEGF) receptors (e.g., neurophilin (NRP)
receptors, such as NRP1, NRP2). More generally, the targeting
moiety can be a therapeutic agent, such as an anti-cancer agent
(e.g., trastuzumab, cetuximab) or an anti-inflammatory agent.
[0043] Biological binding domain. Disclosed herein are
bi-functional allosteric protein-drug molecules comprising a
targeting moiety, one or more biological binding domains, wherein
the biological binding domain comprises an ATP binding domain
and/or a taxane binding domain, and one or more therapeutic agents,
wherein the therapeutic agent is allosterically bound to the
biological binding domain. The biological binding domain disclosed
herein, can be any molecule, compound, enzyme or nucleic acid
capable of forming an allosteric binding pocket. The disclosure
features, for example, one of more of the biological binding
domains capable of allosterically binding to one or more
therapeutic agents. The biological binding domain can be selected
based on its ability to form a binding pocket to permit the capture
of one or more therapeutic agents via allosterically binding.
Examples of one or more therapeutic agents to which a biological
binding domain can be selected include without limitation are
monomethyl auristatin E (MMAE), calicheamicins (e.g., calicheamicin
gammal, enediyne esperamicin), maytansine (also referred to as
maitansine), doxorubicin (also referred to as Adriamycin.RTM.,
Myocet.RTM., Rubex.RTM.; also known as hydroxydaunorubicin,
hydroxydaunocycin), camptothecin and/or duocarmycins, including
synthetic analogs adozelesin, bizelesin, and carzelesin; and any
derivatives or analogues thereof. The biological binding domain can
also be therapeutic agent and/or have therapeutic properties, such
as, ATP, for example.
[0044] The disclosure further features bi-functional allosteric
protein-drug molecules as described herein comprising two or more
biological binding domains that are different (i.e., the biological
binding domains of a single bi-functional allosteric protein-drug
molecule can be a combination of more than one type of biological
binding domain). Such bi-functional allosteric protein-drug
molecules, comprising more than one type of biological binding
domains, can capture different types of therapeutic agents in a
single formulation, and thus, can deliver different therapeutic
agents. For example, a bi-functional allosteric protein-drug
molecule comprising an ATP binding domain and a taxane binding
domain can capture and thus deliver 17-AAG and paclitaxel,
respectively. Also, the bi-functional allosteric protein-drug
molecules described herein can comprise two or more biological
binding domains that are the same but are allosterically bound to
different therapeutic agents.
[0045] Therapeutic agents. A wide variety of therapeutic agents or
cytotoxic agents can be incorporated into the bi-functional
allosteric protein-drug molecule. The therapeutic agents or
cytotoxic agents can be a chemical compound or a protein. In some
aspects, one or more of the therapeutic agents can be an
anti-cancer agent. The anti-cancer agent can be an agent or drug
that has anti-cancer properties. In some embodiments, the
anti-cancer agent can be a derivative of geldanamycin (a naturally
occurring ansamycin antibiotic), a taxane or a heat shock protein
90 (HSP90) inhibitor. Examples of geldanamycin derivatives include
but are not limited to 17-allylamino-17-demethoxygeldanamycin
(17-AAG), 17-dimethylaminoethylamino-17-demethoxygeldanamycin
(17-DMAG) or any analogues thereof. Examples of a taxane include
but are not limited to paclitaxel and docetaxel and any analogues
thereof. Examples of HSP90 inhibitors include but are not limited
to geldanamycin, geldanamycin derivatives (17-DMAG), radicicol
(also called monorden), retaspimycin hydrochloride (also known as
IPI-504), non-ansamycin compounds (BIIB021), SNX-5422
(PF-04929113), ganetespib (also known as STA-9090) and NVP-AUY922.
The anti-cancer agent can also be an EGFR inhibitor. Examples of
EGFR inhibitors include but are not limited to erlotinib
(Tarceva.RTM.) and afatinib (Gilotrif.RTM.).
[0046] The bi-functional allosteric protein-drug molecules
disclosed herein can be a therapeutic agent including but not
limited to one or more molecular chaperone inhibitors (e.g., a
HSP90 inhibitor), or tublin inhibitors (e.g., taxane) and/or
stabilizers or DNA replication inhibitors or any anti-cancer agent
or any derivatives and/or analogues thereof to provide an
additional therapeutic benefit.
[0047] Cytotoxins that target microtubules include, without
limitation, the taxanes, and in particular, the taxanes including,
paclitaxel (Taxol.RTM.), docetaxel (Taxotere.RTM.), and cabazitaxel
(Jevtana.RTM.). Nontaxane microtubule-targeting agents such as an
epothilone (e.g., epothilone A, B, C, D, E, or F) and eribulin can
also be incorporated. Other useful cytotoxic agents include the
alkaloids (e.g., a vinca alkaloid such as vincristine, vinblastine,
vindesine, and vinorelbine), an alkylating agent (e.g.,
cyclophosphamide, mechlorethamine, chlorambucil, or melphan) an
anthracycline (e.g., daunorubicin, doxorubicin, epirubicin,
idarubicin, mitoxantrone, and valrubicin), an auristatin (e.g.
monomethyl auristatin E (MMAE), an antifolate (e.g., methotrexate
or aminopterin), a calicheamicin (e.g., calicheamicin .gamma.1), a
duocarmycin (e.g., adozelesin, bizelesin, or carzelesin); a
mitomycin (e.g., mitomycin C), a pyrimidine analog (e.g.,
fluorouracil), or a derivative of mytansine (e.g., a mytansinoid
such as ansamitocin, mertansine, or emtansine).
Linker-Free Technology
[0048] Although advances in linker technology have led to the
production of highly potent antibody-drug conjugates with enhanced
stability in the bloodstream, which in turn has improved the
targeted delivery of cytotoxic agents, they still have limited
clinical use because of poor therapeutic efficacy in human
patients. These limitations include antibody accumulation in normal
tissues, immunogenicity and difficulties in the chemical
conjugation of cytotoxic drugs to the required antibody. In
addition, cytotoxins have limited therapeutic use in cancer
patients because of their toxicity profiles. Factors that limit the
number of drug molecules delivered into cancer cells include the
small number of antigens present on the cell surface; the rate of
internalization of the antibody-drug conjugate; and intracellular
processing to release the drug from the antibody. Further, several
steps are involved beginning from the administration of the
antibody-drug conjugate to the release of the drug (or cytotoxic
agent) in cancer cells. These steps include: 1) the antibody-drug
conjugate reaching the tumor; 2) binding of the antibody-drug
conjugate to the surface of the cell; 3) internalization of the
antibody-drug conjugate; 4) cleavage of the linker; 5)
endolysosomal escape; and 6) the drug reaching its intra-cellular
target.
[0049] Disclosed herein, are compositions comprising bi-functional
allosteric protein-drug molecules and methods of making said
molecules using linker-free technology. For example, one or more of
the therapeutic agents are incorporated into the biological binding
domain spontaneously without altering the structure or activity of
the therapeutic agent or the affinity of the bi-functional
allosteric protein (e.g., scFv), to its target (e.g., antigen). In
other words, the formation of the bi-functional allosteric
protein-drug molecules described herein relies on the biological
binding affinity of the therapeutic agent (e.g., anti-cancer agent)
and does not require chemical conjugation to join molecules.
Accordingly, in some aspects, the bi-functional allosteric
protein-drug molecule comprises a targeting moiety (e.g.,
recombinant scFv), one or more biological binding domains (e.g.,
ATP) and one or more therapeutic agents such that a single binding
domain (e.g., ATP) captures a single therapeutic molecule (e.g.,
17-AAG). The association of the biological binding domain and the
therapeutic agent occurs because of the binding affinity of the
therapeutic molecule (e.g., 17-AAG) to the binding domain (e.g.,
ATP). Another feature disclosed herein is that the incorporation of
the therapeutic agent (e.g., 17-AAG) into the biological domain
(for example, scFv-ABD), results in a bi-functional allosteric
protein-drug molecule (e.g., scFv-ATP-17-AAG) that is ready to use.
In some instances, the incorporation of the therapeutic agent into
the biological domain can be within one hour of incubation.
[0050] Additional advantages of using linker-free technology
compared to the drugs conjugated to an antibody with a synthetic
linker are as follows. In current antibody-drug conjugate
technologies, the optimum number of drug molecules is limited to 3
to 4 drug molecules per antibody. The bi-functional allosteric
protein-drug molecule described herein can be homogenous and the
number of therapeutic molecules incorporated into the bi-functional
allosteric protein can be controlled by adding more biological
binding domains. Moreover, the number of steps from administration
to drug release listed above is reduced from six steps to four
(e.g., steps 4 and 6 are omitted). And, other steps can be
minimized (e.g., step 3). For example, the bi-functional allosteric
protein (e.g., scFv) is smaller than the monoclonal antibody used
in current antibody-drug conjugates and thus, facilitates its
internalization into cancer cells and thereby minimizing the time
for the therapeutic agent to reach its intracellular target. And,
because the release of the therapeutic agent is driven by its
inherent affinity for its target, chemical cleavage of a synthetic
linker is not required. Further, when the therapeutic agent is a
drug that targets a protein, for example, a cytosolic protein, that
is important in cancer cell survival, such as 17-AAG, for instance,
the therapeutic agent does not need to migrate into the nucleus and
can exert its anti-cancer activity even in cancer cells that are
not dividing. Accordingly, in some embodiments, the therapeutic
agent is a drug that targets onco-proteins present in the cytosol.
Because the methods of producing bi-functional allosteric
protein-drug molecules, as disclosed herein, has fewer steps from
administration to drug activity compared to antibody-drug conjugate
therapies, more of the dose delivered can reach its target site to
exert its anti-cancer activity. In some embodiments, the dose
delivered to the target site can be more than 6-fold higher
compared to current antibody-drug conjugate technologies.
[0051] The methods described herein can serve as a platform for the
design of other binding domains to anchor other highly potent drugs
to a targeting moiety without the need for a linker or other forms
of chemical conjugation. Further, by using the platform described
herein, chemotherapeutic agents currently in clinical use as well
as therapeutic agents and cytotoxins are candidates for the
development and/or production of bi-functional allosteric
protein-drug molecules.
Methods of Making Bi-Functional Allosteric Protein-Drug
Molecules
[0052] Disclosed herein are techniques that can be used to produce
the bi-functional allosteric protein-drug molecules described
herein.
[0053] Bi-functional allosteric proteins. In some aspects, the
scFv-ABD or the scFv-TBD disclosed herein is a recombinant fusion
protein that is expressed in living cells (e.g., mammalian cells).
Briefly, a plasmid DNA encoding the recombinant fusion protein,
bound to one or more biologically binding domains (e.g., scFv-ABD
or scFv-TBD), amino acid sequences is transfected into mammalian
cells (e.g., HEK293 cells or CHO cells). The transfected cells are
incubated to express the scFv-ABD or the scFv-TBD for at least a
week or in some cases more than one week. After the incubation
period, the cells are lysed and the whole protein is collected. The
expressed scFv-ABD or the scFv-TBD is purified using a fast protein
liquid chromatography (see Example 1). The purified protein, the
scFv-ABD or the scFv-TBD, is then lyophilized and stored at
-80.degree. C. until use.
[0054] Bi-functional allosteric protein-drug molecules. Disclosed
herein are methods of producing the bi-functional allosteric
protein-drug molecule (e.g., scFv-ABD-17-AAG) comprising the
following steps: (1) dissolving a bi-functional scFv-ABD in
physiological buffer at a predetermined concentration; (2)
dissolving 17-AAG in an appropriate solvent at a predetermined
concentration; (3) mixing the solutions as a result of steps (1)
and (2) at an optimized ratio, i.e., the ratio that yields the
highest incorporation of a therapeutic agent into a bi-functional
scFv-ABD; and (4) diluting the solution of step (3) in
physiological buffer to make a final concentration for
administration (e.g., infusion, injection). One of ordinary skill
in the art can determine the proper solvent(s) required and
calculate the concentrations of any of the ingredients involved in
each step.
[0055] Antibodies. As noted above, the bi-functional allosteric
protein-drug molecules as disclosed herein, can include an antibody
or a biologically active variant thereof. As is well known in the
art, monoclonal antibodies can be made by recombinant DNA. DNA
encoding monoclonal antibodies can be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
Libraries of antibodies or active antibody fragments can also be
generated and screened using phage display techniques.
[0056] In vitro methods are also suitable for preparing monovalent
antibodies. As it is well known in the art, some types of antibody
fragments can be produced through enzymatic treatment of a
full-length antibody. Digestion of antibodies to produce fragments
thereof, particularly, Fab fragments, can be accomplished using
routine techniques known in the art. For instance, digestion can be
performed using papain. Papain digestion of antibodies typically
produces two identical antigen binding fragments, called Fab
fragments, each with a single antigen binding site, and a residual
Fc fragment. Pepsin treatment yields a fragment that has two
antigen combining sites and is still capable of cross-linking
antigen. Antibodies incorporated into the present bi-functional
allosteric protein-drug molecules can be generated by digestion
with these enzymes or produced by other methods.
[0057] The fragments, whether attached to other sequences or not,
can also include insertions, deletions, substitutions, or other
selected modifications of particular regions or specific amino
acids residues, provided the activity of the antibody or antibody
fragment is not significantly altered or impaired compared to the
non-modified antibody or antibody fragment. These modifications can
provide for some additional property, such as to remove/add amino
acids capable of disulfide bonding, to increase its bio-longevity,
to alter its secretory characteristics, etc. In any case, the
antibody or antibody fragment must possess a bioactive property,
such as specific binding to its cognate antigen. Functional or
active regions of the antibody or antibody fragment can be
identified by mutagenesis of a specific region of the protein,
followed by expression and testing of the expressed polypeptide.
Such methods are readily apparent to a skilled practitioner in the
art and can include site-specific mutagenesis of the nucleic acid
encoding the antibody or antibody fragment.
[0058] As used herein, the term "antibody" or "antibodies" can also
refer to a human antibody and/or a humanized antibody. Many
non-human antibodies (e.g., those derived from mice, rats, or
rabbits) are naturally antigenic in humans, and thus can give rise
to undesirable immune responses when administered to humans.
Therefore, the use of human or humanized antibodies in the methods
serves to lessen the chance that an antibody administered to a
human will evoke an undesirable immune response.
[0059] Antibody humanization techniques generally involve the use
of recombinant DNA technology to manipulate the DNA sequence
encoding one or more polypeptide chains of an antibody molecule.
Accordingly, a humanized form of a non-human antibody (or a
fragment thereof) is a chimeric antibody or antibody chain (or a
fragment thereof, such as an Fv, Fab, Fab', or other antigen
binding portion of an antibody) which contains a portion of an
antigen binding site from a non-human (donor) antibody integrated
into the framework of a human (recipient) antibody.
[0060] The Fv region is a minimal fragment containing a complete
antigen-recognition and binding site consisting of one heavy chain
and one light chain variable domain. The three CDRs of each
variable domain interact to define an antigen-biding site on the
surface of the Vh-Vl dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. As well known in the
art, a "single-chain" antibody or "scFv" fragment is a single chain
Fv variant formed when the VH and Vl domains of an antibody are
included in a single polypeptide chain that recognizes and binds an
antigen. Typically, single-chain antibodies include a polypeptide
linker between the Vh and Vl domains that enables the scFv to form
a desired three-dimensional structure for antigen binding.
[0061] To generate a humanized antibody, residues from one or more
complementarity determining regions (CDRs) of a recipient (human)
antibody molecule are replaced by residues from one or more CDRs of
a donor (non-human) antibody molecule that is known to have desired
antigen binding characteristics (e.g., a certain level of
specificity and affinity for the target antigen). In some
instances, Fv framework (FR) residues of the human antibody are
replaced by corresponding non-human residues. Humanized antibodies
can also contain residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. Generally,
a humanized antibody has one or more amino acid residues introduced
into it from a source which is non-human. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies. Humanized antibodies
generally contain at least a portion of an antibody constant region
(Fc), typically that of a human antibody.
[0062] Methods for humanizing non-human antibodies are well known
in the art. For example, humanized antibodies can be generated by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Methods that can be used to produce
humanized antibodies are also well known in the art.
[0063] Biological binding domains. The term "binding affinity" in
the context of further describing the biological binding domain,
refers to the interaction of a biological binding domain with a
therapeutic agent. In general, the binding affinity of the
biological binding domain to a therapeutic agent should be
maintained at a level that promotes an interaction between the
biological binding domain and a therapeutic agent that is stable in
systemic circulation (i.e., the therapeutic agent remains
allosterically bound to the biological binding domain in the
presence of serum proteins), and facilitates the delivery or
unloading of a therapeutic agent to its target (e.g., cancer cell).
It can be this difference in the binding affinities of a
therapeutic agent to serum proteins compared to a given target
molecule in a cell that provides a mechanism for the release of a
therapeutic agent from the biological binding domain in a cell. For
example, the binding affinity of a therapeutic agent is generally
greater for the biological binding domain compared to any serum
protein, but low enough to permit its release once it reaches its
target.
[0064] Assessment. The bi-functional allosteric protein-drug
molecules themselves or parts of the bi-functional allosteric
protein-drug molecule can be assessed in any number of ways. For
example, the binding of the therapeutic agent with the biological
binding domain can be confirmed (by using a magnetic bead-based
pull down assay); cellular or kinetic uptake of the therapeutic
agent can be evaluated (using fluorescence techniques); levels of
the unbound antibody and low levels of free drug can be confirmed
(by gel separation and Western blotting), function of the
biological binding domain as a reducer of cellular ATP and an
inducer of apoptosis (by performing live/dead assay using FACS);
the binding of, for example, scFv-ABD to different numbers of
therapeutic molecules (by binding assays using a biotinylated GM
probe); and for stability and tissue distribution in vivo (e.g., by
measuring plasma levels over time and tissue distribution by
imaging assays).
[0065] Configurations. Each part of a given bi-functional
allosteric protein-drug molecule, including the targeting moiety,
biological binding domain and therapeutic agent, can be selected
independently. One of ordinary skill in the art would understand
that the component parts need to be associated in a compatible
manner. The bi-functional allosteric protein-drug molecules can be
used to deliver antibody moieties and therapeutic agents to a
patient for the treatment of cancer. The targeting moiety can be
referred to as a "first agent," the therapeutic called the "second
agent" and the biological binding domain, referred to as a "third
agent." And, thus, the bi-functional allosteric protein-drug
molecules can be a combination therapy for a disease (e.g., a
cancer). Different binding domains can carry different therapeutic
agents. Thus, a bi-functional allosteric protein-drug molecule can
deliver two or more different therapeutic agents. With the
inclusion of a detectable marker, the bi-functional allosteric
protein-drug molecule or the bi-functional allosteric protein as
described herein can also be used to map the distribution of
targets to which the targeting moieties bind. The number of
therapeutic molecules per bi-functional allosteric protein (e.g.,
scFv) can be controlled by adding more binding domains. For
example, more binding domains can be added to the C-terminus end of
the bi-functional allosteric protein.
[0066] Accordingly, in some aspects, the biological binding domain
can be two or more. In other embodiments, the biological binding
domain and the therapeutic agent are present in a ratio of 1:1
(binding domain:therapeutic agent). The binding domain:therapeutic
agent ratio can also be 2:2, 3:3, 4:4 or 5:5 or any other
combination thereof. Each binding domain is capable of capturing a
single therapeutic agent or drug molecule. For example, the one or
more taxane binding domains can allosterically bind to one or more
taxane molecules, in which the taxane molecules can be either the
same or different. In some aspects, the biological binding domain
can be different in a single formulation. Thus, the biological
binding domains capture different therapeutic agents or drug
molecules.
[0067] In addition, the biological binding domain (e.g., ATP)
serves not only as a carrier of the therapeutic agent, but can also
act as a therapeutic agent or possess one or more therapeutic
properties (i.e., reduce cellular ATP levels). For instance, after
the release of the therapeutic agent, the ATP binding domain can
bind to ATP molecules in cancer cells, thereby leading to a
decrease in cellular free ATP levels, cell cycle arrest, reduced
proliferation, and/or apoptosis. Thus, the bi-functional allosteric
protein-drug molecules described herein can be multi-functional and
thus, the target delivery of, for example, an anti-cancer agent or
molecular chaperone inhibitor, such as an HSP 90 inhibitor (e.g.,
17-AAG) by the bi-functional allosteric protein-drug molecule can
be a synergistic therapy for the treatment of disease, such as
cancer (e.g., breast cancer). Also, the ATP biological binding
domain can bind more than one type or class of therapeutic agent.
Similarly, the biological binding domain can be designed to
accommodate many types of therapeutics, including but not limited
to monomethyl auristatin E (MMAE), calicheamicins (e.g.,
calicheamicin gammal, enediyne esperamicin), maytansine (also
referred to as maitansine), doxorubicin (also referred to as
Adriamycin.RTM., Myocet.RTM., Rubex.RTM.; also known as
hydroxydaunorubicin, hydroxydaunocycin), camptothecin and/or
duocarmycins, including synthetic analogs adozelesin, bizelesin,
and carzelesin; and any derivatives or analogues thereof.
[0068] Using the linker-free technology to join molecules for
targeted drug delivery as described herein, chemotherapeutic agents
currently used in the clinic to treat cancers, are also considered
candidates for the development of novel bi-functional allosteric
protein-drug molecules. Moreover, because binding domains can be
added to a bi-functional allosteric protein or recombinant fusion
protein comprising a scFv antibody that has a high binding affinity
for a particular drug candidate, the number, for example, of 17-AAG
molecules per scFv can be controlled by adding more ATP binding
domains, for instance, at the C-terminus end of the scFv. The
Protein Data Bank provides the information necessary for protein
structural analysis, a key to finding a binding domain that can
capture a particular ligand drug. With the completion of the Human
Genome Project, DNA sequences encoding these binding domains have
been identified and cDNAs that contain these DNA sequences are
commercially available. In addition, protein crystallography and
NMR-based protein structural analysis further assist in identifying
which amino acids are involved in the actual interaction between
the drug molecule and the binding pocket. Using the technology
described herein, the number of potential chemotherapeutic agents
that can be used in the development of novel bi-functional
allosteric protein-drug molecules is significant. The tailored
linker-free technology including biological binding domain
selection, site-specific chemistry, and the introduction of
functional groups to the drug can be optimized for each drug
candidate.
[0069] The methods disclosed herein related to the process of
producing the bi-functional allosteric protein-drug molecules as
disclosed can be readily modified to produce a pharmaceutically
acceptable salt of the bi-functional allosteric protein-drug
molecules. Pharmaceutical compositions including such salts and
methods of administering them are accordingly within the scope of
the present disclosure.
Pharmaceutical Compositions
[0070] As disclosed herein, are pharmaceutical compositions,
comprising the bi-functional allosteric protein-drug molecule and a
pharmaceutical acceptable carrier described above. In some aspects,
the therapeutic agent is an anti-cancer agent and the
pharmaceutical composition is formulated for intravenous
administration. The compositions of the present disclosure also
contain a therapeutically effective amount of a bi-functional
allosteric protein-drug molecule as described herein. The
compositions can be formulated for administration by any of a
variety of routes of administration, and can include one or more
physiologically acceptable excipients, which can vary depending on
the route of administration. As used herein, the term "excipient"
means any compound or substance, including those that can also be
referred to as "carriers" or "diluents." Preparing pharmaceutical
and physiologically acceptable compositions is considered routine
in the art, and thus, one of ordinary skill in the art can consult
numerous authorities for guidance if needed.
[0071] The pharmaceutical compositions as disclosed herein can be
prepared for oral or parenteral administration. Pharmaceutical
compositions prepared for parenteral administration include those
prepared for intravenous (or intra-arterial), intramuscular,
subcutaneous, intraperitoneal, transmucosal (e.g., intranasal,
intravaginal, or rectal), or transdermal (e.g., topical)
administration. Aerosol inhalation can also be used to deliver the
bi-functional allosteric protein-drug molecules. Thus, compositions
can be prepared for parenteral administration that includes
bi-functional allosteric protein-drug molecules dissolved or
suspended in an acceptable carrier, including but not limited to an
aqueous carrier, such as water, buffered water, saline, buffered
saline (e.g., PBS), and the like. One or more of the excipients
included can help approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents, detergents, and the like. Where the compositions include a
solid component (as they may for oral administration), one or more
of the excipients can act as a binder or filler (e.g., for the
formulation of a tablet, a capsule, and the like). Where the
compositions are formulated for application to the skin or to a
mucosal surface, one or more of the excipients can be a solvent or
emulsifier for the formulation of a cream, an ointment, and the
like.
[0072] The pharmaceutical compositions can be sterile and
sterilized by conventional sterilization techniques or sterile
filtered. Aqueous solutions can be packaged for use as is, or
lyophilized, the lyophilized preparation, which is encompassed by
the present disclosure, can be combined with a sterile aqueous
carrier prior to administration. The pH of the pharmaceutical
compositions typically will be between 3 and 11 (e.g., between
about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8).
The resulting compositions in solid form can be packaged in
multiple single dose units, each containing a fixed amount of the
above-mentioned agent or agents, such as in a sealed package of
tablets or capsules. The composition in solid form can also be
packaged in a container for a flexible quantity, such as in a
squeezable tube designed for a topically applicable cream or
ointment.
Methods of Treatment
[0073] Disclosed herein, are methods of treating a patient with
cancer, the method comprising: (a) identifying a patient in need of
treatment; and (b) administering to the patient a therapeutically
effective amount of the pharmaceutical composition comprising
bi-functional allosteric protein-drug molecules comprising a
targeting moiety, one or more biological binding domains, and one
or more therapeutic agents, wherein the therapeutic agent is
allosterically bound to the biological binding domain, and a
pharmaceutically acceptable carrier.
[0074] The pharmaceutical compositions described above can be
formulated to include a therapeutically effective amount of a
bi-functional allosteric protein-drug molecule. Therapeutic
administration encompasses prophylactic applications. Based on
genetic testing and other prognostic methods, a physician in
consultation with their patient can choose a prophylactic
administration where the patient has a clinically determined
predisposition or increased susceptibility (in some cases, a
greatly increased susceptibility) to a type of cancer.
[0075] The pharmaceutical compositions described herein can be
administered to the subject (e.g., a human patient) in an amount
sufficient to delay, reduce, or preferably prevent the onset of
clinical disease. Accordingly, in some aspects, the patient is a
human patient. In therapeutic applications, compositions are
administered to a subject (e.g., a human patient) already with or
diagnosed with cancer in an amount sufficient to at least partially
improve a sign or symptom or to inhibit the progression of (and
preferably arrest) the symptoms of the condition, its
complications, and consequences. An amount adequate to accomplish
this is defined as a "therapeutically effective amount." A
therapeutically effective amount of a pharmaceutical composition
can be an amount that achieves a cure, but that outcome is only one
among several that can be achieved. As noted, a therapeutically
effective amount includes amounts that provide a treatment in which
the onset or progression of the cancer is delayed, hindered, or
prevented, or the cancer or a symptom of the cancer is ameliorated.
One or more of the symptoms can be less severe. Recovery can be
accelerated in an individual who has been treated.
[0076] In some aspects, the cancer is a primary or secondary tumor.
In other aspects, the primary or secondary tumor is within the
patient's breast or lung. In yet other aspects, the cancer is
associated with the expression of HER2 and/or the expression of an
epidermal growth factor receptor.
[0077] Disclosed herein, are methods of treating a patient with
cancer. The cancer can be any cancer. In some aspects, the cancer
is breast cancer, ovarian cancer, lung cancer, or gastric
cancer.
[0078] Amounts effective for this use can depend on the severity of
the cancer and the weight and general state and health of the
subject, but generally range from about 0.05 .mu.g to about 1000
.mu.g (e.g., 0.5-100 .mu.g) of an equivalent amount of the
bi-functional allosteric protein-drug molecule per dose per
subject. Suitable regimes for initial administration and booster
administrations are typified by an initial administration followed
by repeated doses at one or more hourly, daily, weekly, or monthly
intervals by a subsequent administration. For example, a subject
can receive a bi-functional allosteric protein-drug molecule in the
range of about 0.05 to 1,000 .mu.g equivalent dose as compared to
unbound or free therapeutic agent(s) per dose one or more times per
week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week). For
example, a subject may receive 0.1 to 2,500 .mu.g (e.g., 2,000,
1,500, 1,000, 500, 100, 10, 1, 0.5, or 0.1 .mu.g) dose per week. A
subject can also receive a bi-functional allosteric protein-drug
molecule in the range of 0.1 to 3,000 .mu.g per dose once every two
or three weeks. A subject can also receive 2 mg/kg every week (with
the weight calculated based on the weight of the bi-functional
allosteric protein-drug molecule or any part or component of the
bi-functional allosteric protein-drug molecule).
[0079] The total effective amount of a bi-functional allosteric
protein-drug molecule in the pharmaceutical compositions disclosed
herein can be administered to a mammal as a single dose, either as
a bolus or by infusion over a relatively short period of time, or
can be administered using a fractionated treatment protocol in
which multiple doses are administered over a more prolonged period
of time (e.g., a dose every 4-6, 8-12, 14-16, or 18-24 hours, or
every 2-4 days, 1-2 weeks, or once a month). Alternatively,
continuous intravenous infusions sufficient to maintain
therapeutically effective concentrations in the blood are also
within the scope of the present disclosure.
[0080] The therapeutically effective amount of one or more of the
therapeutic agents present within the compositions described herein
and used in the methods as disclosed herein applied to mammals
(e.g., humans) can be determined by one of ordinary skill in the
art with consideration of individual differences in age, weight,
and other general conditions (as mentioned above). Because the
bi-functional allosteric protein-drug molecules of the present
disclosure can be stable in serum and the bloodstream and in some
cases more specific, the dosage of the bi-functional allosteric
protein-drug molecule including any individual component can be
lower (or higher) than an effective dose of any of the individual
components when unbound. Accordingly, in some aspects, the
anti-cancer agent administered has increased efficacy or reduced
side effects when administered as part of a bi-functional
allosteric protein-drug molecule as compared to when the
anti-cancer agent is administered alone or not as part of a
bi-functional allosteric protein-drug molecule.
EXAMPLES
Example 1
Synthesis of the Bi-Functional Allosteric Protein-Drug Molecule
[0081] A scheme for constructing a bi-functional allosteric
protein-drug molecule is shown in FIGS. 10 and 11. A recombinant
fusion protein (e.g., bi-functional allosteric protein) comprising
a protein transduction domain (PTD) and an ATP binding domain
(ABD), (PTD-ABD), was used as a model to simulate the anti-cancer
effects of 17-AAG delivered by the scFv-ABD because the PTD is
capable of delivering cargo into cells. Geldanamycin (GM), an
antibiotic that inhibits the function of HSP90, was used to develop
a model bi-functional allosteric protein-drug molecule. The
GM-binding domain of HSP90, located at the N-terminus of HSP90,
contains the ATP binding pocket. The DNA fragment (0.7 kb) encoding
the ATP binding domain was excised from the N-terminus of HSP90
(residues 9-232) and sub-cloned into the pET-28a expression vector.
To prepare PTD-ABD fusion protein, a short DNA fragment encoding
TAT was inserted at the N-terminus of the coding region for the ATP
binding domain with a glycine spacer. The recombinant PTD-ABD was
expressed in E. coli BL21 and purified by using fast protein liquid
chromatography (FPLC) equipped with a Ni.sup.2+-NTA column. The
molecular weight of the final purified protein was determined by
SDS-PAGE as shown in FIG. 1.
[0082] To confirm that the purified PTD-ABD fusion protein has a
binding affinity for 17-AAG, a magnetic bead-based pull down assay
was performed. GM was used instead of 17-AAG because biotin-17-AAG
is not commercially available. Biotin-GM was mixed with the
recombinant PTD-ABD fusion protein at 4.degree. C. for 1 hour and
streptavidin-magnetic beads were added to the mixture to pull down
the bound protein. The PTD-ABD fusion protein was eluted from the
beads (FIG. 1). A breast cancer cell lysate was added to the
extracted sample (PTD-ABD+GM+beads) to verify that native HSP90 in
the cancer cell lysate could replace the PTD-ABD that was already
bound to GM+beads. In the PTD-ABD+GM beads+cell lysate group, the
band of PTD-ABD became thinner compared to the PTD-ABD+GM beads
group (FIG. 1, middle lane) and a new band representing native
HSP90 (FIG. 1, right lane) was observed. These data show that the
PTD-ABD can capture GM and that GM is released from the PTD-ABD
spontaneously in the presence of native HSP90. This observation is
explainable because, in a cancer cell, HSP90 has a higher binding
affinity to GM than it has to the recombinant ATP binding domain.
These results demonstrate that the ATP binding domain retains its
binding affinity to GM when fused with another protein domain,
confirming that the scFv-ATP binding domain will have the same
binding affinity to 17-AAG.
Example 2
Enhanced Anti-Cancer Efficacy of 17-AAG Delivered by the PTD-ABD
Fusion Protein in Breast Cancer Cells
[0083] To test the therapeutic efficacy of the formulation of
PTD-ABD-17-AAG in MCF-7 breast cancer cells, the IC.sub.50 value
was determined in a dose-dependent manner. The IC.sub.50 value is a
measure of how effective a drug or therapeutic is and is defined as
the concentration required to inhibit a biological process or
component of a biological process (e.g., enzyme, cell, cell
receptor) in half. The PTD-ABD-17-AAG was prepared as described
above (Example 1) and administered to cancer cells. MCF-7 cells
were exposed to one of the following formulations: 1)
PTD-ABD-17-AAG; 2) 17-AAG; or 3) PTD-ABD. The IC.sub.50 values were
10 .mu.M and 15 .mu.M for the PTD-ABD-17-AAG and 17-AAG treatment
groups, respectively. These results show that delivery of 17-AAG by
the PTD-ABD enhanced the anti-cancer efficacy of 17-AAG (FIG. 2).
Further, the PTD-ABD fusion protein administered without 17-AAG
resulted in a decrease in cell viability to 80%, confirming that
the ATP binding domain also has anti-cancer efficacy/therapeutic
properties.
[0084] To test whether the PTD-ABD could improve the uptake
kinetics of 17-AAG into cells, FITC-GM bound with the PTD-ABD or
ABD alone was administered to MCF-7 cells in a time-dependent
manner. At 2 hours post-treatment, the PTD-ABD internalized GM into
100% of the cell population, while 60% of the cell population was
positive to FITC in the groups administered free GM and the ABD-GM
(FIG. 3a). In addition, the mean fluorescence intensity (MFI),
indicating the amount of GM internalized, increased continuously in
a time-dependent manner in the PTD-ABD-GM group, whereas the free
GM group and the ABD-GM group did not show this same pattern (FIG.
3b). Finally, 10 .mu.g/ml of GM was sufficient to achieve
internalization into 100% of the cell population when delivered by
the PTD-ABD protein (FIG. 3c). On the other hand, both free GM and
the ABD-GM did not achieve 100% cellular uptake even at a GM
concentration of 25 .mu.g/ml. These results indicate that the
formation of the PTD-ABD-17-AAG improves the uptake kinetics of
17-AAG, reducing the effective dose of 17-AAG required to achieve
anti-cancer efficacy.
[0085] To examine whether the improved cellular uptake of 17-AAG by
the PTD-ABD leads to enhanced efficacy of 17-AAG, breast cancer
cells were exposed to 17-AAG or the PTD-ABD-17-AAG for 4 hours in a
dose-dependent manner. The IC.sub.50 value of the PTD-ABD-17-AAG
was determined to be approximately 5 .mu.g/ml, while that of free
17-AAG was approximately 25 .mu.g/ml (FIG. 4). Due to the
facilitated uptake of 17-AAG bound to the PTD-ABD, the short-term
treatment of 17-AAG at low concentrations can increase the cellular
concentration of 17-AAG, which results in the enhanced anti-cancer
efficacy of 17-AAG. Free 17-AAG was not sufficiently internalized
into cells during the short incubation time because of the poor
uptake kinetics of free 17-AAG.
[0086] To test whether the ATP binding domain is available to
capture cellular ATP upon release of 17-AAG, thereby resulting in a
reduction in the intracellular levels of ATP, an ATP assay
(luciferase ATP detection kit; Invitrogen) was carried out. The ATP
level in the PTD-ABD treatment group was decreased by 20% relative
to control. This result indicates that the PTD-ABD fusion protein
is capable of reducing cellular ATP levels and inhibiting cellular
proliferation. Although the total number of viable cells was
decreased to 80% compared to control (FIG. 2), it is unknown
whether the decreased cell viability was due to cell death or to an
inhibition of cellular proliferation caused by the reduced levels
of intracellular ATP. To this end, a live/dead assay was performed,
which verified an increase in the apoptotic cell population in the
PTD-ABD group, meaning that the decreased cell viability was due to
the increased cellular apoptosis (FIG. 5). Consequently, the
intracellular deposit of PTD-ABD leads to a decrease in cellular
ATP, which in turn induces apoptosis and cell death. This
observation provides evidence that the PTD-ABD can reduce
intracellular levels of ATP and induce apoptosis.
[0087] Through the above experiments, the following findings have
been confirmed: 1) an ATP binding domain with a high binding
affinity to 17-AAG can be produced; 2) the PTD-ABD, as a model
system, is capable of delivering 17-AAG into cells; 3)
PTD-ABD-17-AAG improves the uptake kinetics of 17-AAG; 4) the
improved cellular uptake of 17-AAG leads to enhanced anti-cancer
efficacy of 17-AAG; 5) the ATP binding domain can reduce
intracellular levels of ATP; and 6) the reduced intracellular
levels of ATP induces apoptosis. In addition, the ATP binding
domain when fused with another protein domain retains its binding
affinity to 17-AAG, demonstrating that the scFv-ABD likely retains
the dual functionalities of targeting HER2 and binding 17-AAG in
the formation of a bi-functional allosteric protein.
Example 3
Stability of ABD-GM in Serum
[0088] To determine the stability of ABD-GM in serum, the
ATP-binding domain (ABD) was first incubated with GM-biotin to form
ABD-GM. Next, ABD-GM was mixed with serum at the final serum
concentration of 67% followed by incubation at 37.degree. C. The
bound protein was isolated by streptavidin-magnetic beads and
eluted for SDS-PAGE. The results show that the ABD bands remained
the same size as control and ABD-GM remained bound in the presence
of serum for 4 hours of incubation at 37.degree. C. (FIG. 6).
ABD-GM did not dissociate, nor did the GM bind to other proteins in
the serum, as no other bands were observed. These data indicate
that ABD-GM is stable in serum for an extended period of time.
Example 4
Determination of the Dissociation Kinetics of GM from the scFv-ABD
in Breast Cancer Cells
[0089] The ability of GM to be released from the PTD-ATP-binding
domain and then bind to native HSP90 in cancer cells was studied
using recombinant PTD-ABD protein and cancer cell lysate. Four
different breast cancer cell lines were used: two cell lines that
over express HER2, BT-474 (FIG. 7A) and SK-BR-3 (FIG. 8B); and two
normal cell lines, MCF-7 (FIG. 7B) and MDA-MB-231 (FIG. 8A;
2.times.10.sup.5 cells/well). As described in Example 1, the
PTD-ABD fusion protein was prepared as follows, a short DNA
fragment encoding TAT (100 .mu.g) was inserted at the N-terminus of
the coding region for the ATP binding domain with a glycine spacer
(FIG. 7, 8, lane 1). Cancer cell lysate (FIG. 7, lane 2) was also
incubated with GM-biotin (FIG. 7, 8, lane 3). The results show that
the bound protein to GM is native HSP90 (MW=.about.70 kDa) and that
GM specifically binds to HSP90 in a cancer cell.
[0090] Then, a mixture of cancer cell lysate and ABD was incubated
with biotin-GM (FIG. 7, 8, lane 4) at 4.degree. C. for 1 hour
showing that the primary binding protein for GM is HSP90 because a
very thick HSP90 band is observed, while a thin and weak ABD band
is detected. Next, the cancer cells were incubated with ABD, then,
lysed, and the cell lysate was incubated with biotin-GM (FIG. 7, 8,
lane 5). The results show that GM mostly binds to native HSP90.
Cancer cells were then incubated with ABD-GM for 2 hours (FIG. 7,
8, lane 6) or 4 hours (FIG. 7, 8, lane 7) and then lysed. ABD was
not detected, while native HSP90 was observed to be the bound
protein to GM. These results indicate that GM bound to ABD in fact
internalized into cells and was subsequently released from ABD and
migrated to bind to native HSP90.
[0091] Taken together, the results show that ABD-GM dissociates to
release GM, which in turn binds to native HSP90 in cancer cells.
The mechanism of this dissociation is likely due to the fact that
GM has a weak binding affinity to recombinant ABD (micromolar
affinity) compared to native HSP90 (nanomolar affinity).
Example 5
Construction of a Recombinant HER2-scFv-ABD Fusion Protein
[0092] A series of experiments were carried out to confirm the
binding between HER2-ABD and biotin-GM. First, antibody activity
was examined in comparison with HER monoclonal Ab (mAb). The
results show that about 40% of the cell population was HER2
positive in SK-BR-3 cells using HER mAb alone and about 10% of the
cell population was HER2 positive in SK-BR-3 with exposure to
HER2-scFv-ABD, whereas only 1% of the cell population was positive
in MDA-MB-231, a HER-2 normal breast cancer cell line. Although
these data suggest that an increased amount of the HER2-scFv-ABD
fusion protein is needed to detect an increased number of
HER2-positive cells, the results show that the HER2-scFv-ABD does
in fact, retain the HER2 antibody activity.
[0093] The function of the ABD in the HER2-scFv-ABD was examined
through a GM binding assay (FIG. 9; two trials (A, B)). Lane 4
shows the binding of the HER2-scFv-ABD to GM while lane 3 shows the
HER2-scFv-ABD protein. A clear band appeared in lane 4 at the same
location as lane 3, indicating that the bound protein to GM is the
HER2-scFv-ABD fusion protein. All of the other groups served as
controls. These results confirm that the HER2-scFv-ABD binds to GM.
Taken together, these observations indicate that the HER2-scFv-ABD
is bi-functional because it binds to the HER2 antibody while ABD
captures 17-AAG.
Example 6
Synthesis of the scFv-TBD-Taxane Molecule
[0094] A scheme for constructing a bi-functional allosteric
protein-drug molecule comprising a single-chain fragment (scFv)
antibody as a targeting moiety and a .beta.-tubulin luminal site
domain, referred to as a taxane-binding domain (TBD) to incorporate
paclitaxel (PTX), a microtubule-stabilizing agent is shown in FIG.
11. An important component in producing scFv-TBD-taxane is the TBD.
The TBD permits stable transport of taxane in the bloodstream and
can release taxane (e.g., PTX) in the presence of microtubules
within the target cells. With Abraxane (paclitaxel protein-bound
particles), the hydrophobic pockets of human serum albumin (HSA)
bind a wide range of endogenous and exogenous compounds to adsorb
and solubilize PTX. The overall PTX-HSA binding affinity is
calculated as K=1.43.times.10.sup.4 M.sup.-1 and continuous binding
studies have identified two binding sites in HSA, one with a high
binging affinity of K.sub.a=2.4.times.10.sup.6 M.sup.-1 and the
other with an intermediate binding affinity of K=1.0.times.10.sup.5
M.sup.-1. The binding affinity of PTX to microtubules is
approximately K.sub.a=6.0.times.10.sup.7 M.sup.-1. This difference
in the binding affinities of PTX to HSA versus microtubules
provides the mechanism for the release of PTX from HSA in cancer
cells. These differences in binding affinities serve as the basis
for a site-directed mutagenesis approach, i.e., to increase the
affinity of PTX to the taxane-binding domain so that it is high
enough to provide stability in the circulation such that it is
superior to HSA, but low enough to facilitate release of PTX to
reach the microtubules within the target cancer cells.
Example 7
Construction of a Recombinant HER2-scFv-ABD Bi-Functional
Allosteric Protein Capturing Multiple Therapeutic Molecules
[0095] A set of experiments were carried out to confirm that the
bi-functional allosteric protein-drug molecule can accommodate one
or more biological binding domains (e.g., ABDs), and thus carry a
greater number of (one or more) therapeutic agents (e.g., 17-AAG)
than a bi-functional allosteric protein-drug molecule comprising a
single (e.g., one) biological binding domain (e.g., ABD). For this,
the DNA fragment encoding the ABD located at the N-terminus of
HSP90 was excised from the N-terminus of HSP90 (residues 9-232) and
sub-cloned into the HER2-scFv expression vector with a non-flexible
hinge. To develop a bi-functional allosteric protein-drug molecule
comprising three ABDs, three ABDs were connected by a linker, GGGS,
and then the full sequence containing the three ABDs and two
linkers was inserted into the expression vector. This expression
vector was transfected into HEK293 cells by using a commercially
available transfection kit.
[0096] The cells were cultured for 3 days and the culture media was
collected to purify the recombinant bi-functional allosteric
protein-drug molecule. These bi-functional allosteric protein-drug
molecules were purified using a FPLC equipped with Protein L
column. The purified bi-functional allosteric protein-drug molecule
were dialyzed, concentrated, and kept frozen until use. The
bi-functional allosteric protein-drug molecule was verified by
SDS-PAGE and western blot (anti-His tag antibody) as shown in FIG.
12. Binding of the bi-functional allosteric protein-drug molecule
with GM was confirmed by a magnetic bead-based pull down assay.
Each of the bi-functional allosteric protein-drug molecules was
mixed with biotin-GM and incubated for 30 minutes at 4.degree. C.
After the incubation, the biotin-GM was captured by using
streptavidin-magnetic beads. The protein bound to the biotin-GM was
eluted by heating in the presence of SDS. The eluted sample was
electrophoresed on a SDS-PAGE gel. Western blot was conducted
following the SDS-PAGE in order to confirm the specific binding of
the recombinant proteins to GM.
[0097] The results show that both the HER2 scFv-ABD and the HER2
scFv-ABD-ABD-ABD can capture 17-AAG, whereas the HER2 scFv-MD
(middle domain; control) has no affinity for 17-AAG (See FIG. 12A,
lanes 5-7). These data verify that the biological affinity of
17-AAG to ABD is specific.
Example 8
Determine the Internalization of Geldanamycin
[0098] To verify that the TAT-ABD facilitates the internalization
of geldanamycin (GA) into cells, dual fluorescence-based detection
was used. The TAT-ABD protein was labeled with Rhodamine, mixed
with FITC-GA, incubated for 30 minutes at 4.degree. C., and then
contacted with one of the following cell lines; BT-474 (HER2+),
SKBR-3 (HER2+), MDA-MB-231 (HER2-), and MCF-7 (HER2-).
Free-FITC-GA-treated, non-treated, or rhodamine-TAT-ABD-treated
cells were prepared as control groups. After 4 hours, the cells
were washed and harvested for FACS analysis. FACSCanto II was used
to analyze the cells. The results are as follows.
[0099] BT-474 cell line: Intracellular uptake of FITC-GA was
approximately 20% without the TAT-ABD protein (FIG. 13; green bar).
TAT-ABD internalized into 100% of the cells (FIG. 13; R-tABD).
TAT-ABD plus FITC-GA internalized into approximately 70% of the
cells (FIG. 13; R-tABD-FGA;). Approximately 25% the cells were
positive to solely Rhodamine.
[0100] MCF-7 cell line: Intracellular uptake of FITC-GA was
approximately 15% without the TAT-ABD protein (FIG. 14; FGA).
TAT-ABD internalized into 100% of the cells (FIG. 14; R-tABD).
TAT-ABD plus FITC-GA internalized into approximately 95% of the
cells (FIG. 14; R-tABD-FGA). Approximately 5% cells were positive
to solely Rhodamine.
[0101] MDA-MB-231 cell line: Intracellular uptake of FITC-GA was
approximately 10% without the TAT-ABD protein (FIG. 15; FGA).
TAT-ABD internalized into 100% of the cells (FIG. 15; R-tABD).
TAT-ABD plus FITC-GA internalized into approximately 70% of the
cells (FIG. 15; R-tABD-FGA). Approximately 30% cells were positive
to solely Rhodamine.
[0102] MDA-SK-BR-3 cell line: Intracellular uptake of FITC-GA was
approximately 10% without the TAT-ABD protein (FIG. 16; FGA).
TAT-ABD internalized into 100% of the cells (FIG. 16; R-tABD).
TAT-ABD plus FITC-GA internalized into approximately 70% of the
cells (FIG. 16; R-tABD-FGA). Approximately 30% cells were positive
to solely Rhodamine.
Example 9
Targeted Anti-Cancer Activity of HER2-scFv-ABD-17-AAG
[0103] To verify the targeted anticancer activity of
HER2-scFv-ABD-17-AAG, SKBR-3 (HER2+) and MDA-MB-231 (HER2-) cells
were exposed to free-17-AAG (17A), free HER2-scFv-ABD protein (HA),
or HER2-scFv-ABD-17-AAG (HA+17A). HA was mixed with 17-AAG at the
molar ratio of 1:1 and incubated for 30 minutes at 4.degree. C.
prior to the contact with the cells. The cells were treated with
17-AAG or HA+17-AAG at different concentrations of 17-AAG ranging
from 0.001 .mu.g/ml to 1 ug/ml (final concentration). As a negative
control, cells were exposed to equivalent amounts of free HA. After
5 hours of incubation, the cells were washed to remove unbound
proteins and non-internalized 17-AAG and then further incubated for
up to 72 hours. Cell viability was determined by MTT assay.
[0104] Exposure of the cells to HER2-scFv-ABD-17-AAG (HA+17A)
increased the cytotoxicity of 17-AAG by facilitating uptake of
17-AAG into the target SKBR-3 cancer cells, whereas no difference
in the cell viability was observed in MDA-MB-231 cells (FIG. 17).
These results provide evidence that the formation of a
bi-functional allosteric protein-drug molecule (e.g.,
HER2-ABD/17-AAG) can improve the anti-cancer activity of 17-AAG in
HER2-positive cancer cells through specific binding and facilitated
internalization of the therapeutic agent.
[0105] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
aspects of the invention will be apparent to those skilled in the
art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *