U.S. patent application number 14/380423 was filed with the patent office on 2015-02-12 for modified microbial toxin receptor for delivering agents into cells.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to John R. Collier, Andrew J. McCluskey, Adva Mechaly.
Application Number | 20150044210 14/380423 |
Document ID | / |
Family ID | 49006232 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150044210 |
Kind Code |
A1 |
Mechaly; Adva ; et
al. |
February 12, 2015 |
MODIFIED MICROBIAL TOXIN RECEPTOR FOR DELIVERING AGENTS INTO
CELLS
Abstract
We described a novel system of targeted cell therapy with a
protein toxin, such as anthrax toxin, that has been modified to
re-direct it to a desired cell target instead of its natural cell
target. The system can be used for, e.g., targeted killing of
undesired cells in a population of cells, such as cancer or overly
active immune system cells.
Inventors: |
Mechaly; Adva; (Ness-Ziona,
IL) ; McCluskey; Andrew J.; (Boston, MA) ;
Collier; John R.; (Wellesley Hills, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
49006232 |
Appl. No.: |
14/380423 |
Filed: |
February 22, 2013 |
PCT Filed: |
February 22, 2013 |
PCT NO: |
PCT/US13/27307 |
371 Date: |
August 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61602218 |
Feb 23, 2012 |
|
|
|
Current U.S.
Class: |
424/134.1 |
Current CPC
Class: |
C07K 2317/73 20130101;
C07K 16/2863 20130101; C07K 14/25 20130101; C07K 2318/00 20130101;
C07K 16/32 20130101; A61P 35/00 20180101; C07K 16/40 20130101; C07K
2319/00 20130101; C07K 2319/55 20130101; C07K 14/33 20130101; C07K
14/28 20130101; C07K 2319/33 20130101; C07K 2319/75 20130101; C07K
14/34 20130101; C07K 14/21 20130101; C07K 14/32 20130101 |
Class at
Publication: |
424/134.1 |
International
Class: |
C07K 14/32 20060101
C07K014/32; C07K 16/32 20060101 C07K016/32 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under AI
022021 awarded by National Institutes of Health. The government has
certain rights in the invention.
Claims
1. An altered binary toxin system for delivery of an active
molecule to a target cell comprising: a fusion protein comprising a
receptor-ablated pore-forming AB toxin unit fused to a
non-toxin-associated receptor-binding ligand specific for a target
cell, and a complementary toxin unit capable of associating with
the pore-forming toxin unit for delivery of a therapeutic protein
to the cytosol of the target cell.
2-21. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e)
of a U.S. provisional application Ser. No. 61/602,218, filed on
Feb. 23, 2012, the content of which is incorporated herein by
reference in its entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 21, 2013, is named 002806-073301-PCT_SL.txt and is 17,750
bytes in size.
FIELD
[0004] The present invention relates to molecular genetics and
molecular biology. More specifically, the present embodiments
provide for compositions and methods for restructuring a binary
bacterial toxin, such as anthrax toxin, to bind with a targeted
cellular receptor, form a pore, and deliver proteinaceous
molecules, such as cytotoxins, into the targeted cell.
BACKGROUND
[0005] An important goal for researchers and pharmaceutical
companies is to identify ways to use proteinaceous delivery
vehicles to introduce novel molecules into the cytosol of cells,
particularly into mammalian cells. Although there are a number of
methods for the delivery of bioactive peptides and proteins into
mammalian cells for therapeutic and biotechnological purposes,
there is still a specific need for methods to deliver larger
molecules, such as proteins, enzymes or cytotoxins, that cannot
traverse the plasma membrane by a simple diffusive process.
[0006] The current technologies used to gain therapeutic access to
the cytosol are limited in that they require large quantities of
sample, have limited selectivity, and tend to not escape the
intracellular endosome. Hence, efficient delivery of the novel
therapeutics remains a hurdle in drug development.
SUMMARY
[0007] We provide for a cell-specific, efficient delivery of
bioactive molecules into cells. More specifically we have designed,
binary "AB" toxins such that the B component binds to a
heterologous, specific cell receptor on a target cell, and the A
component interacts with the B component to deliver a biologically
active molecule to the target cell cytosol via translocation
through the cell membrane. For example, the receptor specificity of
the transport protein of anthrax toxin, PA, can be altered as a
means to deliver active toxin, such as anthrax toxin, to the
cytosol of targeted cancer cells. Any cell, such as a cancer cell
can be targeted so long as the cell expresses a specific marker or
a marker that is significantly enriched in such cell. The present
system is useful for both research purposes and medical
applications calling for modification or eradication of selected
populations of cells. For example, the systems and compositions
described, can be used in reducing the number or eradicating cancer
cells or reducing the number of over-reactive immune cells, e.g.,
in a human subject.
[0008] The present invention harnesses a major subclass of
bacterial AB toxins, termed binary toxins, which use a transporter
protein (B or binding unit or B-component) that actively
translocates the catalytic portion of the toxin (A unit or
A-component) into the cell. Although separate, the proteins having
the A and B functions interact during the intoxication of cells.
Examples of binary toxins include anthrax toxin, Clostridium
perfringens Iota-toxin, Clostridium botulinum C2 toxin, and
Clostridium spiroforme Iota-like toxin.
[0009] In the present embodiments, the native receptor-binding
ligand of the B unit is typically ablated but not replaced, and the
B unit fused with a ligand that binds specifically to a receptor on
a target cell, e.g., a cancer cell or an immune cell. Thus, the B
unit retains determinants needed for the cytoplasmic delivery of
the A units, but specific cell targeting can be selected. The
native A-component contains the catalytic activity, and
translocates to the target cell cytosol via the B-component.
[0010] In the present embodiments, the A-component can also be
altered, e.g., fused to a cytotoxin. Further cytotoxic domains of
enzymatic protein toxins produced by bacteria, plants and animals,
that can be harnessed using the delivery systems of the present
embodiments include anthrax toxin, shiga toxin, shiga-like toxin 1
and 2, ricin, abrin, gelonin, pokeweed antiviral protein, saporin,
trichsanthin, pepcin, maize RIP, alpha-sarcin, Clostridium
perfringens epsiolon toxin, Botulinum neurotoxins, Staphylococcus
enterotoxins, Clostridium difficile toxins, pertussis toxins, or
pseudomonas exotoxins.
[0011] A variety of specific cell receptors can be targeted using
the compositions and methods of the present embodiments, as long as
the receptor is one of those that internalize their ligands and
traffic them to an acidic intracellular compartment, which
facilitates proper folding of the translocated components.
Receptors that can be targeted by the engineered binary toxins
according to the present invention include, for example, HER1,
HER2, HER3 and HER4 EGF receptors; vascular endothelial growth
factor receptors VEGFR-1, VEGFR-2 and VEGFR-3; insulin-like growth
factor 1 receptors; fibroblast growth factor receptors;
thrombospondin 1 receptors; estrogen receptors; urokinase
receptors; progesterone receptors; testosterone receptors;
carcinoembryonic antigens; prostate-specific antigens; farnesoid X
receptors; transforming growth factor receptors; transferrin
receptors; hepatocyte growth factor receptors; or vasoactive
intestinal polypeptide receptors 1 and 2.
[0012] Further delivery systems comprising altered binary or AB,
pore-forming protein toxins can be selected from, for example,
Clostridium perfringens toxins (alpha, beta, epsilon, iota);
Clostridium botulinum C2 toxin; or Clostridium spiroforme Iota-like
toxin.
[0013] For example, anthrax toxin is a member of the so-called
binary toxins, a class in which the A and B functions inhabit
separate proteins. Anthrax toxin uses a homopolymeric pore
structure formed by the B moiety, protective antigen (PA), for the
delivery of two alternative A moieties, edema factor (EF) and
lethal factor (LF) into the cytoplasm. The receptor-targeted PA
variants of the present embodiments can deliver a wide variety of
therapeutic proteins, both nontoxic and toxic, to chosen class or
classes of cells including the toxic native A-moieties (EF and LF).
For example, the therapeutic protein is fused to the N-terminal
portion of the lethal factor of anthrax toxin (LF.sub.N), and
undergoes unfolding during translocation through the PA variant to
the target cell cytosol. Example toxins that can be fused to
LF.sub.N for use according to the present embodiments include the
catalytic domain of diphtheria toxin (DTA), the catalytic domain of
shiga toxin, and the catalytic domain of pseudomonas exotoxin A.
Some nontoxic proteins that can be fused to LF.sub.N for use
according to the present embodiments include beta-lactamase,
dihydrofolate reductase (DHFR), and ciliary neurotrophic
factor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows a composite representation of the heptameric
prepore formed by PA63 (PDB#1TZO) with EGF (PDB#1JL9) linked to the
C terminus. Axial view, with domains 1, 2, 3, and 4 in a single
subunit of PA63. Broken lines represent an 8 amino acid linker
(SPGHKTQP) (SEQ ID NO:1) connecting the N terminus of EGF to the C
terminus of PA63.
[0015] FIG. 2 shows characterization of purified mPA-EGF. (2A)
Western blot analysis with anti-PA and anti-EGF antibodies,
demonstrates the presence of both the PA and EGF proteins in the
purified fusion protein. (2B) Conversion of PA63 oligomers from the
SDS-dissociable prepore state (black arrow) to the SDS-resistant
pore state (arrow) at different pH values. Samples (5 .mu.g) of
native (83) and proteolytically-activated ([63]7) forms of WT PA,
mPA, and mPA-EGF were separated by SDS-PAGE and visualized by
Coomassie blue staining.
[0016] FIG. 3 reflects cytotoxicity assays demonstrating
receptor-specific cell targeting of mPA-EGF. (3A) A431 or CHOK1
cells (3.5.times.10.sup.4) were incubated with 10 nM PA or PA
variant plus LF.sub.N-DTA at the concentrations indicated. After a
4-hour (A431 cells) or overnight (CHOK1) incubation, the medium was
replaced with medium containing 1 .mu.Ci of 3H-leucine/ml.
Following a 1-hour incubation, incorporated .sup.3H-leucine was
determined by scintillation counting. (3B,3 C) Assays were
performed as described for panel 3A, but soluble EGF (500 nM) or
PA-binding VWA domain of ANTRX2 (ANTRX2; 100 nM) was present during
a 4 hour incubation with A431 cells (Panel B) or an overnight
incubation period with CHOK1 cells (Panel C). Each point on the
curves represents the average of three experiments.
[0017] FIG. 4 demonstrates that mPA-EGF transports LF and EF into
receptor-bearing cells. (4A) A431 cells (1.times.10.sup.6) were
treated with 100 nM LF plus 10 nM PA or PA variant for 3 hours.
Cell lysates were prepared, fractionated by SDS-PAGE and
transferred to PVDF membrane, and MEK1 cleavage was evaluated by
Western blot with anti-MEK1 antibody. As a control, GAPDH was
monitored with anti-GAPDH antibodies. (4B) A431 cells
(3.5.times.10.sup.4) were exposed to 50 nM EF plus 10 nM PA or PA
variant for 1 hour. A competition enzyme-linked immunoassay was
performed to detect the intracellular concentration of cAMP, based
on a standard curve, following the manufacturers protocol (Cell
Signaling Technology). The column designated "Control" corresponds
to A431 cells treated with EF in the absence of PA. Each bar
represents the average of experiments performed in
quadruplicate.
[0018] FIG. 5 shows the characterization of mPA-DTR. FIG. 5A shows
Western blot analysis with anti-PA and anti-DTR antibodies
demonstrating the presence of both PA and DTR in the purified
mPA-DTR fusion. In FIG. 5B, CHOK1 cells (3.5.times.10.sup.4) were
exposed overnight to a range of concentrations of LF.sub.N-DTA in
the presence of WT PA or mPA-DTR, in the presence or absence of
excess soluble DTR. Protein synthesis was determined by
.sup.3H-leucine incorporation. Each point on the curve corresponds
to the average of three experiments.
[0019] FIG. 6 presents data of cytotoxicity assays that confirm the
receptor-specific targeting of mPA-ZHER2. HER2 receptor-positive
(SKBR-3, A431, and MCF-7) and -negative (CHOK1, HeLA, and
MDA-MB-468) cell lines (3.5.times.10.sup.4 cells) were exposed to a
range of LF.sub.N-DTA concentrations plus a constant concentration
(20 nM) of the chimeric mPA-ZHER2 fusion protein. After a 4-hour
incubation, the medium was replaced with medium containing 1 .mu.Ci
of .sup.3H-leucine/mL. Following a 1-hour incubation, the amount of
radiolabeled leucine was determined by scintillation counting. Each
point on the curves corresponds to the average of four
experiments.
[0020] FIGS. 7A-7D show HER2-dependent killing of cell lines by
mPA-ZHER2 plus LF.sub.N-DTA. In FIG. 7 A, cells were incubated with
a fixed concentration of mPA-ZHER2 (20 nM) plus various
concentrations of LF.sub.N-DTA for 4 h and then with medium
containing [.sup.3H]-leucine for 1 h. Protein synthesis was
measured by scintillation counting and normalized against cells
treated with mPA-ZHER2 alone. In FIG. 7B, HER2 receptor levels were
determined by flow cytometry with a FITC-labeled anti-HER2
Affibody. Mean fluorescence intensity was calculated using the
FloJo software package and plotted versus the logEC.sub.50 for
[LF.sub.N-DTA]. In FIG. 7C, cells were exposed to the same
conditions as FIG. 7A. After 48 h, cell viability was measured by
XTT cytotoxicity assay and normalized against cells treated with
mPA-ZHER2 alone. In FIG. 7D, apoptosis was assessed after exposing
cells to either mPA-ZHER2 alone (minus sign "-"; open bars) or
mPA-ZHER2 plus 10 nM LF.sub.N-DTA (plus sign "+"; filled bars) for
24 h and measuring caspase 3/7 activation. Values corresponding to
relative light units (RLU), generated by caspase 3/7 cleavage of a
pre-luminescent substrate were extracted from dose-response curves.
In all panels, cell lines with high, moderate, low, and no
detectable HER2 receptor levels are indicated solid square, solid
and open circle, solid triangle, and solid and open diamonds,
respectively. Each point on the graphs represents the average of
four experiments.
[0021] FIGS. 8A-8B show competition by high- and low-affinity ZHER2
Affibodies for mPA-ZHER2-dependent killing. Cells were exposed to a
lethal dose of mPA-ZHER2 and LFN-DTA in the presence of increasing
amounts of a high (Z.sub.HER2:342, FIG. 8A) or lower (Z.sub.HER2:4,
FIG. 8B) affinity HER2 Affibody for 4 h, and the incorporation of
[.sup.3H]-leucine was measured and graphed as described in FIG. 7.
High, moderate, and low HER2 expressing cell lines are shown in
square, circle, and triangle, respectively. Each point on the
curves represents the average of four experiments.
[0022] FIGS. 9A-9B show mPA-ZHER2- and mPA-EGF-directed killing of
cell lines by LF.sub.N-RTA. Cells were exposed to mPA-ZHER2 (FIG.
9A) or mPA-EGF (FIG. 9B) in combination with LFN-RTA, at the
indicated concentrations for 4 h, and the level of protein
synthesis was measured by scintillation counting. Cells expressing
high, moderate, low, or no detectable levels of HER2 (epidermal
growth factor 2) or EGFR (epidermal growth factor 1, or HER1) are
indicated with square, circle, triangle and diamond in FIG. 9A; and
square, circle, triangle and diamond in FIG. 9B, respectively.
[0023] FIGS. 10A-10D show killing of a HER2-positive,
trastuzumab-resistant tumor cell line by mPA-ZHER2 plus
LF.sub.N-DTA or LF.sub.N-RTA. In FIG. 10A, the JIMT-1 tumor cell
line was incubated with mPA-ZHER2 in combination with increasing
amounts of LF.sub.N-DTA (circle) or LF.sub.N-RTA (square) for 4 h,
and the effects on [.sup.3H]-leucine incorporation were measured as
described in FIG. 7. In FIG. 10B, FACS analysis using a
FITC-conjugated HER2 Affibody confirms the expression of HER2 on
the surface of JIMT-1 cells. The mean fluorescence was calculated
using the FlowJo software package and plotted in the GRAPHPAD
PRISM.RTM. software package (left panel) from the raw data
presented in the histogram (right panel), which displays the shift
in fluorescence (solid peak on the right of the histogram) compared
to unstained cells (dashed peak on the left of the histogram). In
FIG. 10C, JIMT-1 cells were exposed to the same conditions as FIG.
10A. After 48 or 72 h, cell viability was measured by XTT assay and
plotted as percent cell viability normalized against control cells
treated with mPA-ZHER2 alone. In FIG. 10D, Caspase 3/7 activation,
an indicator of apoptosis, was measured after a 24 and 48 h
exposure to 20 nM mPA-ZHER2 and LF.sub.N-DTA, at the indicated
concentrations. The cleavage of a pre-luminescent caspase 3/7
substrate generated RLU's that are plotted versus LF.sub.N-DTA
concentration. Control cells treated with mPA-ZHER2 alone are
indicated with an X.
[0024] FIGS. 11A-11B shows that mPA-ZHER2 mediates specific killing
of HER2-positive cells in a heterogeneous population. Fluorescent
cells shown to be sensitive to the actions of mPA-ZHER2 and
LF.sub.N-DTA (A431.sup.CFP and SKBR3.sup.RFP) were mixed equally
with resistant cells (CHO-K1 and MDA-MB-468.sup.GFP) and incubated
with mPA-ZHER2 plus LF.sub.N-DTA or with mPA plus LF.sub.N-DTA
(control; the control FACS data are identical to those in FIGS. 7C
and 8A, as all of the experiments were conducted simultaneously).
After 24 h, cells were detached with trypsin and quantified by FACS
or washed with PBS and imaged with a fluorescence microscope
(microscope slide FACS color photos not included). Each bar
represents the average of experiments performed in triplicate.
Control is shown in FIG. 11A and exposure to the re-directed fusion
toxin is shown in FIG. 11B.
[0025] FIG. 12 shows that mPA-ZHER2-mediated killing in a
heterogeneous cell population. Tumor cells were plated in separate
compartments of a chambered slide (right panel) and incubated at
37.degree. C. The following day, the chambers were removed, and the
slide was incubated with mPA-ZHER2 plus LF.sub.N-DTA. After 4 h,
cells were incubated with medium supplemented with
[.sup.3H]-leucine for 1 h and dissolved in 6 M guanidine-HCl, and
the incorporated radiolabel was quantified by scintillation
counting. Percent protein synthesis was normalized against cells
treated with mPA+LF.sub.N-DTA.
[0026] FIGS. 13A-13C show that mPA-EGF specifically kills
EGF-expressing cells in a heterogeneous population. In FIG. 13A,
cells were exposed to 20 nM mPA-EGF and LF.sub.N-DTA at the
concentrations indicated for 4 h and protein synthesis was measured
as in experiments described above. Percent protein synthesis was
normalized against cells treated with mPA-EGF alone. Cell lines
expressing high amount of EGFR are MDA-MB-468, A431 and MDA-MB-231;
low amount of EGFR is BT-474; and substantially no EGFR are SKBR-3
and CHO-K1. Each point on the curves represents the average of four
experiments. In FIG. 13B, populations of fluorescent cells were
mixed and exposed to a lethal dose of mPA-EGF and LF.sub.N-DTA or
mPA+LFN-DTA as a control; the control FACS data are identical to
those in FIGS. 11A and 14A, as all of the experiments were
conducted simultaneously. After 24 h, cells were washed with PBS
and imaged with a fluorescence microscope or detached with trypsin
and quantified by FACS (FIG. 13B). Each bar represents the average
of experiments performed in triplicate. In FIG. 13C, a panel of
cancer cell lines were plated in chambered slides overnight. The
following day the chambers were removed and cells were exposed to
the same treatments as described in Fig. B. Following intoxication
for 4 h, cells were processed, and protein synthesis was quantified
as described in FIG. 13A.
[0027] FIGS. 14A-14C show that re-directed mPA variants act
together to eliminate heterogeneous tumor cell populations. In
FIGS. 14A (control) and 14B (cells exposed to the re-directed
fusion toxin), various fluorescent cells were mixed in equal
numbers and exposed to LF.sub.N-DTA plus an equimolar mixture of
mPA-ZHER2 and mPA-EGF. LF.sub.N-DTA plus mPA was used as control
(the control FACS data are identical to those in FIGS. 11A and 13C,
as all of the experiments were conducted simultaneously). After 24
h, cell populations were detached with trypsin and quantified by
FACS (FIGS. 14A and 14B) or washed with PBS and imaged with a
fluorescence microscope (data not shown). Each bar represents the
average of experiments performed in triplicate using SKBR3 (red),
A431 (cyan), MDA-MB-468 (green), and CHO-K1 (unlabeled) cells. In
FIG. 14C, a larger panel of cancer cell lines were plated in
separate compartments of a chambered slide overnight. The following
day, the partition was removed and cells were exposed to the same
treatments as described above. Following intoxication for 4-hours,
cells were incubated with medium supplemented with [3H]-leucine for
1-hour, and protein synthesis was quantified by scintillation
counting. Percent protein synthesis was normalized against cells
treated with mPA and LFN-DTA.
DETAILED DESCRIPTION
[0028] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0029] As used herein and in the claims, the singular forms include
the plural reference and vice versa unless the context clearly
indicates otherwise. The term "or" is inclusive unless modified,
for example, by "either." Other than in the operating examples, or
where otherwise indicated, all numbers expressing quantities of
ingredients or reaction conditions used herein should be understood
as modified in all instances by the term "about."
[0030] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of the invention, the methods, devices, and
materials in this regard are described herein.
[0032] Many pathogenic bacteria have evolved protein machinery that
efficiently delivers potent enzymes to the cytosol of mammalian
cells. Some infectious bacteria secrete protein toxins that reach
the cytosolic compartment of host cells and disrupt homeostasis. A
major class of bacterial toxins, termed AB toxins, use a
receptor-binding domain (B or binding unit) that, in the case of
some binary toxins, can actively translocate the catalytic portion
of the toxin (A unit) into the cell. More specifically, the "A"
component is usually the "active" portion, and the "B" component is
usually the "binding" portion of the toxin. Thus, the A moiety or
component contains the catalytic activity, while the B moiety or
component possesses determinants needed for the cytoplasmic
delivery of the A moieties into target cells. These delivery
determinants include receptor binding activity, and often, but not
always, membrane penetration activity, such the formation of a pore
which translocates the A moiety. Examples of AB toxins include
anthrax toxin, botulinum neurotoxin, diphtheria toxin, shiga toxin,
shiga like toxin, exotoxin A, and cholera toxin. The A and B
components of these and a variety of other toxins are well known.
See, e.g., PCT US2012/20731. The nucleic acid sequences encoding
these toxins and well as the amino acid sequences of these toxins
are also known.
[0033] For example, anthrax toxin is one member of the so-called
binary toxins, a class in which the A and B functions inhabit
separate proteins. Although separate, the proteins having the A and
B functions interact during the intoxication of cells. Anthrax
toxin uses a single B moiety, protective antigen (PA; 83 kDa), for
the delivery of two alternative A moieties, edema factor (EF; 89
kDa) and lethal factor (LF; 89 kDa) into the cytoplasm.
[0034] Bacterial toxin B components, in general, can be used to
deliver bioactive moieties into the cytosol of the cells when the
bioactive moiety is attached to the A component or a surrogate A
component of the bacterial toxin, as long as the bioactive moiety
unfolds correctly (if such is required for activity) during
translocation. In addition to the anthrax B component, PA, the B
components of Clostridium perfringens toxins (alpha, beta, epsilon,
iota), C. botulinum C2 toxin, and C. spiroforme Iota-like toxins
can be used in the systems, compositions and methods as described
herein.
[0035] A bioactive peptide or cytotoxic domain can be attached to
an A component of the binary system, such as the nontoxic
PA-binding domain of LF (LF.sub.N), and the fusion protein thus
formed passes through the pore into the cytosol of a cell. See PCT
US2012/20731. Cytotoxic domains can be derived from shiga toxin,
shiga-like toxin 1 and 2, ricin, abrin, gelonin, pokeweed antiviral
protein, saporin, trichsanthin, pepcin, maize RIP, alpha-sarcin,
Clostridium perfringens epsiolon toxin, Botulinum neurotoxins,
Staphylococcus enterotoxins, difficile toxins, pertussis toxins, or
pseudomonas exotoxins.
[0036] The actions of the binary toxins depend on their ability to
bind to one or more cell-surface receptors. Anthrax toxin acts by a
sequence of events that begins when the Protective-Antigen (PA)
moiety of the toxin binds to either of two cell-surface proteins,
ANTXR1 and ANTXR2, and is proteolytically activated. The activated
PA self-associates to form oligomeric pore precursors, which, in
turn, bind the enzymatic moieties of the toxin and transport them
to the cytosol. More specifically, the PA63 prepore binds up to
three or four molecules of LF, forming complexes that are then
endocytosed. Upon acidification of the endosome, protective antigen
prepore undergoes a conformational rearrangement to form a
membrane-spanning, ion-conductive pore, which transports lethal
factor from the endosome to the cytosol. LF.sub.N, the N-terminal
domain of lethal factor, has nanomolar binding affinity for the
pore, and this domain alone can be used for translocation of
chemical moieties. Additionally, small positively charged peptide
segments that mimic LF.sub.N can be used to aid in translocating
these molecules through PA pore. These mimics may comprise at least
one non-natural amino acid. See PCT US2012/20731. Engineered binary
toxin B components, such as PA fusion proteins with altered
receptor specificity, are useful in biological research and have
practical applications, including perturbation or ablation of
selected populations of cells in vivo.
[0037] An embodiment of the present invention provides for a
genetically modified PA, carrying a double mutation into domain 4
of PA to ablate its native receptor-binding function and fused
epidermal growth factor (EGF) to the C terminus of the mutated
protein. The resulting fusion protein transported enzymatic
effector proteins into a cell line that expressed the EGF receptor
(A431 cells), but not into a line lacking this receptor (CHO-K1
cells). Addition of excess free EGF blocked transport of effector
proteins into A431 cells via the fusion protein, but not via native
PA. Additionally, fusing the diphtheria toxin receptor-binding
domain to the C terminus of the mutated PA channeled
effector-protein transport through the diphtheria toxin
receptor.
[0038] Based on our examples, receptor binding domain of any of the
AB toxins can be modified to ablate the native receptor binding
domain and to fuse them with a desired receptor binding domain.
[0039] Accordingly, we provide a system or a composition comprising
an altered binary toxin system for delivery of an active molecule
to a target cell comprising: a fusion protein comprising a
receptor-ablated pore-forming binary toxin unit fused to a
non-toxin-associated receptor-binding ligand specific for a target
cell, and a complementary toxin unit capable of associating with
the pore-forming toxin unit for delivery of a therapeutic protein
to the cytosol of the target cell.
[0040] Additional cell receptors that can be targeted and that are
useful according to the present invention include HER1, HER2, HER3
and HER4 EGF receptors; vascular endothelial growth factor
receptors VEGFR-1, VEGFR-2 and VEGFR-3; insulin-like growth factor
1 receptor; fibroblast growth factor receptors; thrombospondin 1
receptor; estrogen receptor; urokinase receptor; progesterone
receptor; testosterone receptor; carcinoembryonic antigen;
prostate-specific antigen; farnesoid X receptor; transforming
growth factor receptors; transferring receptor; hepatocyte growth
factor receptor; or vasoactive intestinal polypeptide receptor 1
and 2.
[0041] The targeting moieties can be, e.g., ligands, antibodies or
Affibodies that specifically bind to any one of the receptors. Such
ligands, antibodies and affibodies are either well known, or can be
made using routine methods known to one of ordinary skill in the
art.
[0042] Targeting of toxic proteins to specific classes of mammalian
cells has been studied extensively, often with the goal of
developing new treatments for malignancies. One approach to
targeting involves replacing the receptor-binding domain of a toxin
with a heterologous protein, such as a growth factor or antibody
that binds to a specific cell-surface receptor.
[0043] Another approach is to link a heterologous protein to an
altered form of the toxin in which the native receptor-binding
function has been disrupted. Herein, the latter approach was used
in the context of a binary toxin to redirect the receptor
specificity of the transport moiety of anthrax toxin to
heterologous receptors.
[0044] Accordingly, in some aspects of all the embodiments of the
invention, the system or a composition comprising an altered binary
toxin system for delivery of an active molecule to a target cell
comprising: a fusion protein comprising a receptor-ablated
pore-forming anthrax toxin unit fused to a
non-anthrax-toxin-associated receptor-binding ligand specific for a
target cell, and a complementary toxin unit capable of associating
with the pore-forming toxin unit for delivery of a therapeutic
protein to the cytosol of the target cell.
[0045] Anthrax toxin (ATx) is an ensemble of three large proteins:
Protective Antigen (PA, 83 kDa), Lethal Factor (LF, 90 kDa), and
Edema Factor (EF, 89 kDa). LF and EF are intracellular effector
proteins: enzymes that modify substrates residing within the
cytosolic compartment of mammalian cells. LF is a metalloprotease
that cleaves most members of the MAP kinase family, and EF is a
calmodulin- and Ca2+-dependent adenylyl cyclase, which elevates the
level of cAMP within the cell. Leppla, 79 PNAS 3162 (1982);
Duesbery et al., 280 Science 734 (1998); Vitale et al., 248
Biochem. Biophys. Res. Commn. 706 (1998). PA, the third component
of the ensemble, is a receptor-binding transporter capable of
forming pores in the endosomal membrane. Miller et al., 38 Biochem.
10432 (1999); Young & Collier, 76 Annu. Rev. Biochem. 243
(2007). These pores mediate the translocation of EF, LF, or various
fusion proteins containing the N-terminal PA-binding domain of EF
or LF, across the endosomal membrane to the cytosol. Collier, 30
Mol. Aspects Med. 413 (2009).
[0046] ATx action at the cellular level is initiated when PA binds
to either of two receptors, ANTXR1 and ANTXR2, and is activated by
a furin-class protease. Scobie, 100 PNAS 5170 (2003); Bradley et
al., 414 Nature 225 (2001); Klimpel et al., 89 PNAS 10277 (1992).
The cleavage yields a 20-kDa fragment, PA20, which is released into
the surrounding medium, and a 63-kDa fragment, PA63, which remains
bound to the receptor. Receptor-bound PA63 spontaneously
self-associates to form ring-shaped heptameric and octameric
oligomers (prepores), which are capable of binding LF and/or EF
with nM affinity. Kintzer et al., 392 J. Mol. Biol. 614 (2009);
Milne et al., 269 J. Biol. Chem. 20607 (1994); Mogridge et al., 99
PNAS 7045 (2002); Cunningham et al., 99 PNAS 7049 (2002). The
resulting heterooligomeric complexes are endocytosed and delivered
to the endosomal compartment, where acidic pH induces the prepores
to undergo a major conformational rearrangement that allows them to
form pores in the endosomal membrane. Young & Collier, 2007.
These pores serve as protein translocases, which unfold bound LF
and EF molecules and transport them across the endosomal membrane,
where they refold and modify their respective intracellular
targets.
[0047] The two known PA receptors, ANTXR1 (also called TEM8) and
ANTXR2 (also called CMG2), are type 1 membrane proteins containing
a von Willebrand/Integrin A (VWA) MIDAS domain. Within PA, both
domain 4, the so-called receptor-binding domain, and domain 2, the
pore-forming domain, participate in binding to the MIDAS domain of
the receptors. Lacy et al., 101 PNAS 13147 (2004). ANTXR1 and
ANTXR2 have differences in affinity for PA (Scobie et al., 2005;
Wigelsworth, 279 J. Biol. Chem. 23349 (2004)), but both of these
receptors bind PA in a manner that allows it to be activated and to
oligomerize; and both receptors mediate trafficking of
prepore:effector complexes to the endosomal compartment and
translocation across the endosomal membrane.
[0048] In particular embodiments of the present invention, the
receptor-binding activity of PA was ablated by mutating two
residues of domain 4, and then fusing the C terminus of the mutated
protein with heterologous receptor-binding proteins: human
epidermal growth factor (EGF) (see FIG. 1), HER2 affibody (ZHER2),
or the receptor-binding domain of diphtheria toxin (DTR). The
resulting fusion proteins mediated the entry of effector enzymes,
and entry was dependent on the cellular receptors for EGF, ZHER2,
and DTR.
[0049] In one example, two mutations in the domain 4 of the PA,
N682A and D683A, were introduced into PA to ablate its native
receptor-binding function (Rosovitz et al., 278 J. Biol. Chem.
30936 (2003)), and the mutated protein (mPA) was expressed in E.
coli BL21 (DE3). SEQ ID NO: 10 provides the amino acid reference
sequence for these mutants. The purified product failed to promote
entry of LF.sub.N-DTA into either CHO-K1 cells or A431 cells at the
highest concentration tested (10 nM), as measured by the inhibition
of protein synthesis in the presence of LF.sub.N-DTA. LF.sub.N-DTA
is a fusion between LF.sub.N, the N-terminal PA63-binding domain of
LF, and DTA, the catalytic domain of diphtheria toxin. See PCT
US2012/20731. The DTA moiety catalyzes the ADP-ribosylation of
eukaryotic elongation factor-2 (eEF-2) within the cytosol, blocking
protein synthesis and causing cell death. Collier & Cole, 164
Science 1179 (1969); Collier, 25 J. Mol. Biol. 83 (1967). The
proteolytically activated form of PA, mPA63, was able to form
SDS-resistant, high molecular weight aggregates, characteristic of
pores, although pH dependence of pore formation was somewhat
altered (FIG. 2B).
[0050] Then, the PA N682A/D683A double mutant (mPA), with its
virtually ablated the receptor-binding function, was fused to human
EGF to the C-terminus of the mutated protein. Purified monomeric
mPA-EGF was stable and ran slightly slower than native PA on SDS
polyacrylamide gels, consistent with its higher molecular weight
(FIG. 2A). Western blots showed that the product reacted with both
anti-PA and anti-EGF antibodies. Also, it was also shown the
mPA63-EGF fragment derived by trypsin treatment formed high
molecular weight aggregates on SDS-PAGE similar to those seen with
mPA63 (FIG. 2B). PA 63 refers to amino acids 197-764 of SEQ ID NO:
9.
[0051] Although the complete anthrax PA amino acid sequence well
known, it is provided herein for reference. The sequence includes a
29 amino acid signal peptide marked with bold and italized:
TABLE-US-00001 (SEQ ID NO: 9) VKQENRLLNE SESSSQGLLG YYFSDLNFQA
PMVVTSSTTG DLSIPSSELE NIPSENQYFQ SAIWSGFIKV KKSDEYTFAT SADNHVTMWV
DDQEVINKAS NSNKIRLEKG RLYQIKIQYQ RENPTEKGLD FKLYWTDSQN KKEVISSDNL
QLPELKQKSS NSRKKRSTSA GPTVPDRDND GIPDSLEVEG YTVDVKNKRT FLSPWISNIH
EKKGLTKYKS SPEKWSTASD PYSDFEKVTG RIDKNVSPEA RHPLVAAYPI VHVDMENIIL
SKNEDQSTQN TDSQTRTISK NTSTSRTHTS EVHGNAEVHA SFFDIGGSVS AGFSNSNSST
VAIDHSLSLA GERTWAETMG LNTADTARLN ANIRYVNTGT APIYNVLPTT SLVLGKNQTL
ATIKAKENQL SQILAPNNYY PSKNLAPIAL NAQDDFSSTP ITMNYNQFLE LEKTKQLRLD
TDQVYGNIAT YNFENGRVRV DTGSNWSEVL PQIQETTARI IFNGKDLNLV ERRIAAVNPS
DPLETTKPDM TLKEALKIAF GFNEPNGNLQ YQGKDITEFD FNFDQQTSQN IKNQLAELNA
TNIYTVLDKI KLNAKMNILI RDKRFHYDRN NIAVGADESV VKEAHREVIN SSTEGLLLNI
DKDIRKILSG YIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKY NDKLPLYISN
PNYKVNVYAV TKENTIINPS ENGDTSTNGI KKILIFSKKG YEIG,
Anthrax Protective antigen, with 29 aa signal peptide; UniProtKB
NO. P13423 (PAG_BACAN)
[0052] The following shows the anthrax PA amino acid sequence
without the 29 amino acid signal peptide. The numbering references
to the mutants throughout this specification relate to the sequence
without the signal peptide. In the following, the N682A/D683A
mutant is indicated with bold:
TABLE-US-00002 (SEQ ID NO: 10) E VKQENRLLNE SESSSQGLLG YYFSDLNFQA
PMVVTSSTTG DLSIPSSELE NIPSENQYFQ SAIWSGFIKV KKSDEYTFAT SADNHVTMWV
DDQEVINKAS NSNKIRLEKG RLYQIKIQYQ RENPTEKGLD FKLYWTDSQN KKEVISSDNL
QLPELKQKSS NSRKKRSTSA GPTVPDRDND GIPDSLEVEG YTVDVKNKRT FLSPWISNIH
EKKGLTKYKS SPEKWSTASD PYSDFEKVTG RIDKNVSPEA RHPLVAAYPI VHVDMENIIL
SKNEDQSTQN TDSQTRTISK NTSTSRTHTS EVHGNAEVHA SFFDIGGSVS AGFSNSNSST
VAIDHSLSLA GERTWAETMG LNTADTARLN ANIRYVNTGT APIYNVLPTT SLVLGKNQTL
ATIKAKENQL SQILAPNNYY PSKNLAPIAL NAQDDFSSTP ITMNYNQFLE LEKTKQLRLD
TDQVYGNIAT YNFENGRVRV DTGSNWSEVL PQIQETTARI IFNGKDLNLV ERRIAAVNPS
DPLETTKPDM TLKEALKIAF GFNEPNGNLQ YQGKDITEFD FNFDQQTSQN IKNQLAELNA
TNIYTVLDKI KLNAKMNILI RDKRFHYDRN NIAVGADESV VKEAHREVIN SSTEGLLLNI
DKDIRKILSG YIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKY NDKLPLYISN
PNYKVNVYAV TKENTIINPS ENGDTSTNGI KKILIFSKKG YEIG.
[0053] A431 cells, which express high levels of the EGF receptor
(Lin et al., 224 Science 843 (1984); Ullrich et al., 309 Nature 418
(1984)), were killed by LF.sub.N-DTA (EC50.about.10 pM) in the
presence of mPA-EGF, whereas CHO-K1 cells, which do not express the
EGF receptor, were not killed (FIG. 3A). Wild-type PA also mediated
the inhibition of protein synthesis in A431 cells, but a higher
concentration of LF.sub.N-DTA (EC50.about.100 pM) was needed,
suggesting that these cells express a low level of ANTXR1, ANTXR2,
or both. The translocation-deficient PA mutant, PAF427H (Krantz,
309 Science 777 (2005)), did not mediate killing on either A431 or
CHO-K1 cells.
[0054] If the entry of LF.sub.N-DTA into A431 cells mediated by
mPA-EGF was dependent on binding to the EGF receptor, then addition
of free EGF should compete for binding and block toxicity. As shown
in FIG. 3B, a 50-fold excess of EGF completely protected the cells
from the cytotoxic effects of LF.sub.N-DTA, whereas the same
concentration of the PA-binding VWA domain of ANTXR2 had no effect.
In contrast, cytotoxicity mediated by wild-type PA on A431 cells
was ablated by the ANTXR2 domain, but was not inhibited to a
significant degree by EGF (FIG. 3C).
[0055] The ability the mPA-EGF to translocate LF and EF, the native
effector moieties of anthrax toxin, into A431 cells was also
demonstrated in an exemplary system. LF inactivates
mitogen-activated protein kinase kinases (MEKs) by cleaving near
their N-termini (Duesbery et al., 1998; Vitale et al., 1998), and
LF entry was charactarized by Western blotting of cell lysates with
an anti-MEK1 antibody after incubating cells with LF plus PA or a
variant thereof. MEK1 was cleaved completely with LF in combination
with PA or mPA-EGF, but not in combination with the
translocation-deficient mutant PA F427H (FIG. 4A). Entry of EF was
measured using an enzyme-linked competition assay to determine the
intracellular level of cyclic AMP (cAMP) and with mPA-EGF as the
translocation vehicle observed a 400-fold elevation of cAMP (FIG.
4B). This level was .about.100.times. higher than that with WT PA,
and the level observed with mPA or PAF427H was at background. The
strong elevation observed with mPA-EGF was likely due in part to
the high level of EGFR on the A431 cells.
[0056] The following mutations in PA are known to reduce toxicity
by reducing cell binding, and can thus be used alone or in
combination to ablate PA receptor binding.
TABLE-US-00003 MUTATION LOCATION IN SEQ ID NO: 9 Effect on receptor
binding 686 N .fwdarw. A: Decrease in cell binding. 710 Y .fwdarw.
A: Decrease in cell binding. 711 N .fwdarw. A: Decrease in cell
binding. 712 D .fwdarw. A: Decrease in cell binding. 715 P .fwdarw.
A: Decrease in cell binding. 716 L .fwdarw. A: Decrease in cell
binding. 718 I .fwdarw. A: Decrease in cell binding.
[0057] In addition to LF.sub.N, analogues of bacterial toxins such
as diphtheria toxin and cholera toxin can be used to deliver the
therapeutic proteins. Thus, in one embodiment, the invention
provides a method of treating a subject by contacting cells of the
subject either in vivo or ex vivo with a composition comprising a
fusion molecule comprising the component A or a surrogate A
component attached to the therapeutic moiety. See PCT
US2012/20731.
[0058] In another example, the 150-residue receptor-binding domain
of diphtheria toxin (DTR) was fused to the C-terminus of mPA. The
purified mPA-DTR fusion reacted with both anti-PA and
anti-diphtheria toxin antibodies (FIG. 5) and retained the ability
to oligomerize and form pores, and to bind and translocate cargo
LFN-DTA in a planar bilayer system. The mPA-DTR variant delivered
LFN-DTA into CHOK1 cells, inhibiting protein synthesis, and soluble
DTR competitively blocked this inhibition (FIG. 5).
[0059] In yet another example, to specifically target HER2-positive
cells, an Affibody (ZHER2; .about.58 amino acids), known to bind
the HER2 receptor with high affinity, was fused to the C-terminus
of a receptor-recognition-deficient, mutated form of PA. Cell
cytotoxicity assays using mPA-ZHER2 demonstrated that LFN-DTA
inhibited protein synthesis in the cytosol of cells expressing the
HER2 receptor, whereas HER-negative cells were unaffected (FIG. 6).
Because amplification of the HER2 gene or overexpression of HER2
occurs in 20% to 30% of early stage breast cancer patients, and
because patients overexpressing the HER2 receptor have decreased
overall survival, PA-based targeting of HER2 receptor-positive
cells is an important example of cancer cells that can be targeted
by the present strategy.
[0060] HER2 is a receptor tyrosine kinase belonging to the same
family as EGFR. Unlike EGFR, however, HER2 has no known natural
ligand. In the present study we developed a redirected binary toxin
by fusing a high affinity Affibody specific for the HER2 receptor
(Z.sub.HER2:342) (Orlova et al. 2006) to the C terminus of receptor
recognition-deficient PA (mPA), creating the fusion mPA-ZHER2.
Affibodies represent a class of targeting polypeptides derived from
the Z domain of Staphylococcus aureus protein A. Advantages over
other receptor-targeting ligands derive from the fact that
Affibodies are small (58 amino acids; .about.6 kDa), pH- and
thermo-stable, lack Cys residues, and fold independently and
reversibly (Nord et al. 1997; Lofblom et al. 2010). Further, they
may be rapidly evolved in vitro by phage-display technologies to
affinity levels comparable to those observed with monoclonal
antibodies.
[0061] Our results show that a receptor-recognition ablated B unit,
such as mPA, with the HER2 targeting moiety, such as a
Z.sub.HER2:342 affibody fused to the C terminus of the receptor
ablated B unit, can direct the action of either of two cytocidal
effector proteins to HER2-positive tumor cells. These cells,
including a HER2-positive trastuzumab-resistant tumor cell line,
were ablated, and specific killing was observed regardless of
whether the cultures consisted of a homogeneous population or had
been mixed with cells lacking the HER2 marker. The amino acid
sequence of the Z.sub.HER2:342 affibody used in the examples is as
follows:
TABLE-US-00004 (SEQ ID NO: 11)
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKK LNDAQAPK.
[0062] Although the inventors used a specific AFFIBODY described in
the examples to show proof of concept, in view of the results, any
AFFIBODY or antibody that specifically binds to a cell surface
protein of choice can be used.
[0063] In some aspects of all the embodiments of the invention, the
cell non-toxin-associated receptor-binding ligand specific for a
target cell comprises an antibody and/or an antigen-binding portion
or fragment of an antibody.
[0064] An example antibody to target HER2 is trastuzumab, a
recombinant monoclonal antibody used in therapeutics (HERCEPTIN).
Another monoclonal antibody that targets HER2 positive cells and
prevents dimerization of HER2 and HER3 is Pertuzumab (also called
2C4, trade name PERJETA). Either one of these antibodies can be
used to fuse with the receptor ablated PA86 subunit, such as the
mPA.
[0065] The term "antibody" refers to an immunoglobulin (e.g., IgG,
IgM, IgA, IgE, IgD, etc.).
[0066] The basic functional unit of each antibody is an
immunoglobulin (Ig) monomer (containing only one immunoglobulin
("Ig") unit). Included within this definition are monoclonal
antibodies, chimeric antibodies, recombinant antibodies, and
humanized antibodies.
[0067] In one embodiment, the invention's antibodies are monoclonal
antibodies produced by hybridoma cells.
[0068] In particular, the invention contemplates antibody fragments
that contain the idiotype ("antigen-binding fragment") of the
antibody molecule. For example, such fragments include, but are not
limited to, the Fab region, F(ab')2 fragment, pFc' fragment, and
Fab' fragments.
[0069] The "Fab region" and "fragment, antigen binding region,"
interchangeably refer to portion of the antibody arms of the
immunoglobulm "Y" that function in binding antigen. The Fab region
is composed of one constant and one variable domain from each heavy
and light chain of the antibody. Methods are known in the art for
the construction of Fab expression libraries (Huse et al., Science,
246: 1275-1281 (1989)) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity. In another
embodiment, Fc and Fab fragments can be generated by using the
enzyme papain to cleave an immunoglobulin monomer into two Fab
fragments and an Fc fragment. The enzyme pepsin cleaves below the
hinge region, so a "F(ab')2 fragment" and a "pFc' fragment" is
formed. The F(ab')2 fragment can be split into two "Fab' fragments"
by mild reduction.
[0070] The invention also contemplates a "single-chain antibody"
fragment, i.e., an amino acid sequence having at least one of the
variable or complementarity determining regions (CDRs) of the whole
antibody, and lacking some or all of the constant domains of the
antibody. These constant domains are not necessary for antigen
binding, but constitute a major portion of the structure of whole
antibodies. Single-chain antibody fragments are smaller than whole
antibodies and may therefore have greater capillary permeability
than whole antibodies, allowing single-chain antibody fragments to
localize and bind to target antigen-binding sites more efficiently.
Also, antibody fragments can be produced on a relatively large
scale in prokaryotic cells, thus facilitating their production.
Furthermore, the relatively small size of single-chain antibody
fragments makes them less likely to provoke an immune response in a
recipient than whole antibodies. Techniques for the production of
single-chain antibodies are known (U.S. Pat. No. 4,946,778). The
variable regions of the heavy and light chains can be fused
together to form a "single-chain variable fragment" ("scFv
fragment"), which is only half the size of the Fab fragment, yet
retains the original specificity of the parent immunoglobulm.
[0071] The "Fc" and "Fragment, crystallizable" region
interchangeably refer to portion of the base of the immunoglobulin
"Y" that function in role in modulating immune cell activity. The
Fc region is composed of two heavy chains that contribute two or
three constant domains depending on the class of the antibody. By
binding to specific proteins, the Fc region ensures that each
antibody generates an appropriate immune response for a given
antigen. The Fc region also binds to various cell receptors, such
as Fc receptors, and other immune molecules, such as complement
proteins. By doing this, it mediates different physiological
effects including opsonization, cell lysis, and degranulation of
mast cells, basophils and eosinophils. In an experimental setting,
Fc and Fab fragments can be generated in the laboratory by cleaving
an immunoglobulin monomer with the enzyme papain into two Fab
fragments and an Fc fragment.
[0072] In some aspects of all the embodiment of the invention, the
non-toxin-associated receptor-binding ligand specific for a target
cell comprises an affibody.
[0073] "Affinity body," "Affibody.RTM.," and "affibody" molecules
are antibody mimetic proteins that, like antibodies, can
specifically bind target antigens (Nord, K., et al. (1997) Nature
Biotechnol. 15: 772-777). Affibody molecules can be designed and
used like aptamers. In one embodiment, Affibody molecules comprise
a backbone derived from an IgG-binding domain of Staphylococcal
Protein A (Protein A produced by S. aureus). The backbone can be
derived from an IgG binding domain comprising the three alpha
helices of the IgG-binding domain of Staphlococcal Protein A termed
the B domain. The amino acid sequence of the B domain is described
in Uhlen et al, J. Biol. Chem. 259: 1695-1702 (1984).
Alternatively, the backbone can be derived from the three alpha
helices of the synthetic IgG-binding domain known in the art as the
Z domain, which is described in Nilsson et al., Protein Eng. 1:
107-113 (1987). The backbone of an affibody comprises the amino
acid sequences of the IgG binding domain with amino acid
substitutions at one or more amino acid positions. The affibody,
for example, comprises the 58 amino acid sequence of the Z
domain
TABLE-US-00005 (VDNKFDKEXXXAXXEIXXLPNLNXXQXXAFIXSLXDDPSQSADLLAEAK
KLDDAQAPK, SEQ ID NO: 12),
wherein X at each of positions 9, 10, 11, 13, 14, 17, 18, 24, 25,
27, 28, 32, and 35 is any amino acid (Capala et al, U.S. Pat. Appl.
No. US20100254899).
[0074] The affibody molecule constitutes a highly suitable carrier
for directing molecules of interest (e.g., toxins, radioisotopes,
therapeutic peptides) to, e.g., tumor cells due to specific target
binding and lack of irrelevant interactions, such as the Fc
receptor binding displayed by some antibodies.
[0075] Common advantages of AFFIBODY.RTM. molecules over antibodies
are better solubility, tissue penetration, stability towards heat
and enzymes, and comparatively low production costs.
[0076] Affibodies are exemplified by, but not limited to,
Anti-ErbB2 AFFIBODY.RTM. (also referred to as anti-HER2
AFFIBODY.RTM.), Anti-EGFR AFFIBODY.RTM., Anti-TNF alpha
AFFIBODY.RTM., Anti-fibrinogen AFFIBODY.RTM., Anti-transferrin
AFFIBODY.RTM., Anti-HSA AFFIBODY.RTM., Anti-Insulin AFFIBODY.RTM.,
Anti-IgG AFFIBODY.RTM., Anti-IgM AFFIBODY.RTM., Anti-IgA
AFFIBODY.RTM., and Anti-IgE AFFIBODY.RTM. (e.g., from Abceam,
Cambridge, Mass.).
[0077] Affibodies with an affinity of down to sub-nanomolar have
been obtained from naive library selections, and affibodies with
picomolar affinity have been obtained following affinity maturation
(Orlova et al. (2006). "Tumor imaging using a picomolar affinity
HER2 binding affibody molecule". Cancer Res. 66 (8): 4339-48. PMID
16618759). Affibodies conjugated to weak electrophiles bind their
targets covalently (Holm et al., Electrophilic affibodies forming
covalent bonds to protein targets, J Biol Chem. 2009 Nov. 20;
284(47):32906-13. PMID 19759009).
[0078] Affibody molecules can be synthesized chemically or in
bacteria or purchased from a commercial source (e.g., Affibody AB,
Bromma, Sweden; Abeam, Cambridge, Mass.).
[0079] Affibody molecules can also be obtained by constructing a
library of affibodies as described in U.S. Pat. No. 5,831,012,
which is incorporated herein by reference. The affibody library can
then be screened for affibodies which bind to target antigens of
interest (e.g., HER-2, EGFR) by methods known in the art.
[0080] Affibody molecules are based on a three-helix bundle domain,
which can be expressed in soluble and proteolytically stable forms
in various host cells on its own or via fusion with other protein
partners (Stahl et al. (1997). "The use of gene fusions to protein
A and protein G in immunology and biotechnology". Pathol. Biol.
(Paris) 45: 66-76. PMID 9097850."
[0081] Affibodies tolerate modification and are independently
folding when incorporated into fusion proteins. Head-to-tail
fusions of Affibody molecules of the same specificity have proven
to give avidity effects in target binding, and head-to-tail fusion
of Affibody molecules of different specificities makes it possible
to get bi-specific or multi-specific affinity proteins. Fusions
with other proteins can also be created (Ronnmark et al. (2002)
"Construction and characterization of affibody-Fc chimeras produced
in Escherichia coli," J. Immunol. Methods 261: 199-211. PMID
11861078; Ronnmark et al. (2003) "Affibody-beta-galactosidase
immunoconjugates produced as soluble fusion proteins in the
Escherichia coli cytosol," J. Immunol. Methods 281: 149-160. PMID
14580889). A site for site-specific conjugation is facilitated by
introduction of a single cysteine at a desired position.
[0082] A number of different Affibody molecules have been produced
by chemical synthesis. Since they do not contain cysteines or
disulfide bridges, they fold spontaneously and reversibly into the
correct three-dimensional structures when the protection groups are
removed after synthesis (Nord et al. (2001) "Recombinant human
factor V111-specific affinity ligands selected from phage-displayed
combinatorial libraries of protein A," Eur. J. Biochem. 268: 1-10.
PMID 11488921; Engfeldt et al. (2005) "Chemical synthesis of
triple-labeled three-helix bundle binding proteins for specific
fluorescent detection of unlabeled protein," Chem. BioChem. 6:
1043-1050. PMID 15880677).
[0083] We hypothesized that the ability of PA to transport two
structurally disparate enzymes, LF and EF, into cells might suggest
it is be capable of delivering heterologous proteins. Delivery of
several such proteins and peptides has been demonstrated following
their fusion to the PA63-binding domain of LF. Pentelute et al., 5
ACS Chem. Biol. 359 (2010); Pentelute et al., 2011; Arora &
Leppla, 62 Infect. & Immun. 4955 (1994); Arora et al., 267 J.
Biol. Chem. 15542 (1992); Hu & Leppla, 4 PLoS ONE 4 e7946
(2009a); Milne et al., 15 Molec. Microbiol. 661 (1995). A second
mode of adaptability is illustrated by studies in which the furin
activation site within PA was replaced with sites specific for
other proteases for the purpose of tumor targeting. Liu et al., 60
Cancer Res. 6061 (2000); Abi-Habib et al., 5 Molec. Cancer Therap.
2556 (2006). The current study demonstrates that a third mode of
adaptability, namely that the protein transport activity of PA, can
be readily channeled through heterologous cell-surface
receptors.
[0084] Given that the toxins have very specific receptors, it was
not sure if the new target receptors would be able to allow pore
formation and transport of the complexes to the cells. However, we
showed that the exemplary mPA-EGF fusion construct was surprisingly
able to transport LF, EF, and the LF.sub.N-DTA fusion protein to
the cytosol, suggesting that the essential oligomerization and
transport functions of PA were not perturbed by channeling entry
through surrogate receptors. One of the surrogate binding domains
examined, DTR, performs an analogous function in an unrelated toxin
(Louie et al., 1 Molec. Cell 67 (1997)), whereas the other, EGF,
has no apparent relationship to bacterial toxin action. Both
proteins bind to receptors that, like ANTXR1 and ANTXR2,
internalize their ligands and traffic them to an acidic
intracellular compartment. It is likely that entry into an acidic
compartment is important for proper functioning of PA fusion
proteins, because (a) acidic intravesicular pH plays a crucial role
in promoting conversion of the PA prepore to the pore (Miller et
al., 38 Biochem. 10432 (1999); Collier, 2009); and (b) the pH
gradient across the endosomal membrane is essential for protein
translocation (Krantz et al., 2006).
[0085] In view of the above, we can conclude that any novel
receptor-targeting domain should function similarly when added to a
receptor ablated PA83, for example, mPA as described herein. In
some aspects of all the embodiments of the invention, the
receptor-targeting domain is added to the C-terminus of the
receptor-ablated PA83. Thus, in some aspects, the invention
provides a composition comprising a fusion protein comprising a
receptor-ablated pore-forming PA, such as mPA fused to a
non-toxin-associated receptor-binding ligand specific for a target
cell, wherein the non-toxin-associated receptor-binding ligand
specific for a target cell is added to the C-terminus of the
receptor-ablated PA. The composition may further comprise a
complementary toxin unit capable of associating with the
pore-forming toxin unit for delivery of a therapeutic protein to
the cytosol of the target cell, such as LF or a fusion protein
comprising the N-terminal PA-binding portion of the LF
(LF.sub.N).
[0086] The decision to fuse surrogate receptor ligands to the C
terminus of mPA, instead of replacing domain 4 with these ligands,
was based on results indicating that domain 4 stabilizes the
prepore. Katayama et al., 107 PNAS 3453 (2010). Domain 4 must pivot
away from domain 2 to allow the pore-forming loop to be relocated
to the base of the structure, so that the transmembrane
.beta.-barrel stem of the pore can be formed. Three-dimensional
structure of the anthrax toxin pore inserted into lipid nanodiscs
and lipid vesicles with the remainder of PA63 inhibit this pivoting
and prevent premature conversion of the prepore to the pore
(Katayama H, Wang J, Tama F, Chollet L, Gogol E P, Collier R J,
Fisher M T. Interactions of domain 4, Proc Natl Acad Sci USA. 2010
Feb. 23; 107(8):3453-7). Thus, retaining domain 4 in mutated form
allowed modification of receptor specificity while minimizing the
likelihood that the process of prepore-to-pore conversion would be
perturbed. That said, the double mutation used to ablate the
receptor binding activity of domain 4 apparently slightly perturbed
stability of the mPA63 prepore, as mPA prepore, unlike native
prepore, underwent some degree of conversion to pore at pH 8.5
(FIG. 2A).
[0087] In addition to the recombinant technologies employed herein
to fuse the receptor-specific ligand of the target cell to the B
unit or create a cytotoxic A unit fusion, these constructs can also
be produced by obtaining isolated components and conjugating them
using chemical ligation or other conjugation techniques. See, e.g.,
Dawson et al., Synthesis of Proteins by Native Chemical Ligation,
266 Science 776 (1994); Muir et al., Expressed Protein Ligation: A
General Method for Protein Engineering, 95 PNAS 6705 (1998);
Nilsson et al., Chemical Synthesis of Proteins, 34 Ann. Rev.
Biophys. Biomol. Struct. 91 (2005).
[0088] Redirecting PA-dependent protein transport through
heterologous cellular receptors has applications both in
experimental science and medicine. Leppla and coworkers have
explored targeting of PA to tumor cells by changing the proteolytic
activation site. Modified forms of PA were used to deliver FP59, a
cytotoxic fusion protein similar to LF.sub.N-DTA, to the cytosol of
cells enriched in urokinase- or matrix metalloprotease. Abi-Habib
et al., 5 Molec. Cancer Therap. 2556 (2006); Liu et al., 2000. Like
these proteases, EGFR is also enriched on several tumors
(Ciardiello & Tortora, 358 N. Engl. J. Med. 1160 (2008)). Thus
mPA-EGF can also serve as an alternative means of targeting. Other
ligands whose receptors are enriched on target cells, including
cancer cells or virus-infected cells, would also be candidates for
fusion to mPA or other B components of AB toxins.
[0089] Examples of other receptors that may be targeted for the
treatment of cancer by fusing a receptor-binding moiety, e.g., an
agonist, with the pore-forming portion of an AB toxin include
estrogen receptors, e.g., in certain breast cancers, progesterone
receptors, insulin-like growth factor, e.g., in certain prostate
cancers.
[0090] Based on our examples, one can envision use of
receptor-targeted PA variants to deliver a wide variety of
proteins, nontoxic, as well as toxic, to chosen classes of cells.
Fusion to LFN does not necessarily render all proteins
transportable by PA, however. Like DTA, the catalytic domains of
shiga toxin and pseudomonas exotoxin A, and some nontoxic proteins,
including beta-lactamase, dihydrofolate reductase (DHFR), and
ciliary neurotrophic factor, were found to be transported by PA
when fused to LFN (Arora & Leppla, 1994; Arora et al., 1992; Hu
& Leppla, 2009b; Wesche et al., 37 Biochem. 15737 (1998)); but
LFN fusions of others, including tetanus toxin light chain,
botulinum toxin E light chain, acidic fibroblast growth factor,
basic fibroblast growth factor, and HIV Tat protein, were not
transported. Introduction of an artificial disulfide into the DTA
moiety of LFN-DTA blocked translocation, as did liganding of
LFN-DTA and LFN-DHFR by adenine and methotrexate, respectively.
Wesche et al., 1998. These findings are consistent with a
requirement that proteins unfold in order to be translocated
through the PA pore, and the propensity to unfold under acidic
conditions may therefore be a major determinant of ability of a
protein to be translocated. Nevertheless, analogues of bacterial
toxins such as diphtheria toxin and cholera toxin, can be used to
deliver other chemical entities or proteinaceous therapeutics.
[0091] Thus, in one embodiment, the invention provides a method of
treating a subject by contacting cells of the subject either in
vivo or ex vivo with a composition comprising a therapeutic
intended to the delivered into the targeted cells of the subject
with a fusion molecule comprising the component A or a surrogate A
component attached to the therapeutic agent.
[0092] In some aspects of all the embodiments, the subject has
cancer. In some aspects of all the embodiments, the subject has
cancer wherein the cancer cells express HER2, and the toxin system
delivered to the cancer patient comprises a fusion of protein as
exemplified in Example 8, except that the targeting moiety can be
changed to another HER2-specific affibody or an antibody specific
to HER2.
[0093] Amplification or over-expression of the HER2 gene occurs in
approximately 30% of breast cancers. It is strongly associated with
increased disease recurrence and a worse prognosis (Roy V, Perez E
A (November 2009). "Beyond trastuzumab: small molecule tyrosine
kinase inhibitors in HER-2-positive breast cancer". Oncologist 14
(11): 1061-9). Over-expression is also known to occur in ovarian,
stomach, and aggressive forms of uterine cancer, such as uterine
serous endometrial carcinoma (Tan M, Yu D (2007). "Molecular
mechanisms of HER2-mediated breast cancer chemoresistance". Adv.
Exp. Med. Biol. 608: 119-29). Accordingly, the systems and methods
described herein are useful in treatment of at least HER2
overexpressing breast cancer, ovarian cancer, stomach cancer, and
aggressive forms of uterine cancer, such as uterine serous
endometrial carcinoma. Uses of the altered binary toxin system for
the treatment of these cancers are thus also provided.
[0094] The compositions, systems and uses of the present invention
can be delivered in a pharmaceutically acceptable carrier. As used
herein, the terms "pharmaceutically acceptable" refers to
compositions, carriers, diluents and reagents, are used
interchangeably and represent that the materials are capable of
administration to or upon a mammal without the production of
undesirable physiological effects such as nausea, dizziness,
gastric upset and the like. A pharmaceutically acceptable carrier
will not promote the raising of an immune response to an agent with
which it is admixed, unless so desired. The preparation of a
pharmacological composition that contains active ingredients
dissolved or dispersed therein is well understood in the art and
need not be limited based on formulation.
[0095] Typically such compositions are prepared as injectable
either as liquid solutions or suspensions, however, solid forms
suitable for solution, or suspensions, in liquid prior to use can
also be prepared. The preparation can also be emulsified or
presented as a liposome composition. The active ingredient can be
mixed with excipients which are pharmaceutically acceptable and
compatible with the active ingredient and in amounts suitable for
use in the therapeutic methods described herein. Suitable
excipients include, for example, water, saline, dextrose, glycerol,
ethanol or the like and combinations thereof. In addition, if
desired, the composition can contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like which enhance the effectiveness of the active
ingredient. The therapeutic composition of the present invention
can include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, tartaric, mandelic and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine and the
like. Physiologically tolerable carriers are well known in the art.
Exemplary liquid carriers are sterile aqueous solutions that
contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH value, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other
solutes. Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of such
additional liquid phases are glycerin, vegetable oils such as
cottonseed oil, and water-oil emulsions. The amount of an active
agent used in the methods described herein that will be effective
in the treatment of a particular disorder or condition will depend
on the nature of the disorder or condition, and can be determined
by standard clinical techniques.
[0096] The term "subject" as used herein and throughout the
specification is intended to include organisms with eukaryotic
cells, including mammals, such as humans and domestic animals,
laboratory animal models, including rodent, canine, and primate
models.
[0097] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
[0098] Some embodiments of the invention are listed in the
following paragraphs:
1. An altered binary toxin system for delivery of an active
molecule to a target cell comprising: [0099] a fusion protein
comprising a receptor-ablated pore-forming binary or AB toxin unit
fused to a non-toxin-associated receptor-binding ligand specific
for a target cell, and [0100] a complementary toxin unit capable of
associating with the pore-forming toxin unit for delivery of a
therapeutic protein to the cytosol of the target cell. 2. The
altered binary toxin system of paragraph 1, wherein the
receptor-ablated pore-forming unit is anthrax toxin protective
antigen (PA). 3. The altered binary toxin system of paragraph 2,
wherein the PA is PA.sup.N682AD683A. 4. The altered binary toxin
system of any one of paragraphs 1-3, wherein the non-toxin
associated specific target receptor-binding ligand is an epidermal
growth factor-1 or epidermal growth factor 2; or wherein the
non-toxin associates specific receptor binding ligand targets
epidermal growth factor receptor 1 (EGFR) or epidermal growth
factor receptor 2 (HER2). 5. The altered binary toxin system of any
one of paragraphs 1-4, wherein the complementary toxin unit is
anthrax toxin lethal factor (LF or EF). 6. The altered binary toxin
system of any one of paragraphs 1-5, wherein the complementary
toxin unit the amino terminal portion of anthrax toxin lethal
factor (LF.sub.N or EF.sub.N). 7. The altered binary toxin system
of any one of paragraphs 1-6, wherein the therapeutic protein is
the catalytic domain of diphtheria toxin (DTA), ricin, shiga toxin,
or pseudomonas exotoxin A. 8. The altered binary toxin system of
any one of paragraphs 1-7, wherein the AB toxin is botulinum
neurotoxin, anthrax toxin, diphtheria toxin, ricin, shiga toxin,
shiga like toxin, exotoxin A, or cholera toxin. 9. The altered
binary toxin system of paragraph 8, wherein the binary toxin is
Clostridium perfringens toxins alpha, beta, epsilon or iota;
Clostridium botulinum C2 toxin; or Clostridium spiroforme Iota-like
toxin. 10. The altered binary toxin system of any one of paragraphs
1-9, wherein the receptor-binding ligand binds to a receptor
selected from epidermal growth factor receptors HER1, HER2, HER3 or
HER4; vascular endothelial growth factor receptors VEGFR-1, VEGFR-2
or VEGFR-3; insulin-like growth factor 1 receptor; fibroblast
growth factor receptors; thrombospondin 1 receptor; estrogen
receptors; urokinase receptors; progesterone receptors;
testosterone receptors; carcinoembryonic antigens;
prostate-specific antigens; farnesoid X receptors; transforming
growth factor receptors; transferrin receptors; hepatocyte growth
factor receptors; or vasoactive intestinal polypeptide receptors 1
and 2. 11. The altered binary toxin system of any one of paragraphs
1-10, wherein the receptor-binding ligand is selected from an
antibody or an affibody. 12. The altered binary toxin system of
paragraph 11, wherein the affibody is a HER2 affibody. 13. The
altered binary toxin system of paragraph 12, wherein the HER2
affibody is ZHER2. 14. The altered binary toxin system of any one
of paragraphs 1-13, wherein the therapeutic protein is the
cytotoxic domain of shiga toxin, shiga-like toxin 1 and 2, ricin,
ricin toxin A chain, abrin, gelonin, pokeweed antiviral protein,
saporin, trichsanthin, pepcin, maize RIP, alpha-sarcin, Clostridium
perfringens epsilon toxin, Botulinum neurotoxins, Staphylococcus
enterotoxins, Clostridium difficile toxins, pertussis toxins, or
pseudomonas exotoxin. 15. A kit for delivering bioactive molecules
to a eukaryotic cell, comprising an altered binary toxin system of
any one of paragraphs 1-14. 16. A method for treating cancer
comprising administering to a subject diagnosed with cancer the
altered binary toxin system of any one of the paragraphs 1-14. 17.
The method of paragraph 16, wherein the altered binary toxin system
comprises a receptor-redirected anthrax protective antigen. 18. The
method of any one of paragraphs 16-17, wherein the cancer is a HER2
positive cancer and the anthrax protective antigen is fused with a
HER2 binding ligand. 19. The method of paragraph 18, wherein the
HER2 binding ligand is an antibody or an affibody. 20. Use of a
binary toxin system of any one of the paragraphs 1-14 for the
treatment of cancer. 21. Use of a receptor-redirected anthrax
protective antigen for the treatment of cancer. 22. A
pharmaceutical composition comprising the toxin system of any of
the paragraphs 1-14 and a pharmaceutically acceptable carrier.
Examples
[0101] The following describes examples that were performed to show
proof of concept of the general invention which outlines using a
receptor-ablated pore forming subunit of AB toxins fused with a
non-native (i.e., not the natural receptor for the toxin) receptor
binding molecule to deliver toxic or non-toxic therapeutic drugs to
a cell expressing the receptor. The specific examples can be
expanded to more broadly encompass classes of toxins, ablation
mutations, receptor binding domains and such with the knowledge in
the art and the instructions provided herein.
Example 1
Generation of PA Expression Plasmids
[0102] One can construct the expression plasmids with any known
sequences for the toxins according to routine methods and
following, e.g., the principles used to make the below-described
exemplary expression plasmid. In our examples we made the two PA
chimeras used in this work: PAN682AD683A-EGF (mPA-EGF) and
PAN682AD683A-DTR (mPA-DTR) were created by overlap extension PCR
using a previously generated PAN682AD683A (mPA) gene coding
sequence. In both cases the first PCR step consisted of two
reactions (a) using a forward primer
(PAFor--GATTTAGTAATTCGAATTCAAGTACGG) (SEQ ID NO:2), plus either
PARevEGF (CATTCAGAGTCGCTGTTTGGTTGCGTTTTATG) (SEQ ID NO:3), or
PARevDTR (GTTTTATGCCCCGGAGATCCTATCTCATAGCC) (SEQ ID NO:4) reverse
primers, which contained the EGF and DTR overlapping regions,
respectively; and (b) using forward and reverse primers to amplify
the EGF (EGFFor--CATAAAACGCAACCAAACAGCGACTATGAATG) (SEQ ID NO:5)
and (EGFRev--GGTGGTGCTCGAGTCAACGGAGCTCCCACCATTTC) (SEQ ID NO:6) and
DTR (DTRFor--GGCTATGAGATAGGATCTCCGGGGCATAAAAC) (SEQ ID NO:7) and
(DTRRev--GTGGTGGTGGTGGTGCTCGAGTCAGCTTTTGATTTC) (SEQ ID NO:8)
sequences. The PCR-generated DNA fragments were then subjected to a
second PCR step using forward primer PAFor in combination with
either the EGFRev or DTRRev primer, for PA-EGF and PA-DTR, to
stitch and amplify the two fragments together. In both cases the
full-length PCR products encoded EcoRI and XhoI restriction sites,
in the forward and reverse primers, respectively. The PCR products
were restriction digested and cloned into the pet22b expression
vector following standard protocols. Each clone also coded for an
8-residue linker (SPGHKTQP, SEQ ID NO: 1) between PA and either EGF
or DTR, which is part of the natural linker between the
transmembrane and receptor-binding domains of diphtheria toxin.
[0103] Oligonucleotides were from Integrated DNA Technologies
(Coralville, Iowa). Sigma-Aldrich (St. Louis, Mo.) supplied all
chemicals unless noted otherwise. A synthetic human EGF gene,
adjusted for E. coli expression, was a generous gift from Prof. E.
Joop van Zoelen (Department of Cell Biology and Applied Biology,
Heijendaalseweg, Nijmegen). Soluble EGF was from ProSpec-Tany
Technogene Ltd (East Brunswick, N.J.).
Example 2
Protein Expression and Purification
[0104] Recombinant wild-type PA (WT PA), PAF427H, mPA, mPA-EGF, and
mPA-DTR were overexpressed in the periplasm of the BL21 (DE3) E.
coli strain (Invitrogen, Carlsbad, Calif.). The resulting bacterial
pellets were lysed and purified as described (Miller et al., 1999).
Oligomeric prepores of WT PA and the various PA variants were
produced by limited trypsin digestion at a final trypsin:PA ratio
of 1:1000 (wt:wt) for 30 min at RT. The nicked proteins were
subjected to anion-exchange chromatography, resulting in the
separation of PA63 and PA20 fragments. PA63 spontaneously
oligomerized to form porepore.
[0105] Purified mPA-EGF and mPA-DTR fusions were characterized by
Western blot analysis. PA83 variants along with WT PA were
subjected to SDS-PAGE and transferred to a polyvinylidene
difluoride membrane (PVDF; Invitrogen, Carlsbad, Calif.). The
membranes were blocked with Tris-buffered saline, pH 7.4,
containing 2% BSA and hybridized with either mouse anti-PA (1:4000;
cat. no. MAB8082; Millipore, Billerica, Mass.), rabbit anti-EGF
(1:50000; cat. no. Ab9695; Abcam Cambridge, Mass.), or rabbit
anti-DT antibodies (1:20000; cat. no. Ab53828; Abcam). Primary
antibodies were detected with either goat anti-rabbit IgG (1:20000;
Santa Cruz Biotechnology, Inc, Santa Cruz, Calif., cat. no.
sc-2004) or rabbit anti-mouse IgG conjugated to HRP (1:10000; Santa
Cruz, cat. no. sc-358914) with enhanced chemiluminescence (ECL)
reagents (Pierce, Rockford, Ill.).
[0106] LF, EF, DTR, and LF.sub.N-DTA were expressed in BL21 (DE3)
E. coli (Invitrogen), under induction with 1 mM isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) for 4 hours, using the
Champion pet-SUMO expression system (Invitrogen). Cell pellets were
lysed by sonication in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM
NaCl, 10 mM imidazole, 10 mg lysozyme, 2 mg DNAase I, supplemented
with a complete Roche protease inhibitor tablet). Following
sonication, the lysates were cleared by centrifugation and loaded
onto a 3 ml bed volume of Ni-NTA agarose (Qiagen, Valencia,
Calif.). The resin was washed with 15 column volumes of wash buffer
(20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole) and eluted
with the same buffer supplemented with 250 mM imidazole. The
resulting purified protein was exchanged into 20 mM Tris-HCl, pH
8.0, 150 mM NaCl, and cleaved with SUMO protease (Invitrogen)
overnight at 4.degree. C. Uncleaved His-SUMO fusion and SUMO
protease were removed by a second round of Ni-NTA chromatography,
in which the flow-thru contained the cleaved product of
interest.
Example 3
SDS Resistance
[0107] Exposure to acidic pH causes the structural transformation
from PA prepore to pore, which is marked by the presence of
SDS-resistant oligomers. WT PA, mPA, mPA-EGF, and mPA-DTR prepores
(5 .mu.g) were incubated in pH 5.5 buffer (100 mM KCl, 1 mM EDTA,
and 10 mM each sodium oxalate, potassium phosphate, and MES, pH
5.5) or pH 8.5 buffer (20 mM Tris pH 8.5+150 nM NaCl) for 30 min at
room temperature. Each sample was then exposed to SDS sample buffer
and resolved by SDS-PAGE electrophoresis. Protein bands were
visualized by Coomassie blue staining.
Example 4
Cell Culture
[0108] The CHO-K1 cell line was from the American Type Culture
Collection (cat no. CCL-61, Manassas, Va.). Cells were maintained
in Ham's F-12 medium supplemented with 10% fetal bovine serum
(FBS), 500 units/ml penicillin G and 500 units/ml streptomycin
sulfate (Life Technologies, Inc., Carlsbad, Calif.). The A431 cell
line, also from the American Type Culture Collection (cat no.
CCL-1555) was grown in Dulbecco's Modified Eagle's Medium, with 10%
FBS, 500 units/ml penicillin G, 500 units/ml streptomycin sulfate,
and 1 mM sodium pyruvate (American Type Culture Collection).
Example 5
Cytotoxicity Assays
[0109] Protein synthesis inhibition was used to measure the ability
of WT PA and its derivatives to deliver LFN-DTA to the cytosol.
CHO-K1 and A431 cells (3.5.times.104 per well) were exposed to six
10-fold serial dilutions of LFN-DTA (starting with 10 nM) in
combination with one of the PA83 variants (10 nM). Cells were
either incubated for 4 hours (A431) or overnight (CHO-K1) at
37.degree. C. Toxin containing medium was removed and the cells
were incubated for 1 hr at 37.degree. C. with leucine-deficient
medium supplemented with 1 .mu.Ci of [3H]-leucine/ml (Perkin Elmer,
Billerica, Mass.). The plates were washed twice with cold PBS and
protein synthesis was measured by the amount of 3H-leucine protein,
as determined by scintillation counting. Percent protein synthesis
was plotted versus the log concentration of LFN-DTA where each bar
represents the average of three experiments.
[0110] Competition experiments were performed as described above
but with a 50-fold molar excess of soluble EGF (Prospec, East
Brunswick, N.J.) or 10-fold excess of DTR to compete with mPA-EGF
and mPA-DTR, respectively. Control experiments were also performed
with a 10-fold excess of the PA-binding VWA domain of ANTRX2
(ANTHRX2), which was produced recombinantly as described (Scobie et
al., 2005).
Example 6
MEK Cleavage
[0111] Translocation of LF to the cytosol of A431 cells was
monitored by Western blot against cell lysates for
mitogen-activated protein kinase kinase 1 (MEK1). A431 cells
(1.times.106 cells) were exposed to lethal toxin (10 nM PA83
variant and 100 nM LF) for 3 hr at 37.degree. C. Cells were
harvested in 100 .mu.l of Tris-buffered saline (20 mM Tris-HCl, 150
mM NaCl pH 7.4), suspended in SDS-PAGE sample buffer and
immediately incubated at 100.degree. C. for 20 min. The lysates
were resolved by SDS-PAGE and transferred to a PVDF membrane
(Invitrogen). The membranes were blocked with Tris-buffered saline,
pH 7.4, containing 2% BSA and hybridized with either anti-MEK1
(1:1000; Abcam, cat. No. Ab32071) or anti-GAPDH (1:2500; Abcam cat.
No. Ab9485) antibodies. Primary antibodies were detected with goat
anti-rabbit IgG conjugated to HRP (1:20000; Santa Cruz, cat. no.
sc-2004) and ECL reagents (Pierce).
Example 7
Edema Factor Adenylate Cyclase Assay
[0112] A competition enzyme-linked immunoassay (Cell Signaling
Technology, Danvers, Mass.) was used to determine the amount of
cAMP generated in A431 cells upon exposure to EF. A431 cells
(3.5.times.10.sup.4) were plated in a 96-well tissue culture plate
and incubated with EF (50 nM) in the presence or absence of a PA
variant (10 nM of WT PA, PAF427H, mPA, or mPA-EGF). After one hour
the medium was removed and cells were washed twice with 200 .mu.l
of ice-cold PBS. Adherent cells were lysed with 100 .mu.l 1.times.
cell lysis buffer and incubated on ice for 10 min. Each cell lysis
supernatant (50 .mu.l) was combined with HRP-linked cAMP solution
(50 .mu.l), added to the cAMP assay plate, and incubated at room
temperature for 3 hr. Wells were then washed four times with 200
.mu.l of 1.times. wash buffer, and TMB substrate (100 .mu.l) was
added to each and let stand for 10 min. Following the addition of
STOP solution (100 .mu.l) the absorbance of each well was read at
450 nm and used to estimate cAMP based a standard curve. The amount
of intracellular cAMP produced by EF+/-each PA variant was plotted
as a histogram where each bar represents the average of four
experiments.
Example 8
Targeting HER2-Positive Cancer Cells with Receptor Re-Directed
Anthrax Protective Antigen
[0113] We created a targeted toxin in which the receptor-binding
and pore-forming moiety of anthrax toxin, termed Protective Antigen
(PA), was modified to redirect its receptor specificity to HER2, a
marker expressed at the surface of a significant fraction of breast
and ovarian tumors. The resulting fusion protein (mPA-ZHER2)
delivered cytocidal effectors specifically into HER2-positive tumor
cells, including a trastuzumab-resistant line, causing death of the
cells. No off-target killing of HER2-negative cells was observed,
either with homogeneous populations or with mixtures of
HER2-positive and HER2-negative cells. A mixture of mPA variants
targeting different receptors mediated killing of cells bearing
either receptor, without affecting cells devoid of these receptors.
Anthrax toxin may serve as an effective platform for developing
therapeutics to ablate cells bearing HER2 or other tumor-specific
cell-surface markers.
[0114] Amplification and/or overexpression of the HER2 gene at the
mRNA or protein level occurs in 20-25% of breast, gastric, and
ovarian carcinomas (Berchuck et al. 1990; Gravalos & Jimeno
2008; Arteaga et al. 2012; Slamon et al. 1989). Particularly in
breast cancer, increased expression of HER2 is associated with an
aggressive form of the disease, which shows signs of increased
tumor growth, recurrence, and resistance to therapy, all
contributing to decreased patient survival (Arteaga et al. 2012).
Although the FDA-approved monoclonal antibody, trastuzumab (trade
name, HERCEPTIN.RTM.), is effective at slowing tumor growth, it
remains ineffective at tumor elimination. New therapeutics that
actively kill tumor cells thus remain a major goal of
cancer-related research. A promising example of this strategy is to
target the action of cytocidal protein toxins to specific cancer
cells (Pastan et al. 2007).
[0115] We developed a straightforward way to redirect the receptor
specificity of anthrax toxin (Mechaly et al. 2012). First we
ablated the native receptor-binding activity of protective antigen
(PA), the receptor-binding/pore-forming component of anthrax toxin,
and then appended a heterologous, receptor-binding ligand to the C
terminus of the mutated protein (mPA). Using this approach we
created fusion proteins that direct toxin action specifically to
two different receptors: the diphtheria toxin (DT) receptor
(HB-EGF) and the epidermal growth factor receptor (EGFR) (Mechaly
et al. 2012). In the current study we used this approach to
redirect toxin action to cells bearing the HER2 receptor.
[0116] Anthrax toxin is an ensemble of three nontoxic, monomeric
proteins (Young & Collier 2007). Two of them, the Lethal Factor
and the Edema Factor (LF and EF), are enzymatic "effector
proteins," which covalently modify molecular targets within the
cytosol. LF is a metalloprotease, which inactivates most members of
the mitogen-activated protein kinase kinase (MEK) family (Duesbery
et al. 1998; Vitale et al. 1998), and EF is a calmodulin- and
Ca2+-dependent adenylate cyclase, which increases the intracellular
concentration of cyclic AMP (Leppla 1982). The third protein, PA,
transports LF and EF from the extracellular milieu to the cytosol
by a process that begins with its binding to specific cell-surface
receptors and culminates in its forming pores in the endosomal
membrane (Collier 2009).
[0117] After binding to either of its two known receptors--ANTXR1
(also called TEM8) and ANTXR2 (also called CMG2) (Scobie 2003;
Bradley et al. 2001)--PA is proteolytically activated by a
furin-family protease (Klimpel et al. 1992). The activated form
self-assembles into heptameric (Milne et al. 1994) or octameric
(Kintzer et al. 2009) ring-shaped oligomers (pore precursors, or
"prepores"), which bind effector proteins with high (nM) affinity
(Cunningham et al. 2002; Mogridge et al. 2002). The resulting
complexes are endocytosed and delivered to the endosomal
compartment, where the acidic pH causes a conformation change in
the prepores that enables them to form pores in the endosomal
membrane (Miller et al. 1999). The pores, in turn, actively unfold
the bound effector proteins and transport them across the membrane
to the cytosol (Young & Collier 2007). There they refold into
active enzymes and modify their cytosolic substrates, causing major
perturbations of cellular processes and, in some cases, cell
death.
[0118] HER2 is a receptor tyrosine kinase belonging to the same
family as EGFR. Unlike EGFR, however, HER2 has no known natural
ligand. In the present study we developed a redirected binary toxin
by fusing a high affinity Affibody specific for the HER2 receptor
(Z.sub.HER2:342) (Orlova et al. 2006) to the C terminus of receptor
recognition-deficient PA (mPA), creating the fusion mPA-ZHER2.
Affibodies represent a class of targeting polypeptides derived from
the Z domain of Staphylococcus aureus protein A. Advantages over
other receptor-targeting ligands derive from the fact that
Affibodies are small (58 amino acids; .about.6 kDa), pH- and
thermo-stable, lack Cys residues, and fold independently and
reversibly (Nord et al. 1997; Lofblom et al. 2010). Further, they
may be rapidly evolved in vitro by phage-display technologies to
affinity levels comparable to those observed with monoclonal
antibodies.
[0119] Our results show that mPA with the Z.sub.HER2:342 affibody
fused to the C terminus can direct the action of either of two
cytocidal effector proteins to HER2-positive tumor cells. These
cells, including a HER2-positive trastuzumab-resistant tumor cell
line, were ablated, and specific killing was observed regardless of
whether the cultures consisted of a homogeneous population or had
been mixed with cells lacking the HER2 marker.
Material and Methods
Reagents and Chemicals
[0120] Oligonucleotides and the Z.sub.HER2:342 gene were
synthesized by Integrated DNA Technologies (Coralville, Iowa). The
Z.sub.HER2:4 and Z.sub.HER2:342 expression plasmids were kindly
provided by Dr. Gregory Poon (Washington State University, Pullman,
Wash.). All chemicals were purchased from Sigma-Aldrich (St. Louis,
Mo.), unless noted otherwise.
Generation of LFN-RTA Expression Plasmid
[0121] The A chain of ricin (RTA) was fused to the C terminus of
the N terminal PA-binding domain of LF (LF.sub.N) by overlap
extension PCR and cloned into the pet-SUMO expression vector
(Invitrogen, Carlsbad, Calif.). The first PCR step consisted of two
reactions (i) using a forward primer for LF.sub.N
(LFNFOR--GCGGGCGGTCATGGTGATGTAGGT, SEQ ID NO: 13) and a reverse
primer for LF.sub.N containing a GS spacer (in bold) and an overlap
sequence for RTA (underlined)
(LF.sub.N-RTA.sup.REV--AATTGGGTATTGTTTGGGGAATATACTACCCCGTTGATCTTGAAGTTCTT-
CCAA, SEQ ID NO: 14), and (ii) using a forward primer for RTA with
a GS spacer (bold) and a 5' overlap region with LFN (underlined)
(LF.sub.N-RTA.sup.FOR--TTGGAAGAACTTAAAGATCAACGGGGTAGTATATTCCCCAAACAATACCC-
AATT, SEQ ID NO: 15) and a reverse primer for RTA encoding a double
stop codon (in bold) (RTA.sup.REV--CTATTAAAACTGTGACGATGGTGGAGGTGC,
SEQ ID NO: 16). A final PCR reaction using the two previous
templates was performed with primers LF.sub.N.sup.FOR and
RTA.sup.REV to combine the two PCR products, which was subsequently
ligated into the pet-SUMO expression vector (Invitrogen).
Protein Expression and Purification
[0122] Recombinant WT PA, mPA, mPA-ZHER2, and mPA-EGF were
expressed and purified as described (Miller et al. 1999; Mechaly et
al. 2012). Recombinant LF.sub.N-DTA and LF.sub.N-RTA were expressed
as hexahistidine-SUMO fusions ("hexahistidine" disclosed as SEQ ID
NO: 17) for 4 hours at 30.degree. C. under the induction of 1 mM
Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) in the BL21 (DE3)
Star strain of E. coli (Invitrogen). Cell pellets were suspended in
100 ml of lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM
imidazole, 10 mg lysozyme, 2 mg DNAse I, supplemented with a Roche
complete protease inhibitor tablet per 50 ml) and lysed by
sonication. Cell lysates were loaded onto a Ni2+-NTA agarose
column, washed with 100 ml of wash buffer (20 mM Tris-HCl pH 8.0,
150 mM NaCl, and 20 mM imidazole), and eluted with wash buffer
supplemented with 250 mM imidazole. The resulting purified protein
was exchanged into 20 mM Tris-HCl pH 8.0 and 150 mM NaCl and
cleaved with SUMO protease overnight at 4.degree. C. to separate
the LFN-DTA/RTA from the His6-SUMO protein ("His6" disclosed as SEQ
ID NO: 17). Cleaved proteins were then subjected to a second
Ni2+-NTA column to bind His6-SUMO ("His6" disclosed as SEQ ID NO:
17), leaving the protein of interest (LFN-DTA/RTA) in the flow-thru
fraction.
[0123] Affibodies (Z.sub.HER2:4 and Z.sub.HER2:342) were expressed
from the pet15b expression vector (EMD Millipore, Billerica, Mass.)
and purified in the same manner as LF.sub.N-DTA, without the need
for a cleavage step.
Cell Lines and Maintenance
[0124] The A431 (cat no. CCL-1555) and CHO-K1 (cat. no. CCL-61)
cell lines were purchased from ATCC (Manassas, Va.). BT-474,
MDA-MB-468, and SKBR3 cell lines were generously provided by Dr.
Jean Zhao (Dana Farber Cancer Institute, Boston, Mass.). The
MDA-MB-231 cell line was provided by Dr. Gregory Poon (Washington
State University). The JIMT-1 cell line was purchased from AddexBio
(cat. no. C0006005; San Diego, Calif.).
[0125] A431 and JIMT-1 cells were maintained in DMEM supplemented
with 10% FCS, 500 units/ml penicillin G and 500 units/ml
streptomycin sulfate (Invitrogen). CHO-K1 and all other cell lines
were grown in Ham's F12 or RPMI medium (Invitrogen), respectively,
supplemented with 10% FCS, 500 units/ml penicillin G and 500
units/ml streptomycin sulfate.
[0126] Stable cell lines expressing fluorescent proteins were
produced by puromycin-selectable lentiviral particles coding for
CFP, RFP, or GFP (GenTarget, San Diego, Calif.). Lentiviruses were
transduced (MOI=1) into A431 (CFP), SKBR3 (RFP), and MDA-MB-468
(GFP) cell lines. At 48 hours post-transduction, the medium was
replaced with medium containing 1 .mu.g/ml puromycin to select for
fluorescent cells that were puromycin resistant. Cells were
passaged three more times in medium containing 1-5 .mu.g/ml
puromycin and analyzed by fluorescence-activated cell sorting
(FACS) to ensure a homogenous, fluorescently-labeled population of
cells were selected.
Quantifying Surface HER2 and EGF Receptor Levels
[0127] Cells (1.times.10.sup.5/experiment) were dissociated using a
non-enzymatic reagent (Cellstripper.TM., Cellgro, Herndon, Va.) to
eliminate the potential for receptor cleavage. Cells were
re-suspended in either 200 .mu.l of PBS or PBS with 1 .mu.g/ml
FITC-labeled anti-EGFR (cat. no. ab81872; Abcam, Cambridge, Mass.)
or 2 .mu.g/ml FITC-labeled anti-HER2 (cat. no. ab31891; Abcam)
affibodies. Cells were incubated for 1-hour at 4.degree. C., washed
twice with 200 .mu.l of PBS, and re-suspended in PBS. FACS was
performed using a BD FACSCalibur flow cytometer. FACS histograms
were analyzed using the FlowJo flow cytometry analysis software
(Tree Star Inc., Ashland, Oreg.), while mean fluorescence intensity
(MFI) was plotted using the GRAPHPAD PRISM.RTM. software package
(GraphPad software Inc., La Jolla, Calif.). Each plot corresponds
to three experiments where 50,000 events/condition were
counted.
Cytotoxicity and Competition Assays
[0128] 2.6.1 Protein synthesis inhibition--Cells were plated in
appropriate medium at densities of 2.5.times.10.sup.4 (BT-474) or
3.5.times.10.sup.4 cells/well (all other cell lines) in 96 well
plates and incubated overnight at 37.degree. C. The following day,
cells were exposed to ten 10-fold serial dilutions of LF.sub.N-DTA
or LF.sub.N-RTA (starting with a final concentration of 1 .mu.M) in
medium supplemented with 20 nM mPA variant. After a 4-hour
incubation, toxin-containing medium was removed and replaced with
leucine-deficient medium supplemented with 1 .mu.Ci of
[.sup.3H]-leucine/ml (Perkin Elmer, Billerica, Mass.) and incubated
for an additional hour. Plates were washed twice with cold PBS (200
.mu.l) prior to the addition of 200 .mu.l of scintillation fluid.
The amount of [.sup.3H]-leucine incorporated was determined by
scintillation counting using a Wallac MicroBeta TriLux 1450 LSC
(PerkinElmer, Waltham, Mass.). Percent protein synthesis was
normalized against cells treated with the mPA variant alone and was
plotted versus the concentration of LF.sub.N-DTA or LF.sub.N-RTA in
GraphPad Prism, where each point on the curve corresponds to the
average of four experiments.
[0129] Competition assays were performed as described above with
increasing concentrations of free i) high-affinity (Z.sub.HER2:342)
or (ii) lower-affinity (Z.sub.HER2:4) affibody added to medium
containing 20 nM mPA-ZHER2 and LFN-DTA. MDA-MB-231 cells which
express low levels of HER2 had to be challenged with a higher
concentration of LF.sub.N-DTA (1 .mu.M), compared to all other cell
lines (10 nM). Percent protein synthesis was normalized against
cells treated with mPA-ZHER2 alone and plotted using GRAPHPAD
PRISM, where each point on the curve corresponds to the average of
four experiments.
[0130] Cell viability--Cell viability was measured by an XTT assay,
following the manufacturers protocol (Biotium, Hayward, Calif.).
Cells (10.sup.4/well) were plated in the appropriate medium in 96
well optical bottom plates, incubated overnight at 37.degree. C.,
and exposed to ten 10-fold serial dilutions of LF.sub.N-DTA in
medium supplemented with 20 nM mPA-ZHER2. After 48 or 72 h, 25
.mu.l of XTT (sodium
2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-t-
etrazolium inner salt) reagent was added to each well, and the
absorbance of reduced XTT was measured at 475 nm, using a
SpectraMax M2e microplate reader (Molecular Devices, Sunnyvale,
Calif.). Percent cell viability was normalized against cells
treated with mPA-ZHER2 alone and plotted in GRAPHPAD PRISM, versus
the concentration of LF.sub.N-DTA. Each data point corresponds to
the average of measurements performed in quadruplicate.
Apoptosis Assay
[0131] A cell-based apoptosis assay measuring the activation of
known apoptotic markers, caspase 3/7, was performed according the
supplier's protocol (Caspase-Glo 3/7 Assay; Promega, Madison,
Wis.). Cells (104/well) were seeded in 96 well optical bottom
plates and exposed to eight 10-fold serial dilutions of LFN-DTA in
medium supplemented with 20 nM mPA-ZHER2. After 24 or 48 h, a
proluminescent caspase 3/7 substrate was added to each well,
followed by incubation at room temperature for 30 min. Luminescence
resulting from substrate cleavage by caspase 3/7 was measured with
a Wallac MicroBeta TriLux 1450 LSC (PerkinElmer). Relative
luminescence was plotted versus the concentration of LF.sub.N-DTA
using GRAPHPAD PRISM, where each data point represents the average
of four independent measurements.
Microscopy
[0132] Fluorescent cells were mixed (2.times.10.sup.4 cells each)
as described above and grown on tissue culture treated coverslips
overnight at 37.degree. C. Coverslips were exposed to 10 nM
LF.sub.N-DTA and mPA, mPA-ZHER2, mPA-EGF, or mPA-ZHER2 and mPA-EGF
(20 nM each). After 24 hours, cells were washed twice with PBS,
fixed with 4% formaldehyde, and mounted on glass slides. Images
were taken with a Nikon Eclipse TE2000-U fluorescence inverted
microscope and analyzed using the MetaMorph software package
(Molecular Devices, Sunnyvale, Calif.).
Co-Culture Cytotoxicity Assay (Protein Synthesis)
[0133] Fluorescence--Fluorescent cell lines (A431.sup.CFP,
MDA-MB-468.sup.GFP, and SKBR3.sup.RFP) were mixed equally (10.sup.5
cells each) with unlabeled CHO-K1 cells, seeded into 6-well tissue
culture dishes in RPMI medium, and incubated overnight at
37.degree. C. The next day, cells were treated with 10 nM
LF.sub.N-DTA and either mPA, mPA-ZHER2, mPA-EGF, or mPA-ZHER2 and
mPA-EGF (20 nM each). Cells were incubated an additional 24 hours,
washed 2 times with PBS, and detached with trypsin. Cell
populations were washed again in PBS and sorted based on
fluorescence using a BD FACSCalibur flow cytometer (BD Biosciences,
San Jose, Calif.). Each bar on the graphs corresponds to three
experiments where at least 75,000 events were counted. FACS data
was analyzed using the FlowJo analysis software and plotted using
the GRAPHPAD PRISM.RTM. software package.
[0134] Protein synthesis--A panel of cancer cell lines and CHO-K1
cells were seeded (3.5.times.10.sup.4 cells/well) in partitioned
sections of a chambered tissue culture slide. After an overnight
incubation, the medium was removed, and the partitioning element
was discarded. The slides were washed twice with PBS and incubated
for 4 hours with RPMI containing 10 nM LFN-DTA with 20 nM of either
(i) mPA, (ii) mPA-ZHER2, (iii) mPA-EGF, or (iv) a mixture of both
mPA variants. Slides were removed from the toxin containing medium,
washed with 15 ml of PBS, and incubated for an additional hour in
leucine-deficient medium supplemented with 1 .mu.Ci of
[.sup.3H]-leucine/ml (Perkin Elmer). Slides were removed from the
medium, washed with 30 ml of PBS, and dried. Individual cell
populations were dissolved in 6 M Guanidine-HCl (75 .mu.l) and
added to scintillation fluid. The amount of [.sup.3H]-leucine
incorporated was determined by scintillation counting. The percent
of protein synthesis was normalized against cells treated with mPA
and LF.sub.N-DTA and plotted using the GRAPHPAD PRISM software
package.
Results
[0135] mPA-ZHER2 Mediates the Killing of HER2-Positive Cells
[0136] We fused a high-affinity, 58-residue Affibody,
Z.sub.HER2:342, to the C terminus of mPA, a mutated,
receptor-recognition-deficient form of PA. The resulting fusion
protein (mPA-ZHER2) was tested in combination with the LF.sub.N-DTA
effector protein for ability to kill cancer cell lines displaying
various levels of the HER2 receptor. Because LF and EF are not
cytocidal for most cells, we used LF.sub.N-DTA, a fusion of the
N-terminal PA-binding domain of LF (LF.sub.N) to the catalytic
domain of diphtheria toxin (DTA), as intracellular effector. DTA
ADP-ribosylates eukaryotic elongation factor 2 (eEF-2) in the
cytosol, blocking protein synthesis and causing cell death (Collier
& Cole 1969; Collier 1967; Honjo et al. 1968).
[0137] Various cell lines were incubated 4 h with a constant
concentration of mPA-ZHER2 (20 nM) plus various concentrations of
LFN-DTA, after which protein synthesis over a 1-h period was
measured. The BT-474 cell line, which expressed the highest level
of HER2 among the cell lines tested, was also the most sensitive;
that is, it required the lowest concentration (EC50) of
LF.sub.N-DTA for 50% inhibition of protein synthesis (FIG. 7A). Two
cell lines expressing moderate levels of HER2 (SKBR-3 and A431)
showed intermediate levels of sensitivity; a line with a low level
of HER2 (MDA-MB-231) showed low sensitivity (EC.sub.50.about.10
nM); and two lines with no detectable HER2 (CHO-K1, MDA-MB-468)
were unaffected, even at the highest concentrations of LFN-DTA
tested. Thus, EC.sub.50 was inversely related to the level of HER2
on the cell surface (FIG. 7B). Levels of HER2 on the various cell
lines were determined by FACS analysis after incubation with a
fluorescently labeled anti-HER2 Affibody.
[0138] Cell viability confirmed that inhibition of protein
synthesis by LF.sub.N-DTA caused cell death. Cancer cell lines were
exposed to mPA-ZHER2 (20 nM) and LF.sub.N-DTA, at the indicated
concentrations. After 48 h, cell viability was quantified by a
cytotoxicity assay that quantifies the reduction of XTT reagent by
mitochondrial enzymes that are active in live cells. Protein
synthesis inhibition and cell death directly correlated (compare
FIGS. 7A and 7C), with comparable EC50 values that reflect the
amount of HER2 present on the cell surface (Table 1; FIG. 7B).
Activation of known apoptotic markers, caspase 3/7, confirmed that
cell death resulted from apoptosis (FIG. 7D). Caspase 3/7
activation did not increase after 24 h (data not shown) and was
dose-dependent; cells expressing higher amounts of HER2 receptor
showed caspase 3/7 activation at a lower LF.sub.N-DTA
concentration. The level of caspase 3/7 activation differed among
various cell types and could not be confirmed for the SKBR3 cell
line.
[0139] Free Z.sub.HER2:342 affibody competitively inhibited
mPA-ZHER2-dependent killing of HER2-positive cells (FIG. 8). BT474
cells expressing high levels of HER2 required a higher level of
free Affibody (EC.sub.50.about.400 nM) for toxin blockage relative
to cell lines expressing low or moderate levels of HER2
(EC.sub.50.about.20 nM) (FIG. 8A). A lower-affinity Affibody
(Z.sub.HER2:4) Wikman et al. 2004) was less effective in blocking
toxin action than the higher-affinity Z.sub.HER2:342 Affibody (FIG.
8B).
[0140] Bafilomycin A1 protected A431 cells from
LF.sub.N-DTA-dependent killing mediated by either mPA-ZHER2 or
mPA-EGF, indicating that translocation of effectors by mPA variants
was dependent on the endosomal pH, as is the case with wild-type
PA.
mPA-ZHER2 can Deliver Multiple Cytocidal Effectors
[0141] We tested an analog of LF.sub.N-DTA in which DTA was
replaced with the catalytic domain of ricin (RTA). RTA inhibits
protein synthesis by a different biochemical mechanism than DTA,
namely by depurinating a crucial adenosine residue in the 28S rRNA
of the eukaryotic ribosome (Endo & Tsurugi 1987). LF.sub.N-RTA
combined with mPA-ZHER2 (FIG. 3A) or mPA-EGF (FIG. 9B) killed
HER2-positive or EGFR-positive cells, respectively. Generally
LF.sub.N-RTA was 10-100 fold less efficient than LF.sub.N-DTA in
killing the cell lines tested. An exception was the SKBR-3 cell
line, in which the EC.sub.50 values for LF.sub.N-RTA or
LF.sub.N-DTA combined with mPA-ZHER2 were about the same (compare
FIG. 7A and FIG. 9A). Because SKBR-3 lacks detectable levels of EGF
receptor, it was resistant to mPA-EGF-mediated killing.
HER2-Targeted Anthrax Toxin Kills a Trastuzumab-Resistant Tumor
Cell Line
[0142] The FDA-approved monoclonal antibody trastuzumab has been
effective in prolonging HER2-positive patient survival, but not all
patients respond, and a large percentage develop therapeutic
resistance over time (Arteaga et al. 2012). The JIMT-1 cell line
recently isolated from a patient that had HER2 amplification and
clinically resistant to trastuzumab (Tanner et al. 2004). As in
other HER2-positive cell lines we tested, protein synthesis in
JIMT-1 cells was inhibited in response to mPA-ZHER2 and
LF.sub.N-DTA, resulting in cell death by apoptosis (FIG. 10). The
level of sensitivity was consistent with the HER2 level, and
killing mediated by LF.sub.N-RTA was less efficient than by
LF.sub.N-DTA (FIG. 10A). JIMT-1 cells required a longer duration of
toxin exposure (additional 24 h) to achieve similar cell killing
and caspase 3/7 activation, compared to other HER2-positive cell
lines (FIGS. 10C and 10D).
No Bystander Effect was Seen in Mixtures of HER2-Positive and
HER2-Negative Cells
[0143] To test for a possible bystander effect, we evaluated the
specificity of mPA-ZHER2 in mixtures of HER2-positive and
HER2-negative cells. First, to allow individual cell types to be
distinguished by fluorescence microscopy or FACS, we labeled
selected cell lines by transduction with puromycin-selectable
lentiviruses encoding fluorescent proteins with distinguishable
emission properties. Equal numbers of cells from each of 4 cell
lines (2 HER2-positive and 2 HER2-negative lines) were mixed, and
the resulting mixture was incubated 24 h with mPA-ZHER2 plus
LF.sub.N-DTA. Flow cytometry revealed that the HER2-negative cells,
CHO-K1 (unlabeled) and MDA-MB-468.sup.GFP (green), now comprised
almost the entire population; the HER2-positive A431.sup.CFP (cyan)
and SKBR3.sup.RFP (red) cells had been reduced from .about.50% to
less than 5% of the total (FIG. 11A). Fluorescence microscopy of
adherent cells gave comparable results (FIG. 11B). Because the
small remaining population of SKBR-3 cells (.about.4%) appeared to
be dead by microscopy, we believe that flow cytometry may have
overestimated this population because of an inherently long
half-life of the fluorescent protein used for labeling. Thus, the
mPA-ZHER2/LF.sub.N-DTA combination was able to kill the
HER2-positive cells in a mixed cell population, with no evident
bystander effect on HER2-negative cells.
[0144] We also used another approach to test for bystander effects.
Multiple cell lines were grown in separate wells of a chambered
slide (FIG. 12). The partitioning element was removed from the
slide, and the slide, containing all cell lines, was then incubated
in medium containing mPA-ZHER2 and LF.sub.N-DTA. After a 4-hour
incubation, the slide was washed and transferred to medium
supplemented with [.sup.3H]-leucine. After a further 1-hour
incubation, the cells were washed, individual cell populations were
dissolved with 6M guanidine-HCl, and the incorporated radioactive
leucine was quantified by scintillation counting. FIG. 6 shows that
cells expressing high and moderate levels of HER2 were killed,
MDA-MB-231 cells with low HER2 expression maintained limited
resistance, and cells lacking HER2 (CHO-K1 and MDA-MB-468) were
unaffected.
Mixing mPA-ZHER2 and mPA-EGF Allowed Killing of Both HER2- and
EGFR-Positive Cells in a Heterogeneous Cell Population
[0145] Like mPA-HER2, mPA-EGF in combination with LF.sub.N-DTA was
able to kill cognate cell lines (EGFR-positive in this case) in
both homogeneous (FIG. 13A) and heterogeneous cell populations,
with no effects on cells lacking the EGF receptor (FIG. 13).
Killing a mixed population of fluorescent cells by mPA-EGF
presented the same caveats as those described for mPA-ZHER2, where
a small population of EGFR-positive cells (MDA-MB-468, colored
green) remained (FIGS. 13B and 13C). Once again, a more sensitive
assay measuring protein synthesis by incorporation of radioactive
leucine showed that EGFR-positive cells were killed, and cells with
very low or no EGFR expression survived (FIG. 13D).
[0146] We also tested the ability of a mixture of mPA-ZHER2 and
mPA-EGF to target specific receptor-bearing cells in a mixed
population of cancer cells. As shown in FIG. 14, the combination of
mPA-ZHER2 and mPA-EGF was able to kill both HER2-positive and
EGFR-positive cells in the presence of LF.sub.N-DTA, while CHO-K1
cells, which do not express either receptor, remained
unaffected.
Quantification of HER2 and EGFR Levels on Cell Lines
[0147] Cells (10.sup.5) were incubated with either a
FITC-conjugated HER2- or EGFR-specific Affibody and analyzed by
FACS. (Left panels). The mean fluorescence intensity for 50,000
events was calculated in FlowJo and plotted in the GRAPHPAD
PRISM.RTM. software package. Histograms (not shown) of the raw data
displayed the shift in fluorescence compared to unstained cells for
EGF and HER2 receptors in CHOK1, A431, BT474, MDA-MB-231,
MDA-MB-468, SKBR-3 cells.
Delivery of LFN-DTA Causes Cell Death by Apoptosis
[0148] Apoptosis was measured by caspase 3/7 activation, after
exposing various cell lines (MDA-MB-231, MDA-MB-468, and CHOK1) to
mPA-ZHER2 and LFN-DTA, at concentrations 10.sup.-15 M, 10.sup.-13
M, 10.sup.-11 M, 10.sup.-9 M, 10.sup.-7 M for 24 h. Relative light
units (RLU) generated by caspase 3/7 activation and cleavage of a
pre-luminescent substrate are plotted against LFN-DTA
concentration. Each data point represented the average of 4
experiments. Control cells exposed to mPA-ZHER2 alone. Data not
shown.
Entry of Effectors Mediated by Wild-Type and Redirected mPA
Variants is Dependent on Endosomal pH
[0149] A431 cells (3.times.10.sup.5) were exposed to LF.sub.N-DTA
(1 nM) and either mPA-ZHER2, mPA-EGF, or WT PA (20 nM) in the
absence or presence of bafilomycin A at concentrations 10.sup.-11
M, 10.sup.-10 M, 10.sup.-9 M, 10.sup.-8 M, 10.sup.-7 M. After
4-hours, cells were washed with PBS and incubated with medium
containing [.sup.3H]-leucine. After 1-hour, the level of protein
synthesis was measured by scintillation counting. Percent protein
synthesis was normalized against cells treated with the mPA variant
alone and plotted using the GRAPHPAD PRISM software package. Each
point on the curves represented the average of four experiments.
Data not shown. The level of cell surface HER2, EGFR, and ANTRX1/2
were quantified on A431 cells using either anti-HER2 or EGFR
affibodies or FITC-labeled PA. Data not shown.
DISCUSSION
[0150] Cell-surface markers on aggressive forms of certain cancers
have been an important focus of efforts to develop targeted
anticancer therapies. A prominent example is the monoclonal
anti-HER2 antibody trastuzumab, which is effective in slowing tumor
growth and prolonging patient survival (Vogel et al. 2002).
However, most patients develop resistance to this antibody over
time due to its ineffectiveness in eliminating tumors (Arteaga et
al. 2012). Antibody therapies have been combined with conventional
chemotherapy or radiation to circumvent such resistance, and
antibody-drug conjugates (ADC's), which kill cells through the
action of a linked cytotoxic small molecule compound ("payload"),
have recently emerged as an alternative mode of targeted therapy
(Carter & Senter 2008).
[0151] Modifying intracellularly acting toxins to direct their
actions to tumor cells represents an attractive approach to
targeted therapy, in part because the catalytic mode of action of
the effector moieties renders these toxins so potent. Replacing the
native receptor-binding domain of toxins such as DT or Pseudomonas
exotoxin A (ETA) with a heterologous receptor-binding protein has
been employed effectively to target the cytocidal actions of these
toxins (Pastan et al. 2007). This line of investigation has led to
a licensed treatment for cutaneous T-cell lymphomas, termed
denileukin diftitox (trade name, ONTAK.RTM.) (Foss 2000; Williams
et al. 1987), and other targeted protein toxins are currently under
investigation (Madhumathi J & Verma 2012). ONTAK is a fusion
protein created by replacing the receptor-binding domain of DT with
interleukin-2 (IL-2). The IL-2 moiety binds the fusion toxin to
high-affinity IL-2 receptors on tumor cells, and the catalytic
moiety of DT (DTA) is transported to the cytosol, where it blocks
protein synthesis and causes cell death (Collier & Cole 1969;
Collier 1967).
[0152] Elucidation of the structure and activities of anthrax toxin
in recent years has led to experiments to explore its potential as
a platform for developing anticancer chemotherapeutics. In one
study the furin site of PA was mutated to prevent activation of the
protein, and the native receptor binding activity of the modified
PA was exploited to inhibit vascular endothelial growth
factor-induced and basic fibroblast growth factor-induced
angiogenesis (Rogers et al. 2007). In other studies lethal factor
combined with native PA was found to induce apoptosis in human
melanoma cells, suggesting possible applications for this and other
cancers in which disease progression is due in part to constitutive
activation of MAPK signaling (Duesbery et al. 2001; Koo et al.
2002). One approach to targeting PA has been to replace its furin
cleavage site with a site selective for a different
protease--metalloproteinase or urokinase plasminogen
activator--that is overexpressed on the surface of cancer cells
(Abi-Habib et al. 2006; Liu et al. 2000).
[0153] In the current work we changed the receptor recognition
specificity of PA as an approach to using the protein as a vehicle
for introducing cytotoxic effectors specifically into HER2-positive
cells. mPA-ZHER2 proved to be a highly selective mediator of the
entry of LF.sub.N-DTA and LF.sub.N-RTA into HER2-positive cells.
The EC50 of LFN-DTA showed an inverse relationship to the level of
HER2 on the cell lines tested (FIG. 7). Why LFN-RTA was 10- to
100-fold less potent than LFN-DTA (FIG. 9) is unclear, but may be
related to differences in stability of the effectors in the
cytosol, the kinetics of inactivation of target molecules, or any
of a number of other factors.
[0154] The specificity of mPA-ZHER2 for cells bearing the HER2
receptor was shown by competition assays (FIG. 8) and by its
ability to target only HER2-positive tumor cells in a mixed cell
population (FIGS. 12 and 11). No off-target effects were observed
when HER2-negative cells were mixed with HER2-positive cells before
treatment with LF.sub.N-DTA plus mPA-ZHER2. The affinity of
monomeric ZHER2 Affibody for the HER2 marker rivals that of the
best antibodies (.about.20 pM) (Orlova et al. 2006), and the
natural oligomerization properties of mPA-ZHER2 presumably increase
the avidity of the interaction of the complex for the HER2 receptor
on cells. Once oligomerization takes place, the avidity for the
receptor would be such that effectively no dissociation of toxic
complexes from cells would occur.
[0155] The entry of the cytocidal effectors mediated by either
mPA-ZHER2 or mPA-EGF was pH-dependent, as is the case for wild-type
PA. The difference in the inhibitory concentration of BFA for the
different PA variants is likely due to their respective receptor
abundances', where EGFR>ANTXR1/2>HER2. Alternatively the pH
threshold of PA pore formation may vary for the three receptors, as
the pH threshold of WT PA bound to ANTXR1 is a full unit higher
than when it is bound to ANTXR2, which has higher affinity (Lacy et
al. 2004; Rainey et al. 2005).
[0156] A HER2-positive, trastuzumab-resistant tumor cell line
(JIMT-1) was also susceptible to toxin action (FIG. 10). The JIMT-1
cell line, isolated from a patient clinically-resistant to
trastuzumab, displays properties thought to be associated with the
development of HER2-targeted antibody resistance, including low
expression of HER2 (despite gene amplification), receptor masking
by other cell surface proteins (an event which can mask up to 80%
of the trastuzumab binding sites), low PTEN expression, activation
of the PIK3CA gene, and high expression of neuregulin-1 (NRG-1)
(Tanner et al. 2004; Nagy et al. 2005; Koninki et al. 2010). The
EC50 in relation to HER2 level was consistent with our data on
trastuzumab-sensitive HER2-positive cell lines. LF.sub.N-RTA was
also effective in killing JIMT-1 cells, but higher concentrations
than LF.sub.N-DTA were needed (FIG. 4A). The delivery of
LF.sub.N-DTA, into the cytosol of JIMT-1 cells, led to apoptotic
cell death, as assessed by an XTT cytotoxicity assay and caspase
3/7 activation (FIGS. 10C and 10D). The redirected toxin was able
to kill most cells (>75%) after 48 h and achieved almost
complete elimination (.about.95%) after 72 h (FIG. 10C). The
greater exposure required to achieve complete cell killing could
have resulted from any of a number of differences that increased
the time to reach caspase 3/7 activation (48 h versus 24 h; FIG.
10D).
[0157] The elimination of a trastuzumab-resistant cell line by
anthrax toxin represents a potential advantage over current
antibody therapies. Some ADC's have been shown to kill
trastuzumab-resistant tumor cell lines (such as JIMT-1), but are
significantly less effective than LF.sub.N-DTA plus mPA-ZHER2, and
require high doses (.mu.g/ml versus pg/ml) to achieve moderate
killing (.about.25% cell death) (Lewis Phillips et al. 2008;
Koninki et al. 2010). This difference in potency (.about.5000 fold)
could result from efficient delivery of a cytocidal enzyme into the
cytosol, reflecting the strength of the interaction between
mPA-ZHER2 and the HER2 receptor, as well as the catalytic
inactivation of the cytosolic substrate. The accessibility of the
mPA-ZHER2 binding site on the surface of JIMT-1 cells compared to
the antibody binding site, estimated to be 20% available, could
also be a factor (Nagy et al. 2005).
[0158] Because tumors are composed of a heterogeneous population of
cells that have different receptor expression levels, it is
unlikely that any single anti-cancer therapy can achieve complete
tumor elimination. Combinations of small molecules, antibodies, and
radiation have been used with some success. The binary nature of
anthrax toxin and the ability of mPA to oligomerize also suggests
that one may be able to combine mPA-ZHER2 with other forms of mPA
targeted to different overexpressed surface tumor markers to
eliminate heterogeneous populations of cells. Consistent with this
notion, as mPA-ZHER2 and mPA-EGF, in combination with LF.sub.N-DTA
completely eliminated a panel of tumor cells with different HER2
and EGF receptor expression levels (FIG. 14).
[0159] The ability of mPA-ZHER2 to act cooperatively with an
analogous mPA-variant targeting a different tumor marker highlights
the adaptability of targeting with mPA. In addition to combining
mPA variants, the ability of the PA pore to translocate any of a
variety of intracellular effector enzymes allows the possibility of
using combinations of effectors that kill by different biochemical
mechanisms. The enzymatic destruction of targeted cells from within
by multiple effectors should minimize the likelihood of resistant
escape mutants arising, a universal problem in chemotherapy.
[0160] Our in vitro data indicate that the targeting of the HER2
receptor by modified, receptor-targeted anthrax toxin is specific
and potent, and displays no off-target toxicity towards
HER2-negative cell lines. The susceptibility of a HER2-positive
trastuzumab-resistant tumor cell line to toxin action highlights a
significant potential advantage of our system over current
FDA-approved antibody therapies. For these reasons and the
advantages described above, the PA-based targeting of distinct
populations of cancer cells represents a promising therapeutic
strategy for cancer treatment.
[0161] Table 1 below shows in vitro activity of mPA-ZHER2 and
LF.sub.N-DTA against various cancer cell lines:
TABLE-US-00006 Cell line EC.sub.50 (M) Assay BT-474 JIMT-1 SKBR-3
A431 MDA-MB-231 MDA-MB-468 CHO-K1 Protein Synthesis 9.4 .times.
10.sup.-14 3.0 .times. 10.sup.-12 1.3 .times. 10.sup.-11 1.5
.times. 10.sup.-11 7.0 .times. 10.sup.-9 >1 .times. 10.sup.-6
>1 .times. 10.sup.-6 Inhibition.sup.a Cell Viability.sup.b 8.0
.times. 10.sup.-14 2.5 .times. 10.sup.-12 1.6 .times. 10.sup.-12
4.1 .times. 10.sup.-11 1.3 .times. 10.sup.-9 >1 .times.
10.sup.-6 >1 .times. 10.sup.-6 Apoptosis.sup.c 7.2 .times.
10.sup.-13 5.1 .times. 10.sup.-11 ND 1.6 .times. 10.sup.-11 1.1
.times. 10.sup.-9 >1 .times. 10.sup.-6 >1 .times. 10.sup.-6
.sup.aMeasured by [3H]-leucine incorporation after 4 hr toxin
exposure .sup.bMeasured by XTT cell viability assay after 48 hr
toxin exposure .sup.cMeasured by caspase 3/7 activation after 24 hr
toxin exposure
[0162] We have also shown that the system works with cytocidal
effector proteins, e.g., LF.sub.N fused to the catalytic domain of
ricin, and demonstrated the killing of a Herceptin resistant cell
line, JIMT-1, using the system as described. For example, killing
by an alternative cytocidal LFN-fusion (LFN fused to the catalytic
domain of ricin; LFN-RTA) is demonstrated in FIGS. 9A and B.
Killing of a Herceptin-resistant cell line is shown, e.g, in FIGS.
10A-D.
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Sequence CWU 1
1
1718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Pro Gly His Lys Thr Gln Pro 1 5
227DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2gatttagtaa ttcgaattca agtacgg 27332DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3cattcagagt cgctgtttgg ttgcgtttta tg 32432DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4gttttatgcc ccggagatcc tatctcatag cc 32532DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5cataaaacgc aaccaaacag cgactatgaa tg 32635DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6ggtggtgctc gagtcaacgg agctcccacc atttc 35732DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7ggctatgaga taggatctcc ggggcataaa ac 32836DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8gtggtggtgg tggtgctcga gtcagctttt gatttc 369764PRTBacillus
anthracis 9Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser
Thr Ile 1 5 10 15 Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln
Ala Glu Val Lys 20 25 30 Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu
Ser Ser Ser Gln Gly Leu 35 40 45 Leu Gly Tyr Tyr Phe Ser Asp Leu
Asn Phe Gln Ala Pro Met Val Val 50 55 60 Thr Ser Ser Thr Thr Gly
Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu 65 70 75 80 Asn Ile Pro Ser
Glu Asn Gln Tyr Phe Gln Ser Ala Ile Trp Ser Gly 85 90 95 Phe Ile
Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala Thr Ser Ala 100 105 110
Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val Ile Asn Lys 115
120 125 Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg Leu Tyr
Gln 130 135 140 Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys
Gly Leu Asp 145 150 155 160 Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn
Lys Lys Glu Val Ile Ser 165 170 175 Ser Asp Asn Leu Gln Leu Pro Glu
Leu Lys Gln Lys Ser Ser Asn Ser 180 185 190 Arg Lys Lys Arg Ser Thr
Ser Ala Gly Pro Thr Val Pro Asp Arg Asp 195 200 205 Asn Asp Gly Ile
Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp 210 215 220 Val Lys
Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His 225 230 235
240 Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser
245 250 255 Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr Gly
Arg Ile 260 265 270 Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu
Val Ala Ala Tyr 275 280 285 Pro Ile Val His Val Asp Met Glu Asn Ile
Ile Leu Ser Lys Asn Glu 290 295 300 Asp Gln Ser Thr Gln Asn Thr Asp
Ser Gln Thr Arg Thr Ile Ser Lys 305 310 315 320 Asn Thr Ser Thr Ser
Arg Thr His Thr Ser Glu Val His Gly Asn Ala 325 330 335 Glu Val His
Ala Ser Phe Phe Asp Ile Gly Gly Ser Val Ser Ala Gly 340 345 350 Phe
Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser Leu Ser 355 360
365 Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu Asn Thr Ala
370 375 380 Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr
Gly Thr 385 390 395 400 Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser
Leu Val Leu Gly Lys 405 410 415 Asn Gln Thr Leu Ala Thr Ile Lys Ala
Lys Glu Asn Gln Leu Ser Gln 420 425 430 Ile Leu Ala Pro Asn Asn Tyr
Tyr Pro Ser Lys Asn Leu Ala Pro Ile 435 440 445 Ala Leu Asn Ala Gln
Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn 450 455 460 Tyr Asn Gln
Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp 465 470 475 480
Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly 485
490 495 Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro
Gln 500 505 510 Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys
Asp Leu Asn 515 520 525 Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro
Ser Asp Pro Leu Glu 530 535 540 Thr Thr Lys Pro Asp Met Thr Leu Lys
Glu Ala Leu Lys Ile Ala Phe 545 550 555 560 Gly Phe Asn Glu Pro Asn
Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile 565 570 575 Thr Glu Phe Asp
Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys 580 585 590 Asn Gln
Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr Val Leu Asp 595 600 605
Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys Arg 610
615 620 Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser
Val 625 630 635 640 Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser
Thr Glu Gly Leu 645 650 655 Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys
Ile Leu Ser Gly Tyr Ile 660 665 670 Val Glu Ile Glu Asp Thr Glu Gly
Leu Lys Glu Val Ile Asn Asp Arg 675 680 685 Tyr Asp Met Leu Asn Ile
Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe 690 695 700 Ile Asp Phe Lys
Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn 705 710 715 720 Pro
Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr Ile 725 730
735 Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys
740 745 750 Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly 755 760
10735PRTBacillus anthracis 10Glu Val Lys Gln Glu Asn Arg Leu Leu
Asn Glu Ser Glu Ser Ser Ser 1 5 10 15 Gln Gly Leu Leu Gly Tyr Tyr
Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30 Met Val Val Thr Ser
Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45 Glu Leu Glu
Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60 Trp
Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala 65 70
75 80 Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu
Val 85 90 95 Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu
Lys Gly Arg 100 105 110 Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu
Asn Pro Thr Glu Lys 115 120 125 Gly Leu Asp Phe Lys Leu Tyr Trp Thr
Asp Ser Gln Asn Lys Lys Glu 130 135 140 Val Ile Ser Ser Asp Asn Leu
Gln Leu Pro Glu Leu Lys Gln Lys Ser 145 150 155 160 Ser Asn Ser Arg
Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175 Asp Arg
Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190
Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195
200 205 Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro
Glu 210 215 220 Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu
Lys Val Thr 225 230 235 240 Gly Arg Ile Asp Lys Asn Val Ser Pro Glu
Ala Arg His Pro Leu Val 245 250 255 Ala Ala Tyr Pro Ile Val His Val
Asp Met Glu Asn Ile Ile Leu Ser 260 265 270 Lys Asn Glu Asp Gln Ser
Thr Gln Asn Thr Asp Ser Gln Thr Arg Thr 275 280 285 Ile Ser Lys Asn
Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His 290 295 300 Gly Asn
Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val 305 310 315
320 Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His
325 330 335 Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met
Gly Leu 340 345 350 Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile
Arg Tyr Val Asn 355 360 365 Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu
Pro Thr Thr Ser Leu Val 370 375 380 Leu Gly Lys Asn Gln Thr Leu Ala
Thr Ile Lys Ala Lys Glu Asn Gln 385 390 395 400 Leu Ser Gln Ile Leu
Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415 Ala Pro Ile
Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425 430 Thr
Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435 440
445 Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe
450 455 460 Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser
Glu Val 465 470 475 480 Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile
Ile Phe Asn Gly Lys 485 490 495 Asp Leu Asn Leu Val Glu Arg Arg Ile
Ala Ala Val Asn Pro Ser Asp 500 505 510 Pro Leu Glu Thr Thr Lys Pro
Asp Met Thr Leu Lys Glu Ala Leu Lys 515 520 525 Ile Ala Phe Gly Phe
Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly 530 535 540 Lys Asp Ile
Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln 545 550 555 560
Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr 565
570 575 Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile
Arg 580 585 590 Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val
Gly Ala Asp 595 600 605 Glu Ser Val Val Lys Glu Ala His Arg Glu Val
Ile Asn Ser Ser Thr 610 615 620 Glu Gly Leu Leu Leu Asn Ile Asp Lys
Asp Ile Arg Lys Ile Leu Ser 625 630 635 640 Gly Tyr Ile Val Glu Ile
Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655 Asn Asp Arg Tyr
Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660 665 670 Lys Thr
Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr 675 680 685
Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu 690
695 700 Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn
Gly 705 710 715 720 Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr
Glu Ile Gly 725 730 735 1158PRTStaphylococcus aureus 11Val Asp Asn
Lys Phe Asn Lys Glu Met Arg Asn Ala Tyr Trp Glu Ile 1 5 10 15 Ala
Leu Leu Pro Asn Leu Asn Asn Gln Gln Lys Arg Ala Phe Ile Arg 20 25
30 Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55
1258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Val Asp Asn Lys Phe Asp Lys Glu Xaa Xaa Xaa
Ala Xaa Xaa Glu Ile 1 5 10 15 Xaa Xaa Leu Pro Asn Leu Asn Xaa Xaa
Gln Xaa Xaa Ala Phe Ile Xaa 20 25 30 Ser Leu Xaa Asp Asp Pro Ser
Gln Ser Ala Asp Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asp Asp
Ala Gln Ala Pro Lys 50 55 1324DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13gcgggcggtc atggtgatgt aggt
241454DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14aattgggtat tgtttgggga atatactacc ccgttgatct
tgaagttctt ccaa 541554DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 15ttggaagaac ttaaagatca
acggggtagt atattcccca aacaataccc aatt 541630DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16ctattaaaac tgtgacgatg gtggaggtgc 30176PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
17His His His His His His 1 5
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References