U.S. patent application number 13/241683 was filed with the patent office on 2012-01-19 for compositions for the treatment of cancer, and methods for testing and using the same.
This patent application is currently assigned to UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY. Invention is credited to Charles S. Kachlany.
Application Number | 20120016111 13/241683 |
Document ID | / |
Family ID | 40467119 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120016111 |
Kind Code |
A1 |
Kachlany; Charles S. |
January 19, 2012 |
Compositions for the treatment of cancer, and methods for testing
and using the same
Abstract
A composition comprising leukotoxin proteins isolated from a
bacterium is provided. In this composition, greater than 85% of the
leukotoxin proteins are chemically modified at a basic amino acid
residue, and the proteins induce cell death in myeloid leukocytes,
while remaining substantially non-toxic to lymphoid leukocytes,
lymphocytes, and red blood cells. Also provided is a method of
selectively inducing cell death in myeloid leukocytes. The method
comprises contacting the myeloid leukocytes with a composition
comprising leukotoxin proteins. These leukotoxin proteins may be
isolated from the NJ4500 strain of Actinobacillus
actinomycetemcomitans. A method of purifying leukotoxin protein
from the NJ4500 strain of Actinobacillus actinomycetemcomitans is
also provided, as well as an assay that allows for the rapid
determination of the activity of a given drug against leukemic
cells either taken from a patient or derived from a cell line. The
assay is performed in the presence of whole blood or serum.
Inventors: |
Kachlany; Charles S.;
(Bridgewater, NJ) |
Assignee: |
UNIVERSITY OF MEDICINE AND
DENTISTRY OF NEW JERSEY
SOMERSET
NJ
|
Family ID: |
40467119 |
Appl. No.: |
13/241683 |
Filed: |
September 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12154843 |
May 27, 2008 |
8053406 |
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13241683 |
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PCT/US2006/045258 |
Nov 25, 2006 |
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12154843 |
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12150038 |
Apr 23, 2008 |
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12154843 |
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60739537 |
Nov 25, 2005 |
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60925794 |
Apr 23, 2007 |
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Current U.S.
Class: |
530/402 ;
530/350 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/00 20130101; G01N 2800/52 20130101; C07K 14/285 20130101;
G01N 33/5011 20130101 |
Class at
Publication: |
530/402 ;
530/350 |
International
Class: |
C07K 14/195 20060101
C07K014/195 |
Goverment Interests
RELATED FEDERALLY SPONSORED RESEARCH
[0002] The work described in this application was sponsored at
least in part, under Grant No. R01 DE16133, from the National
Institute of Dental and Craniofacial Research, and under Grant No.
NIH R01DE16133, from the National Institutes of Health.
Accordingly, the Government has certain rights in the invention.
Claims
1. A composition comprising leukotoxin proteins isolated from a
bacterium, wherein greater than 85% of the leukotoxin proteins are
chemically modified at a basic amino acid residue.
2. The composition of claim 1 wherein the bacterium is
Actinobacillus actinomycetemcomitans.
3. The composition of claim 1 wherein the bacterium is a NJ4500
strain of Actinobacillus actinomycetemcomitans.
4. The composition of claim 1 wherein the proteins induce cell
death in myeloid leukocytes.
5. The composition of claim 1 wherein the protein induces cell
death in anyone of white blood cells, neutrophils, monocytes,
basophils, and eosinophils.
6. The composition of claim 1 that is substantially non-toxic to
lymphoid leukocytes.
7. The composition of claim 6 that is substantially non-toxic to
lymphocytes.
8. The composition of claim 1 that is substantially non-toxic to
red blood cells.
9. The composition of claim 1 wherein the chemical modification is
a fatty acid modification.
10. The composition of claim 1 wherein the basic amino acid residue
is a lysine.
11. The composition of claim 1 wherein greater than 90% of the
leukotoxin proteins are chemically modified at a basic amino acid
residue.
12. A composition comprising leukotoxin proteins, wherein 85% of
the leukotoxin proteins have a pI less than 9.0.
13. The composition of claim 12, wherein 90% of the leukotoxin
proteins have a pI less than 9.0.
14. The composition of claim 13, wherein 95% of the leukotoxin
proteins have a pI less than 9.0.
15. The composition of claim 14, wherein 100% of the leukotoxin
proteins have a pI less than 9.0
16. A composition comprising leukotoxin proteins, wherein 85% of
the leukotoxin proteins have a pI less than 8.5.
17. The composition of claim 16, wherein 90% of the leukotoxin
proteins have a pI less than 8.5.
18. The composition of claim 17, wherein 95% of the leukotoxin
proteins have a pI less than 8.5.
19. The composition of claim 18, wherein 100% of the leukotoxin
proteins have a pI less than 8.5.
20. The compositions of claim 12 wherein the leukotoxin proteins
are isolated from Actinobacillus actinomycetemcomitans.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation In Part of PCT
Application No. PCT/US2006/045258, filed Nov. 25, 2006, which, in
turn, claims priority under 35 U.S.C. .sctn.119(e) from U.S.
Provisional Application Ser. No. 60/739,537, filed Nov. 25, 2005;
and co-pending U.S. Non-Provisional application Ser. No.
12/150,038, filed Apr. 23, 2008, which, in turn, claims priority
under 35 U.S.C..sctn.119(e) from U.S. Provisional Application Ser.
No. 60/925,794, filed Apr. 25, 2007. The entire contents of all
prior listed applications are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The present invention is generally related to agents and
compositions that demonstrate the ability to treat certain cancers,
and to a method and system for the testing of such agents and
compositions under physiological conditions. More particularly, the
agents and compositions comprise a repeat in toxin (RTX) molecule
that demonstrates leukocyte specificity, and that specifically
targets myeloid leukocyte cells, as well as to an assay that allows
for the rapid determination of the activity of a given drug against
cancer cells, such as leukemic cells, either taken from a patient
or derived from a cell line.
BACKGROUND OF THE INVENTION
[0004] Bacteria and their toxins have been investigated for their
anticancer activities. In the 1970s, bacteria (such as
non-pathogenic Clostridium) were used for the treatment of
malignant brain tumors, but the tumors recurred in these brain
tumor patients. More than 100 microorganisms have been studied for
their potential anticancer activities, and many bacteria have
growth specificity for tumors that is 1000 times greater than for
other tissue.
[0005] While their anti-tumor activities make many bacteria
attractive therapeutic agents, there are inherent risks to
administering live bacteria to humans. A safer and more effective
strategy has been to use biological toxins, specifically from
bacteria, as therapeutic agents. Bacterial toxins are not only
toxic, but are also highly specific for certain cell types, or can
be engineered to be specific by fusing the toxin to other
molecules. Many bacterial toxins are able to enter mammalian cells
where they exert their toxic effects. Because of extensive
evolutionary adaptation between bacteria and their hosts, bacteria
have become very good at "developing" highly effective toxins.
[0006] Each year, more than 60,500 people die of hematologic
malignancies (leukemia, lymphoma, myeloma) with more than 110,000
new annual diagnoses in the US alone. Current treatment for these
cancers includes the use of synthetic compounds that target the
cell division process of nearly all cells of the body, not just the
cancerous ones. As a result, devastating side effects are all too
common. Furthermore, a significant percentage of patients
eventually show resistance to many of the drugs, thus rendering
treatment largely ineffective. Indeed, there is an effort to
identify agents that induce cancer cell death by methods other than
damage to DNA or cell division.
[0007] While the drugs currently in use are toxic for cells, they
are not highly specific. A new class of therapeutic agents for the
treatment of hematologic malignancies, and cancer in general,
includes drugs that exhibit specificity for predominantly the
cancerous cell type. Examples of targeted therapeutics include
Rituximab, which is a monoclonal antibody against B-lymphocytes,
and Mylotarg, an antibody-anti-tumor antibiotic fusion directed
against cells of myelomonocytic lineage.
[0008] Actinobacillus actinomycetemcomitans is a Gram negative
pathogen that inhabits the oral cavities of humans. A.
actinomycetemcomitans is the etiologic agent of localized
aggressive periodontitis (LAP), a rapidly progressing and
destructive disease of the gingiva and periodontal ligaments. Among
its many virulence factors, A. actinomycetemcomitans produces an
RTX (repeats in toxin) leukotoxin. A. actinomycetemcomitans
leukotoxin is an approximately 115 kDa protein that kills
specifically leukocytes of humans and Old World Primates.
Leukotoxin is part of the RTX family that includes E. coli
.alpha.-hemolysin (HlyA) and Bordetella pertussis adenylate cyclase
(CyaA). Leukotoxin may play an important role in A.
actinomycetemcomitans pathogenesis by helping the bacterium destroy
gingival crevice polymorphonuclear leukocytes (PMNs) and monocytes,
resulting in the suppression of local immune defenses.
[0009] The initial identification and testing of novel anti-cancer
agents relies on in vitro killing assays using relevant cancer cell
lines. While in vitro assays performed under cell culture
conditions prove useful and necessary for preclinical testing of
new therapeutics, extrapolation to the physiological conditions of
a living organism is often difficult or impossible (27). Because of
the high cost of drug development ($800 million), new drug screens
are constantly being sought to more efficiently eliminate or
identify candidate therapeutic agents (27). Indeed, increasing the
clinical success rate from 1/5 to 1/3 because of more effective
preclinical drug screens would reduce drug development costs by
more than $200 million (27).
[0010] The activity, specificity, or toxicity of a compound in the
physiological environment can vary significantly from cell culture
conditions. While no in vitro assay or screen can represent the
complexity of the human body, several assays have been developed to
more closely mimic in vivo conditions. Several of these assays
include the colony forming cell assay using bone marrow cells
(27,29), hepatic drug biotransformation assays (3), and assays in
whole blood (4,45). Because most chemotherapeutic agents are
administered intravenously and are therefore immediately affected
by blood cell components, screening for potential drugs in the
presence of whole blood would be expected to yield more meaningful
results. Blood contains biological components, such as proteases,
antibody, and blood cells, which can affect the nature of a
compound. For example, red blood cells and plasma proteins are
known to affect the pharmacokinetics of drugs such as the
anti-cancer agents docetaxel and gemcitabine (8,9). Vaidyanathan et
al. (43) also reported that the cardioprotective drug, dexrazoxane,
inhibits binding of the anti-cancer agent, doxorubicin, to red
blood cells and that this interaction alters the pharmacokinetics
of doxorubicin, and Clarke et al. (4) used an in vitro whole blood
assay to study the binding affinity of a surrogate anti-CD 11 a
monoclonal antibody to blood components. In addition, leukocytes
produce a cytochrome P450 isoform (CYP2E1) that is involved in drug
biotransformation (3). Thus, identifying and studying drugs in the
presence of whole blood or blood components can offer a unique
advantage over assays using cells in monoculture.
[0011] For studies on leukemia therapeutics, the cell line HL-60 is
used as a standard target cell line. HL-60 cells were originally
isolated from a 36-year-old female patient with acute promyelocytic
leukemia (13). Testing the efficacy of anti-leukemia therapeutics
against HL-60 cells in whole blood or other biological material is
currently a challenge due to the inefficiency in differentiating
the viability of HL-60 cells from other cells.
[0012] Thus, there remains a need for the identification and
development of therapeutic agents and strategies, for the treatment
of cancers such as leukemia, and for the development of effective
testing methodology, such as efficient screens for therapeutics
such as anti-leukemia agents, and particularly, for the
facilitation of preclinical studies on a highly specific bacterial
leukotoxin as a novel anti-leukemia therapeutic agent.
SUMMARY OF THE INVENTION
[0013] Accordingly, in a first embodiment of the invention, a
composition for the treatment cancers, and particularly,
hematologically related cancers, is disclosed that comprises
leukotoxin proteins isolated from a bacterium. In this composition,
greater than 85% of the leukotoxin proteins are chemically modified
at a basic amino acid residue, and the proteins induce cell death
in myeloid leukocytes, while remaining substantially non-toxic to
lymphoid leukocytes, lymphocytes, and red blood cells.
[0014] Also, in a method of treatment aspect, there is provided a
method of selectively inducing cell death in myeloid leukocytes.
The method comprises contacting the myeloid leukocytes with a
composition comprising leukotoxin proteins. These leukotoxin
proteins may be isolated from the NJ4500 strain of Actinobacillus
actinomycetemcomitans. A method of purifying leukotoxin protein
from the NJ4500 strain of Actinobacillus actinomycetemcomitans is
also provided.
[0015] Accordingly, in a second embodiment of the invention, a
stable bioluminescent HL-60 cell line whose viability can be
rapidly and effectively determined in the presence of whole blood
and live animals has now been developed along with an assay that
allows for the rapid determination of the activity of a given drug
against a cell sample, such as leukemic cells, either taken from a
patient or derived from a cell line. The assay is carried out in
the presence of whole blood or serum. This quantitative assay can
screen thousands of drugs at a time or multiple concentrations of a
drug in a 96- or 384-well format.
[0016] The present assay uses HL-60 cells that have been engineered
that stably express firefly luciferase and produce light, whereby
such bioluminescent HL-60luc cells may be rapidly detected in whole
blood, eg. with a sensitivity of approximately 1000 viable cells.
As demonstrated herein, treatment of HL-60luc cells with a
bacterial leukocyte-specific toxin or the drug chlorambucil reveals
that the bioluminescent viability assay is more sensitive than the
trypan blue dye exclusion assay. HL-60luc cells administered
intraperitoneally (i.p) or intravenously (i.v.) were visualized in
living mice using an in vivo imaging system (IVIS). The rapidity
and ease of detecting HL-60luc cells in biological fluid indicates
that this cell line can be used in high throughput screens for the
identification of drugs with anti-leukemia activity under
physiological conditions.
[0017] Accordingly, it is a principal object of the invention to
provide a series of modified leukotoxin proteins, and compositions
comprising them, that can be used for the treatment of cancers, ad
particularly, cancers that are hematologically related.
[0018] It is a further object of the invention to provide a method
for the preparation of the modified leukotoxin proteins by their
isolation and purification from a bacterium, eg. from the NJ4500
strain of Actinobacillus actinomycetemcomitans.
[0019] It is a yet further principal object of the invention to
provide a an assay and associated methodology, for the testing of
candidate therapeutic agents for the treatment of cancers, that is
rapid and efficient, and that can assess the activity of the
candidate agents under physiological conditions.
[0020] A still further object of this invention is to provide
methods of treating cancers and conditions by the administration of
the leukotoxin proteins and compositions comprising them.
[0021] Other important objects and features of the invention will
be apparent from the following description of the invention taken
in connection with the accompanying drawings in which:
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows fluorescence microscopy images of leukemia
HL-60 cells when exposed to LtxA.
[0023] FIG. 2 is a graph showing activity data of two forms of LtxA
against human red blood cells.
[0024] FIG. 3 is a series of images showing the effectiveness of
NJ4500 LtxA in vivo.
[0025] FIG. 4 a bar graph representation of data showing the
sensitivity of human red blood cells to the JP2 and NJ4500 forms of
LtxA.
[0026] FIG. 5 is a bar graph representing data on the toxicity of
the JP2 and NJ4500 forms of LtxA against HL-60 cells.
[0027] FIG. 6 is a graph of time vs. cell death for various cell
types in whole human blood during incubation with 0.2 .mu.g/ml
NJ4500 LtxA.
[0028] FIG. 7 is a two-dimensional gel electrophoresis of two forms
of LtxA.
[0029] FIGS. 8A and 8B show the construction of a stable
luciferase-expressing HL-60 cell line wherein (A) HL-60 cells were
transfected with pMP1 and then grown in wells with different
concentrations of geneticin. Bioluminescence was detected with the
IVIS 50 instrument and (B) Growth curves for parental HL-60 and
engineered HL-60luc cells. Cells were grown in RPMI as described
and viable cells were counted with the ViCELL cell counter.
[0030] FIGS. 9A-9C shows the detection of HL-60luc cells in whole
blood including (A) Kinetics of BL over time. HL-60luc cells were
mixed with blood and luciferin and then imaged with the IVIS 50
instrument at the indicated time points. The observed pattern was
highly reproducible. (B) Detection limit of HL-60luc cells. Cells
were mixed with blood and luciferin and then incubated for one hour
before imaging. (C) The number of HL-60luc cells shows a linear
correlation with BL.
[0031] FIG. 10 shows effects of LtxA on cells. (A) Lysis of human
red blood cells by LtxA from two different strains of A.
actinomycetemcomitans. (B) HL-60 and HL-60luc cells are equally
sensitive to killing by LtxA from strain NJ4500. Assays were
performed in RPMI medium and viability was determined using the
trypan blue dye exclusion assay.
[0032] FIGS. 11A-11C show the cytotoxicity of LtxA and
chlorambucil. (A) Activity of LtxA against HL60luc cells in whole
human blood and RPMI medium. Viability was measured using BL. (B)
Comparison between BL and trypan blue as viability assays for
LtxA-mediated cytotoxicity. Cells were incubated in RPMI medium
with LtxA or buffer for 4 hours and viability was determined. (C)
Comparison between BL and trypan blue as viability assays for
chlorambucil-mediated cytotoxicity. Cells were incubated in RPMI
medium with chlorambucil or buffer for 24 hours and viability was
determined
[0033] FIG. 12 shows bioluminescent imaging of HL-60luc cells in
living mice. Swiss-Webster mice were anesthesized with XXX and
injected with 106 HL-60luc cells intraperitoneally (i.p.; top) or
intravenously (i.v.; bottom) and followed by luciferin i.p. Mice
were imaged with the IVIS 50 instrument at different times post
luciferin injection. The scale on the right of each image indicates
surface radiance (photons/second/cm.sup.2/steradian).
DETAILED DESCRIPTION OF THE INVENTION
[0034] Leukotoxin is an effective cell-delivery protein, permeating
leukemia cells and penetrating to the inside of specific cells.
Leukotoxin mediated cell-delivery is demonstrated by introducing
fluorescing molecules to specific cells, and measuring
cell-delivery by monitoring the fluorescence by fluorescent
microscopy. As shown in FIG. 1, the leukotoxin LtxA facilitates
delivery of fluorescein into HL-60 leukemia cells. The leukotoxin
forms pores or disruptions in the host cell membranes, and these
openings in the membrane may allow the passage and entry of small
molecules. In FIG. 1, HL-60 cells were treated with fluorescein, a
reagent that can be easily tracked by fluorescence microscopy.
Fluorescein exhibits a green fluorescence color under the
microscope, and is approximately the same molecular weight as many
of the cancer drugs currently in use. The cells treated with
leukotoxin (LtxA) and fluorescein (FIG. 1, bottom panel) exhibited
more intense and abundant fluorescence than the cells treated with
fluorescein alone (FIG. 1, center panel), indicating that
leukotoxin is able to increase the number of fluorescein molecules
that enter the cells.
[0035] Not only is leukotoxin capable of penetrating cells, but
this penetration is toxic and lethal to HL-60 cells. HL-60 cells
were modified to express luciferase genes, and with this HL-60luc
system, it was shown that at certain concentrations, leukotoxin is
quite toxic to the HL-60luc cells. By monitoring the luminescence
of the cells, nearly 80% of the cells were killed by concentrations
of leukotoxin as low as 200 ng/ml.
[0036] Data reflecting the sensitivity of HL-60luc cells to
leukotoxin is shown in FIG. 2. The activity of purified leukotoxin
against HL-60luc cells in vitro is quantified. The leukotoxin used
in this experiemenbt was LtxA isolated from the NJ4500 strain of
Actinobacillus actinomycetemcomitans. The LtxA was mixed with
HL-60luc cells at various concentrations as indicated, and
incubated for two hours, and then imaged with the IVIS 50
instrument. Relative viability was calculated by quantifying the
number of photons produced in each well. Significant cell death was
observed after two hours for concentrations of 2.0 .mu.g/ml, 0.2
and 0.02 .mu.g/ml.
[0037] To determine if the leukotoxin LtxA has activity in vivo,
two Swiss Webster mice were injected i.p. with 10.sup.6 HL-60luc
cells. One of the mice was injected with 8 .mu.g of LtxA i.p.
immediately following HL-60luc cell injection. Both mice then
received an i.p. injection of luciferin substrate. The mice were
monitored by in vivo bioluminescence imaging with an IVIS 50
imaging system immediately following injection of the luciferin.
The luminescent signal was visible and intense in the control
mousee that did not receive the LtxA injection. In contrast, the
mouse that received LtxA showed essentially no luminescent signal,
showing that the LtxA had killed the HL-60luc cells in vivo.
[0038] Forms of LtxA include the JP2 form (isolated from the JP2
strain of Actinobacillus actinomycetemcomitans) and the NJ4500 form
(isolated from the NJ4500 strain of Actinobacillus
actinomycetemcomitans). NJ4500 LtxA is well tolerated by the Swiss
Webster mice. Two mice, weighing approximately 45 grams each, were
injected with 10 .mu.g of NJ4500 LtxA intravenously. These mice
were monitored over a five-month period, and during this period,
the mice remained healthy, did not lose weight, and had no apparent
adverse reaction to the LtxA.
[0039] The individual forms of LtxA show different cell
specificity. The NJ4500 and JP2 forms of LtxA demonstrate
specificity to different types of blood cells, as demonstrated in
FIG. 4. The JP2 form of LtxA is lethal to human red blood cells,
whereas human red blood cells are insensitive to the NJ4500 form of
LtxA. Thus, the NJ4500 form of LtxA is lethal to HL-60 cells (as
shown in FIG. 3), but innocuous to human red blood cells (as shown
in FIG. 4). The JP2 form is quite lethal to human red blood cells,
but as shown in FIG. 5, is less deadly to leukemia cells than the
NJ4500 form. The data displayed in FIG. 4 was collected by a trypan
blue dye excusion assay. Leukotoxin protein LtxA (2 .mu.g/ml) was
added to 1.times.10.sup.6 HL-60 cells, and were incubated for 90
minutes at 37.degree. C. The cells were measured for viability with
the trypan blue dye exclusion assay.
[0040] The activity of the JP2 and NJ4500 forms of LtxA against
HL-60 cells differs dramatically. In FIG. 5, a bar graph
representing the toxicity of the two forms of LtxA against HL-60
cells. As shown by the bar graph, the NJ4500 form of LtxA is much
more lethal to the leukemia cell line than the JP2 form of the
protein. Accordingly, not only is the NJ4500 form of LtxA
non-lethal to human red blood cells (unlike the JP2 strain of
LtxA), but the NJ4500 strain of LtxA is more lethal to leukemia
cells than the JP2 strain of LtxA, thus indicating that the NJ4500
form of LtxA is a desirable leukemia or blood disease treatment as
it is highly toxic to leukemia cells, but not to human red blood
cells. LtxA provides a highly specific approach to treat
hematologic malignancies, such as leukemia, lymphoma, and myeloma,
without damaging other blood cells, such as red blood cells.
[0041] The data displayed in FIG. 5 was collected using a trypan
blue exclusion assay. HL-60 cells were mixed with 2 .mu.g/ml final
concentration of LtxA from JP2 and NJ4500 as indicated and
incubated for 90 minutes at 37.degree. C. Equal amounts of LtxA
from either strain were mixed with approximately 5.times.10.sup.6
cells/ml of HL-60 cells and incubated for ninety minutes. Cell
death was then assayed using the trypan blue dye exclusion assay.
LtxA from NJ4500 was more effective at killing HL-60 cells than was
LtxA from JP2. The toxin from NJ4500 was approximately twice as
active and this result was highly reproducible for even different
preparations of LtxA over four different experiments.
[0042] The NJ4500 strain of LtxA is also active in whole human
blood. Whole human blood was mixed with LtxA (2.0 .mu.g/ml final
conc.) and incubated for 4 hours at 37.degree. C. The mixtures were
then mixed with red blood cell (RBC) lysis buffer (eBioscience) and
the RBCs were lysed according to the manufacturer's protocol. The
remaining white blood cells (WBCs) were then resuspended in PBS and
cells were counted using a ViCell counter (Beckman Coulter), which
employs the trypan blue dye assay to measure viability. The sample
that was not treated with LtxA had 93% viability, while the sample
that was treated with LtxA had a viability of 42%. Because the
RBC's were lysed and removed before viability was measured, the
viability measurement assesses only the viability of the remaining
white blood cells. The NJ4500 LtxA caused death in nearly 60% of
the white blood cells.
[0043] NJ4500 LtxA displays unique sensitivity among the blood
cells found in whole blood. At a concentration of 20 .mu.g/ml, LtxA
from NJ4500 showed a high level of specificity in inducing cell
death in human whole blood. After four hours incubation at 20
.mu.g/ml LtxA, the red blood cells, basophils, and lymphocytes
suffered no significant cell death. In contrast, the 60% of the
white blood cells were killed, and approximately 95% of the
neutrophils, monocytes, and eosinophils were killed by the
LtxA.
[0044] Similar specificity was shown over time at lower doses of
NJ4500 LtxA. FIG. 6 is a graph representing data that was collected
over time, and shows that NJ4500 LtxA specifically target certain
blood cell types. Neutrophil cells appear most sensitive to the
relatively low concentration of 0.2 .mu.g/ml LtxA with nearly 80%
of neutrophils killed in only one hour. Both basophils and white
blood cells (or leukocytes) were killed rapidly by the NJ4500 with
about 60% of the cells dying within an hour. Monocytes are quite
sensitive to NJ4500 LtxA, as nearly 100% of the monocytes died,
however significant amounts of cell death required longer
incubation periods. In contrast, red blood cells (erthrocytes) and
lymphcytes were completely insensitive to 0.2 .mu.g/ml NJ4500 LtxA
over time.
[0045] All histological data presented herein was collected through
histology examinations. The blood samples were smeared onto a glass
slide and then processed and stained on a Coulter LH slide maker,
using a Wright stain for differentials.
[0046] The JP2 and NJ4500 forms of LtxA isolated Actinobacillus
actinomycetemcomitans differ functionally in that the NJ4500 shows
greater toxicity towards leukemia cells, along with greater
specificity. The NJ4500 form of LtxA is highly specific towards
white blood cells (leukocytes). Specifically, the NJ4500 form of
LtxA is highly specific towards basophils, neutrophils, and
monocytes. The NJ4500 form of LtxA is also highly specific towards
eosinophils. This form does not induce significant cell death in
red blood cells (erythrocytes) or lymphocytes. These functional
distinctions may be related to structural modifications.
Specifically, NJ4500 LtxA is highly modified with fatty acids.
[0047] A two-dimensional gel electrophoresis of JP2 and NJ4500 LtxA
is shown in FIG. 7. The gel shown in FIG. 7 shows that the JP2 form
of LtxA contains a significant amount of protein with an
isoelectric point of at least 9.0. To create the 2-D gel, LtxA (20
mg) from JP2 or NJ4500 was separated first by isoelectric point
through a pH gradient of 7-10. LtxA separated by isoelectric
focusing was then separated by mass using polyacrylamide gel
electrophoresis. Protein was visualized with SYPRO ruby stain. The
LtxA samples were prepared for 2-D gel electrophoresis by
processing 20 mg with a 2-D gel clean-up kit according to the
manufacturer's directions (Amersham Biosciences, Piscataway, N.J.).
Following the clean-up, the pelleted protein was resuspended in 182
ml rehydration buffer (Bio-Rad, Hercules, Calif.) and mixed with
3.7 ml DTT (500 mg/ml) and 2 ml 1% bromophenol blue. The sample was
then loaded onto a pH 7-10 IPG strip and processed for isoelectric
focusing according to the manufacturer's protocol (Bio-Rad,
Hercules, Calif.). After isoelectric focusing, the IPG strip was
layered atop a 10% polyacrylamide gel and resolved in SDS buffer
for several hours. Visualization of protein spots was accomplished
by staining the gel in SYPRO ruby protein stain (Bio-Rad, Hercules,
Calif.).
[0048] Two other RTX toxins, E. coli HlyA and B. pertussis CyaA,
are modified covalently with fatty acid moieties at internal lysine
residues. In E. coli, hlyC is a fatty acyl transferase and is
required for modification of HlyA. Based on the presence of an hlyC
homologue in A. actinomycetemcomitans (ltxC), it is predicted that
A. actinomycetemcomitans LtxA is also modified. Modification of
HlyA and CyaA is required for toxin activity and the degree of
modification is directly correlated with toxicity.
[0049] The two forms of LtxA were subjected to two-dimensional gel
electrophoresis to assess whether differential modification of the
proteins accounted for the functional differernces. The
two-dimensional gel electrophoresis showed that LtxA from both
strains exists in multiple isoforms (See FIG. 7). Several
representative spots were excised and subjected to trypsin
digestion and MALDI-TOF MS analysis. MALDI-TOF MS confirmed that
all species were A. actinomycetemcomitans LtxA (data not
shown).
[0050] The predicted pI of LtxA based on primary amino acid
sequence is approximately 9. (Represented by the rightmost spot and
small arrow in FIG. 7). Modification of lysine residues with fatty
acids shifts the pI towards the acidic end, therefore, a greater
fraction of the NJ4500 LtxA is modified compared to LtxA from
JP2.
[0051] Approximately half of the JP2 LtxA is completely unmodified
(as represented by the dense spot at approximately pI 9.). In
contrast, none of the NJ4500 LtxA appears to be completely
unmodified in the 2-D gel shown in FIG. 7.
[0052] Other RTX toxins are modified with fatty acids and this
modification is required for activity. Modification of RTX toxins
may contribute to host and cell type specificity. E. coli,
a-hemolysin, is modified at two internal lysine residues with C14,
C15, or C17 fatty acid residues. Because E. coli can incorporate
into HlyA three different fatty acids at two different lysine
residues, preparations of HlyA are heterogeneous. Based on the
two-dimensional gel electrophoresis data shown in FIG. 7, LtxA is
even more heterogeneous than HlyA.
[0053] LtxA has approximately 100 lysine residues and modification
at several of them with different types of fatty acids accounts for
the relatively large number of different isoforms. LtxA purified
from NJ4500 is more active than LtxA from the JP2 strain. Nearly
all of NJ4500 LtxA existed in some type of modified form whereas
preparations of JP2 LtxA contained a significant amount of
unmodified form (FIG. 7). Consistent with other RTX toxins, the
unmodified toxin from JP2 is inactive against HL-60 cells. Thus,
the percent of active LtxA molecules in preparations from NJ4500 is
greater than from JP2.
[0054] A fatty acid is defined as a long-chain monobasic organic
acid or a hydrocarbon chain. Fatty acids associated with NJ4500
LtxA were analyzed by Mylnefield Research Services, Lt., the Lipid
Analysis Unit, Scotland. The protocol for analysis of myristoylated
proteins described by Neubert, T. A. and Johnson, R. S. (Methods
Enzymol., 250, 487-494 (1995)) was followed. It involves acidic
hydrolysis of the protein, followed by acid catalysed methylation
of the fatty acids for analysis by gas-chromatography.
Approximately 3 mg of LtxA was subjected to this protocol, and
preliminary data shows that at least myristic acid (C14) and
palmitic acid (C16) are present in the LtxA protein.
[0055] The pI values of the two-dimensional gel indicated that
basic residues are modified (making the protein more acidic). Any
basic residue may be chemically modified by a fatty acid to reduce
the pI. Because it is known that the lysine residues of RTX family
members are modified by fatty acids, and that such a chemical
modification increases activity of the RTX familiy members, the
LtxA protein is likely to be modified at lysine residues.
[0056] Based on the data collected to date, the specificity and
increased activity evident in the NJ4500 form of LtxA can be
attributed to the composition of leukotoxin proteins with greater
than 85% of the leukotoxin proteins chemically modified at a basic
amino acid residue. This composition is an LtxA composition
isolated from a bacterium such as Actinobacillus
actinomycetemcomitans, and preferably the NJ4500 strain of
Actinobacillus actinomycetemcomitans.
[0057] This composition as described induces cell death in myeloid
leukocytes. The composition is specific to any of white blood
cells, neutrophils, monocytes, basophils, and eosinophils. Myeloid
cells are cells belonging to the white blood cell lineage, and
consist of granulocytes (basophils, neutrophils, eosinophils),
monocytes, erythrocytes and platelets. The LtxA composition is
specific as it is substantially non-toxic to lymphoid
leukocytes.
[0058] White blood cells are a type of cell formed in the
myelopoietic, lymphoid, and reticular portions of the
reticuloendothelial system. Lymphocytes are a white blood cell
formed in the lymphatic tissue throughout the body (eg. Lymph
nodes, spleen, thymus, tonsils, Peyer patches, and sometimes in the
bone marrow). In normal adults approximately 22-28% of the total
number of white blood cells in the circulating blood are
lymphocytes.
[0059] The specificity of the NJ4500 LtxA is especially useful as a
treatment against acute myeloid leukemia (AML) and chronic myeloid
leukemia (CML) because these are diseases in which only the myeloid
cells are malignant. Thus, LtxA provides a high level of toxicity
to certain myeloid cells, while leaving the red blood cell
population unharmed, as the NJ4500 is substantially non-toxic to
lymphocytes and red blood cells.
[0060] As discussed above, the NJ4500 LtxA composition of the
invention includes chemical modifications, and the chemical
modifications include fatty acid modifications to basic amino acid
residues. Preferably, the basic amino acid residue that is modified
is a lysine residue, and greater than 90% of the leukotoxin
proteins are chemically modified at at least one basic amino acid
residue.
[0061] Also discussed above, the NJ4500 LtxA compositions of the
invention have a pI less than 9. Within the composition, 85% of the
leukotoxin proteins have a pI less than 9.0. In another embodiment,
90% of the leukotoxin proteins have a pI less than 9.0, and in yet
another embodiment, 95% of the leukotoxin proteins have a pI less
than 9.0. In still another embodiment, 100% of the leukotoxin
proteins have a pI less than 9.0.
[0062] As shown in FIG. 7, most of the NJ4500 LtxA proteins have a
pI less than 8.5. In one embodiment of the invention, 85% of the
leukotoxin proteins have a pI less than 8.5, and in another
embodiment, 90% of the leukotoxin proteins have a pI less than 8.5.
The invention includes compositions where 95% of the leukotoxin
proteins have a pI less than 8.5, and in yet another embodiment,
100% of the leukotoxin proteins have a pI less than 8.5. The
leukotoxin proteins are isolated from Actinobacillus
actinomycetemcomitans, and in another embodiments, the leukotoxin
proteins are isolated from the NJ4500 strain of Actinobacillus
actinomycetemcomitans.
[0063] Because NJ4500 demonstrates a unique specificity among RTX
family members, one embodiment of the present invention includes an
RTX family protein that selectively lyses white blood cells more
effectively than red blood cells. The RTX family member is
substantially non-toxic to red blood cells.
[0064] Also provided is a pharmaceutical composition comprising
leukotoxin proteins and a pharmaceutically acceptable carrier,
along with a method of selectively inducing cell death in myeloid
leukocytes comprising contacting the myeloid leukocytes with a
composition comprising leukotoxin proteins.
[0065] In another embodiment, a method of killing a target cell by
contacting the target cell with leukotoxin proteins is provided. In
this method each leukotoxin protein has a pI less than 9.0. In one
embodiment of this method, the myeloid leukocyte cells die at a
faster rate than lymphoid cells.
[0066] In still another embodiment, a method of treating a blood
disease is provided. This method comprises administering a
composition of leukotoxin proteins isolated from the NJ4500 strain
of Actinobacillus actinomycetemcomitans to a subject suffering from
the blood disease. In treating a blood disease, a chemotherapeutic
pharmaceutical may be administered to the subject in conjuction
with the leukotoxin. Some appropriate chemotherapeutic
pharmaceuticals include idarubicin, cytarabine, etosposide,
daunorubicin, mitoxantrone, and melphalan. Other common
chemotherapeutic agents for the treatment of leukemia and lymphoma
include Chlorambucil, Fludarabine phosphate, Cytarabine, and
Daunorubicin hydrochloride. These drugs share the common property
of being highly toxic to humans, affecting many different tissue
and organ systems of the body. Bone marrow suppression, severe
neurologic effects, infertility, pulmonary, and gastrointestinal
effects are some of the adverse effects exhibited by these drugs.
Many of the drugs act by inhibiting DNA synthesis, a process that
all dividing cells carry out. Most cells of the body are targeted
by these non-specific pharmaceuticals. Any suitable pharmaceutical
agent may be used in conjunction with LtxA, and the combination of
a pharmaceutical agent with leukotoxin is intended to reduce the
dose of the pharmaceutical necessary to achieve effective results
in patients.
[0067] In addition to the potential uses as an anti-cancer agent,
Actinobacillus actinomycetemcomitans leukotoxin may serve as a
potent anti-viral. Specifically, HIV replicates and resides inside
macrophages and T-lymphocytes. Viruses are difficult to combat
because they often "hide" from the immune system inside host cells.
Leukotoxin could destroy those macrophages that are infected with
HIV, allowing the virus to be released and attacked by the natural
host immune defenses. This treatment would be different in that the
therapy would not be directed against the virus (which would select
for resistant HIV mutants), but rather against the host cell in
which the virus resides.
[0068] In one embodiment the blood disease is leukemia, lymphoma,
or myeloma, and in another embodiment, leukotoxin is used in a
method of selectively sensitizing myeloid leukocyte cells to
permeates. This method comprises contacting the myeloid leukocyte
cells with a composition comprising leukotoxin proteins, wherein
each leukotoxin protein has a pI less than 9.0, and lymphoid cells
are substantially unsensitized to permeates by the composition.
[0069] In another embodiment, a method of purifying a RTX family
protein from Actinobacillus actinomycetemcomitans comprises: [0070]
a. inoculating a single colony of Actinobacillus
actinomycetemcomitans into a fresh broth and growing cultures;
[0071] b. adding the growing cultures to fresh broth, adding beads
and incubating; [0072] c. centrifuging the incubated culture,
forming a pellet and a supernatant; [0073] d. filtering the
supernatant through a membrane to provided a filtered supernantant:
[0074] e. mixing (NH.sub.4).sub.2SO.sub.4 and the filtered
supernatant together to form a mixture; [0075] f. centrifuging the
mixture to form a mixture pellet; [0076] g. resuspending the
mixture pellet in buffer to form a protein resuspension; [0077] h.
passing the protein resuspension through a column; and [0078] i.
collecting the protein eluting off the column.
[0079] In a further aspect of the invention, an assay system and
method are presented, that enables the testing of anti-cancer drugs
such as the leukotoxins of the present invention, under
physiological conditions, with the consequence and benefit that a
relevant prediction of activity is rapidly and efficiently
obtained.
[0080] Screens for compounds and proteins with anti-cancer activity
employ viability assays using relevant cancer cell lines. For
leukemia studies, the human leukemia cell line, HL-60, is often
used as a model system. To facilitate the discovery and
investigation of anti-leukemia therapeutics under physiological
conditions, and in accordance with this invention, a bioluminescent
HL-60 cell line has been engineered that can be visualized in whole
human blood and living mice and whose viability can be rapidly
determined. A WBC-specific bacterial toxin has been shown to be
active in blood. The engineered HL-60luc cells of the invention
behave similar to the parental HL60 cell line. When used in a
bioluminescence imaging assay (BLI) as discussed in detail herein,
the BLI signal peaked approximately one hour following the addition
of luciferin but remained relatively high for several hours. This
type of in vitro kinetics where an early peak in luminescence is
followed by a slow decline is consistent with other BLI cell lines.
The detection limit of 1000 viable cells is also consistent with
other reports (35,36). Because human blood contains plasma
proteins, such as antibody and proteases, and other cells, that may
affect the activity, availability, or stability of a drug, the
anti-leukemia assays with HL-60luc cells in the presence of blood
can yield more physiological results than with buffer or growth
media alone.
[0081] In vivo bioluminescence imaging (BLI) is a technology that
allows visualization of live bioluminescent cells (mammalian,
bacterial, viruses) in complex biological material and living
animals (24,31). Firefly luciferase has been used extensively in
reporter systems and its expression can be measured quantitatively
using a luminometer or highly sensitive charge coupled device (CCD)
camera. Rocchetta et al. (32) found that the CCD camera was
approximately 25 times more sensitive than a luminometer, and so
the IVIS 50 imaging system (Xenogen, Alameda, Calif.) was used for
the work presented here. Luciferase reacts with its substrate,
luciferin, to produce oxyluciferin and light (11). Because ATP and
oxygen are required for the reaction, photon production has been
used as a quantitative measurement of cellular viability (14).
Animal studies have demonstrated a strong correlation between the
abundance of emitted photons and number of cells present in a given
tissue or animal (5,11).
[0082] In general, the field of oncology has utilized BLI
extensively to study the effects of anti-cancer therapy in vivo
(15,23). However, application of BLI to study hematologic
malignancies has been limited (6,22,44), and to date, there are no
bioluminescent hematologic cell lines commercially available
(Xenogen Corp., Alameda, Calif.). Validation of BLI in preclinical
models has been carried out using currently available methods and
evidence indicates that BLI has excellent sensitivity and offers
unique advantages (5,25,31,33). For example, non-invasive BLI
allows visualization of cells temporally and spatially, thus
permitting small changes in cell number and localization to be
detected over time (24,31). In addition, animals need not be
sacrificed at each sampling time point, decreasing the number of
animals that are required for an experiment and minimizing
inconsistency from animal-to-animal variations.
[0083] There is a significant difference between the sensitivity of
BLI and the trypan blue dye exclusion assay. For a cell to be
detected as nonviable with the trypan blue assay, the dye must
enter the cytoplasm of the cell. Trypan blue is a relatively large
molecule (mw 960.8) and while many cells may be metabolically dead,
their membranes could be sufficiently intact to exclude the dye to
appear viable. In contrast, BLI detects killing sooner because ATP
is no longer available in a metabolically dead cell. The results
are in strong agreement with Kuzmits et al. (17) who found that an
ATP/bioluminescent assay with HL-60 cells indicated nearly complete
killing after a 24 hour incubation with 5.7 .mu.mol/l doxorubicin,
while the trypan blue assay indicated almost no killing after 48
hours with the same drug concentration. Furthermore, Petty et al.
(30) reported that a bioluminescent ATP assay could detect as few
as 1500 viable cells/well while the MTT assay could not detect less
than 25,000 cells/well.
[0084] Bacterial toxins have been investigated for their
anti-cancer therapeutic potential for many years. Several
widely-studied toxins include diphtheriae toxin (DT) and
Pseudomonas exotoxin A (PE) (16). To increase the specificity of
these toxins, their toxic domains are often fused to other
molecules that target the toxin to certain cell types. For example,
ONTAK, a recently approved drug used to treat cutaneous T-cell
lymphoma, is a fusion molecule of DT and IL-2 (10,26).
[0085] As discussed above, the oral bacterium A.
actinomycetemcomitans produces a 113 kDa protein toxin, leukotoxin
(LtxA), which kills only blood cells of humans and Old World
Primates (3739). Furthermore, and in accordance with a first aspect
of the invention, a strain has been identified whose purified LtxA
does not lyse RBCs. LtxA binds to LFA-1 on host cells (19) and
destroys cells by apoptosis or necrosis (18). Because LtxA already
has specificity towards WBCs, it has been proposed that the protein
might serve as an effective targeted therapy for hematologic
malignancies. In addition, the toxin kills host cells by disruption
of the cell membrane (18) and therefore represents a mechanism of
action that is different from other chemotherapeutic agents.
[0086] In an effort to further evaluate the therapeutic potential
of LtxA, a test was conducted with the present in vivo screening
assay. Accordingly, the toxic effects LtxA against HL-60luc cells
in blood were examined. As set forth and demonstrated herein, the
toxin remains highly active in human blood and kills HL60luc cells
as efficiently as in RPMI medium. In addition, bone marrow
progenitor cell proliferation assays indicate that LtxA is active
toward myeloid progenitor cells and has an IC.sub.50 value in the
low ng/ml range. Preliminary studies also suggest that LtxA is
active mice and does not display toxicity when injected at high
doses into mice.
[0087] With the ability to rapidly determine HL-60luc cell
viability in the presence of biological fluids, it is possible to
efficiently screen thousands of different compounds at a time for
anti-leukemia activity. Assays may be performed in 96- or 384-well
dishes in the presence of physiologically-relevant samples such as
blood, plasma, or hepatocytes. Indeed, an important preclinical
screen to study drug biotransformation is performed in the presence
of hepatic material, such as human liver microsomes, human liver
cytosol fractions, and hepatocytes (3). HL-60luc cells could be
used in high-throughput hepatic screens for drugs with
anti-leukemia bioactivity.
[0088] In addition to using the present assay comprising the
HL-60luc cells for drug discovery, the test system can be used to
monitor the condition of a patient undergoing drug therapy.
Accordingly, the condition and behavior of a known drug or
combination of drugs in the presence of blood samples from
different leukemia patients may be measured and determined. For
example, neutralizing antibody in a patient's blood against a
potential drug might allow a clinician to exclude the drug from the
therapeutic regimen. Excluding an otherwise ineffective drug might
greatly reduce unwanted side effects. Indeed several studies have
shown a correlation between in vitro chemosensitivity of tumor
cells and therapy outcome (34,42). Such correlations could allow
the development of assay-directed individualized chemotherapy
regimens. Thus the assay of the invention can be used in the
following ways:
1. Screening novel drugs for anti-leukemia/cancer activity. 2.
Determine the best drug dosage for a leukemia/cancer patient. 3.
Determine which drug might be most effective for a leukemia/cancer
patient.
EXAMPLES
[0089] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way be construed, however, as limiting the broad scope of the
invention.
Example 1
Purification of LtxA from the NJ4500 Strain of A.
actinomyceterncomitans
[0090] The JP2 strain of A. actinomycetemcornitans produces
abundant LtxA, but it does not represent a fresh clinical isolate.
Here, LtxA was purified from the clinical isolate NJ4500 of A.
actinomycetemcomitans. This strain also produces and secretes a
large amount of LtxA, but the cells adhere to surfaces instead of
growing planktonically. This type of adherent growth results in a
relatively low number of cells per volume. The cell density of
adherent cells was increased by increasing the surface area on
which the cells can grow through the addition of spherical glass
beads. Soda lime beads provided the greatest amount of LtxA when
compared to Pyrex glass beads. The amount of LtxA that was purified
from NJ4500 in the presence of soda lime beads was approximately
twice that of JP2.
[0091] It is important to note that growth of A.
actinomycetemcomitans in the presence of both types of glass beads
was similar suggesting that differences in LtxA quantity was not
due variable growth. A. actinomycetemcomitans strains JP2 and
NJ4500 are known in the art. All bacteria were grown in A.
actinomycetemcomitans growth medium (AAGM) as known in the art.
Plates were incubated at 37.degree. C. in 10% CO.sub.2 for 4 days.
Broth cultures were incubated for 24 h unless otherwise noted.
[0092] LtxA was isolated from JP2 by growing cells in 5 ml AAGM
broth for 7-9 h and then diluted into 400 ml fresh AAGM broth.
These cultures were then grown for 13-17 h before harvesting
supernatant. To obtain supernatant, cultureswere centrifuged at
17,000 g for 10 minutes at 4 8 C. The supernatant was filtered
through a 0.22 mm low-protein binding membrane filter. For every
100 ml of filtered supernatant, 32.5 g (NH.sub.4).sub.2SO.sub.4 was
added. The mixture was gently rocked at 4 8 C for 1 h. The
precipitated protein was collected by centrifugation at 10,000 g
for 20 min at 4 8 C. The pellet from 400 ml supernatant was then
resuspended in 2 ml LtxA buffer (20 mM Tris-HCl, pH 6.8, 250 mM
NaCl, and 0.2 mM CaCl.sub.2).
[0093] The resuspended pellet was loaded on a column packed with 40
ml of Sephadex G-100 (Sigma, St. Louis, Mo.). Protein was eluted in
1 ml fractions with LtxA buffer. Protein content in each fraction
was determined with the Bradford reagent. The three fractions with
the highest protein content were combined, aliquoted and stored at
-80.degree. C. The purity of LtxA was determined on a 4-20%
SDS-PAGE gel and the concentration was determined by the BCA assay
according to the manufacturer's protocol (Pierce, Rockford,
Ill.).
[0094] LtxA was purified from the adherent strain NJ4500 by first
growing cells in tubes filled with 5 ml AAGM broth for 14 h and
then transferring 20 ml of growing cultures into 400 ml AAGM broth
in a 500-ml bottle. Prior to adding 400 ml sterile AAGM broth to
the 500 ml-bottle, 300 g of glass beads (or no beads, for controls)
were autoclaved inside the bottle. The soda lime beads were
obtained from Fisher Scientific (cat. 11-312C) and pyrex beads from
Corning Incorporated (cat. 7268-5). The inoculated bottle was grown
for 36-40 h as described above. During growth, the bottle was
inverted several times to allow adherent cells to coat all the
beads. After growth, the broth was removed and centrifuged and
processed as described above for JP2 LtxA. For these experiments,
cells were not removed from the beads.
[0095] Although adherent variants such as NJ4500 retain a greater
amount of LtxA than the nonadherent variants, a large amount of
secreted LtxA from NJ4500 can still be harvested. Because NJ4500
attaches avidly to surfaces, the number of growing cells per volume
can be increased by adding 5 mm glass beads to the growth medium.
In methods using one of two different types of glass beads, Pyrex
and soda lime, the yield of LtxA from cells growing on Pyrex was
significantly reduced when compared to the control of no glass
beads or soda lime beads.
Example 2
Imaging of Mice Injected with HL-60luc
[0096] The images of the mice shown in FIG. 3 were collected using
in vivo bioluminescence imaging. The SCID mouse model has been used
extensively for the study of hematologic malignancies, and the
pattern of leukemia displayed in SCID mice closely resembles human
clinical disease. In the model, leukemia cells are injected into
SCID mice, usually intravenously. A commonly used leukemia cell
line is HL-60, originally isolated from a 36-year-old female
patient with acute promyelocytic leukemia. Animal studies have
shown that HL-60 cells can infiltrate bone marrow, the spleen,
thymus, kidney, liver, lungs, and even the brain. It has been
reported that the mean survival time for SCID mice that were
injected with HL-60 cells was 42.5 days following injection;
however, this time can vary depending on the passage state of the
HL-60 cells being injected.
[0097] In vivo bioluminescence imaging (BLI) is a technology that
allows visualization of live bioluminescent cells (mammalian,
bacterial, viruses) in a living animal, without sacrificing the
animal. Cells to be visualized are engineered to express
luciferase, which reacts with its substrate, to result in light
production. Because the reaction also requires ATP, bioluminescence
has also been used as a measure of viability. In bacteria, the
substrate is encoded within the same operon as the luciferase
enzyme. In the mammalian system, the substrate, luciferin, must be
injected separately into the animal for the light-producing
reaction to take place. Visualization of luminescent cells requires
a highly-sensitive CCD camera that can detect low-level light
within a short period of time. We currently maintain the Xenogen
IVIS 50 imaging system for this purpose (Xenogen Corp., Alameda,
Calif.). The distribution and abundance of luciferase-producing
cells can be quantified by anesthetizing the animals and imaging
them with the IVIS 50 imaging system.
[0098] BLI allows visualization of cells temporally and spatially,
thus allowing small changes in cell number and localization to be
detected over time. In contrast, using standard methods, animals
must be sacrificed and extensive histological examination performed
to localize cells in question. In general, the field of oncology
has utilized BLI extensively, however, application of BLI to study
hematologic malignancies has been limited, and to date, there are
no bioluminescent hematologic cell lines commercially available
(Xenogen Corp., Alameda, Calif.).
[0099] In the oncology models, engineered malignant cells are
injected into animals and the progression of cancer is observed. In
addition, transgenic light-producing mice are available for use in
several oncology models (Xenogen Corp., Alameda, Calif.).
Metastasis and regression can be monitored with great sensitivity
and efficiency with the IVIS instrument. Of great significance, the
effects of anticancer therapy can be determined before the endpoint
of death is reached.
Example 3
Prepareation of Assay System Experimental
Cells and Growth Conditions
[0100] HL-60 cells were obtained from American Type Culture
Collection (ATCC) and maintained in RPMI+10% fetal bovine serum
(FBS) (Invitrogen, Carlsbad, Calif.) at 37.degree. C.+5% CO.sub.2.
Escherichia coli was grown in LB medium at 37.degree. C. A.
actinomycetemcomitans strains were grown in AAGM at 37.degree.
C.+10% CO.sub.2 as previously described (12).
DNA Manipulations
[0101] The luciferase-encoding plasmid for transfecting HL-60 cells
was constructed by cloning luciferase gene from pGL3 (Promega,
Madison, Wis.) into the geneticin resistance gene-containing
plasmid pCI-neo (Promega, Madison, Wis.). Both plasmids were
digested with BglII and XbaI and the Neo-containing fragment was
then ligated to the pGL3 fragment that contained the luciferase
gene. The mixture was transformed into E. coli and the bacteria
were selected on LB+carbenecillin (50 .mu.g/ml). Plasmid from
bacteria was prepared using the plasmid miniprep kit (Qiagen,
Valencia, Calif.). The new plasmid, encoding both luciferase and
geneticin, was designated pMP1.
[0102] The plasmid, pMP1, was transfected into HL-60 cells by
electroporation. Briefly, 106 cells were resuspended in 400 .mu.l
electroporation buffer (20 mM HEPES pH 7.0, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.2HPO.sub.4, 6 mM glucose, 0.1 mM
.beta.-mercaptoethanol). Plasmid pMP1 was added at a concentration
of 12.5 .mu.g/ml and the mixture was incubated for 5 minutes on
ice. The mixture was added to a cuvette and a pulse of 380 V was
administered. Five ml of fresh RPMI medium was added to the cells
and they were grown for 24 hours before geneticin was added.
Preparation of Cytotoxic Agents
[0103] Bacterial leukotoxin (LtxA) was purified from A.
actinomycetemcomitans as previously described (7). LtxA was stored
in 100 .mu.l aliquots at -80.degree. C. until used. A stock
solution of chlorambucil (Sigma, St. Louis, Mo.) was prepared by
dissolving 30 mg into 1 ml of DMSO. The drug was freshly prepared
prior to each experiment.
Bioluminescent Imaging (BLI)
[0104] For detection of bioluminescence (BL) from cultured HL-60luc
cells, 200 .mu.l of cells were mixed with 1 .mu.l luciferin (15
mg/ml) and then imaged with the IVIS 50 imaging system (Xenogen
Corp., Alameda, Calif.). For animal studies, Swiss Webster mice
were first injected with 106 HL-60luc cells (resuspended in PBS) or
PBS control intraperitoneally (i.p.) or intravenously (i.v.). Mice
were then anesthesized with acepromazine (0.3 mg/40 g, i.p.) and a
rodent cocktail [ketamine (20 mg/ml) and xylazine (2.5 mg/ml)] (0.1
ml/25 g, i.p.). Luciferin was then injected (150 mg/kg) i.p and the
mice were imaged with the IVIS 50 instrument at different times.
Images were analyzed using the Living Image Software (Xenogen
Corp., Alameda, Calif.).
Results
Construction of a Stable HL-60 Luciferase-Expressing Cell Line
[0105] To generate an HL-60 cell line that stably expresses
luciferase, a plasmid was constructed by cloning the luciferase
gene from pGL3 into the geneticin resistance gene-containing
plasmid pCI-neo. The modified plasmid, pMP1, was then
electroporated into HL-60 cells (obtained from ATCC) and grown
under geneticin selection. When geneticin was included in the
growth medium to select for the plasmid, bioluminescence (BL) was
observed, indicating that cells received the luciferase-encoding
plasmid. FIG. 8A shows HL-60 cells that were transfected with pMP1
and then grown in wells with different concentrations of geneticin.
Bioluminescence was detected with the IVIS 50 instrument. Cells
were grown for 8 weeks longer to allow the generation of stable
clones. After 8 weeks, geneticin selection was removed to determine
if the luciferase-encoding gene had successfully integrated into
the genome. Even after growing cells for many generations without
selection, the HL-60 cells still emitted light, suggesting that
stable transfectants had been obtained.
[0106] To continue studies, a homogeneous population of cells
derived from a single stable clone was isolated by performing
minimal dilutions with stable transfectants. Cells were diluted to
approximately one cell/well in a 96-well dish and then examined
microscopically to exclude wells that received more than one cell.
Dishes were further incubated and then imaged with the IVIS 50
instrument. Cells were transferred to larger dishes, grown and then
and saved in liquid nitrogen. Viability of these saved cells was
greater than 90%.
[0107] An important property for BLI studies is photon flux per
cell (photons/second/cell). The flux/cell for one specific clone
that was used in all subsequent assays described here was
calculated. The calculated value of 16 photons/second/cell is
consistent with values obtained from other engineered cell lines
(Xenogen Corp., Alameda, Calif.). It is believed that this is the
first HL-60 cell line that has been engineered to stably express
luciferase.
[0108] To confirm that the engineered HL-60 cells maintain basic
growth characteristics, growth studies were performed comparing
HL-60luc cells to parental HL-60 cells. Cells were grown in RPMI
with 10% FBS and then counted with a Vi-CELL cell viability
analyzer (Beckman Coulter, Inc., Miami, Fla.). Growth curve
experiments in RPMI for the two cell lines indicated that HL-60luc
cells behave like the parental cell line. FIG. 8B shows growth
curves for parental HL-60 and engineered HL-60luc cells. Cells were
grown in RPMI as described and viable cells were counted with the
ViCELL cell counter.
Detection of HL-60luc in Blood
[0109] To determine the kinetics of bioluminescence in blood,
6.times.105 HL-60luc cells were mixed with human peripheral blood
and luciferin was added to the mixture. BL was then measured over
time as photons/second from each sample. FIG. 9A shows the kinetics
of BL over time. HL-60luc cells were mixed with blood and luciferin
and then imaged with the IVIS 50 instrument at the indicated time
points. The observed pattern was highly reproducible. The signal
peaked at one hour and was approximately 200 times greater than the
background signal from blood alone. FIG. 9A. These results were
highly reproducible and a similar pattern was obtained when the
same experiment was performed in RPMI. BL values in RPMI were
approximately two-fold greater than in blood likely due to light
absorption by the blood.
[0110] The sensitivity of detection in blood was then determined.
Different numbers of HL-60luc cells were mixed with blood (200
.mu.l total) and luciferin was added to each sample. The mixtures
were incubated at 37.degree. C. for one hour and BL was measured.
FIG. 9B shows detection limit of HL-60luc cells. Cells were mixed
with blood and luciferin and then incubated for one hour before
imaging. Approximately 1000 cells could be detected above the
background level of the blood alone. The signal emitted from the
highest number of cells tested (1.25.times.106) was approximately
2000 times greater than blood alone. The BL signal correlated
strongly with cell number. FIG. 9C shows that the number of
HL-60luc cells shows a linear correlation with BL.
Sensitivity of HL-60luc Cells to a Bacterial Toxin
[0111] The gram negative bacterium, A. actinomycetemcomitans,
produces leukotoxin (LtxA), a protein toxin that kills specifically
white blood cells from humans and Old World Primates (37-39) and
red blood cells (1). Examination of LtxA from a strain of A.
actinomycetemcomitans, NJ4500, revealed that this purified protein
does not lyse erythrocytes in vitro compared to LtxA from the
standard strain, JP2. FIG. 10A shows the lysis of human red blood
cells by LtxA from two different strains of A.
actinomycetemcomitans. Because erythrocyte lysis would be an
undesirable property for a chemotherapeutic agent, studies here
employ LtxA from NJ4500.
[0112] To determine if HL-60luc cells are equally sensitive to LtxA
as parental HL-60 cells, cell killing was assayed by LtxA in RPMI.
HL-60 cells were mixed with LtxA and viability was measured with
the trypan blue dye exclusion assay using the Vi-CELL instrument.
LtxA had an equal toxic effect on both cell lines. FIG. 10B shows
that HL-60 and HL-60luc cells are equally sensitive to killing by
LtxA from strain NJ4500. Assays were performed in RPMI medium and
viability was determined using the trypan blue dye exclusion assay.
This result was highly reproducible. Thus, the HL-60luc cell line
is similar to the parental HL-60 cell line for it sensitivity to a
bacterial toxin.
LtxA Activity in Whole Blood
[0113] To determine if LtxA is active in whole blood and retains
its ability to kill HL-60 cells, HL-60luc cells were resuspended in
blood or RPMI and different concentrations of purified LtxA or LtxA
buffer was added to the HL-6luc-blood mixture and incubated at
37.degree. C. for 4 hours. BLI was then measured and relative
viabilities were determined comparing experimental values to the
buffer-containing sample. LtxA was highly active in whole blood
against HL-60luc cells and this activity was similar to that seen
in RPMI. FIG. 11A shows the activity of LtxA against HL60luc cells
in whole human blood and RPMI medium. Viability was measured using
BL.
[0114] The sensitivity of BL to trypan blue as an assay for cell
viability was also compared. Luminescence was significantly more
sensitive than trypan blue. FIG. 11B shows the comparison between
BL and trypan blue as viability assays for LtxA-mediated
cytotoxicity. Cells were incubated in RPMI medium with LtxA or
buffer for 4 hours and viability was determined. Nearly complete
cell killing was observed with leukotoxin concentrations as low as
10 ng/ml using BL values. In contrast, trypan blue revealed that
only 35% killing had occurred at this concentration.
[0115] To determine if the difference in detection limit between
the two methods was specific for LtxA-mediated cytotoxicity,
another compound, chlorambucil, was used to induce cell death.
Chlorambucil alkylates DNA and induces apoptosis (2,21) and
therefore represents a mechanism of killing different from that of
LtxA. For chlorambucil, it was also observed that BLI was a more
sensitive assay than trypan blue for detecting viability. FIG. 4C
shows the comparison between BL and trypan blue as viability assays
for chlorambucil-mediated cytotoxicity. Cells were incubated in
RPMI medium with chlorambucil or buffer for 24 hours and viability
was determined. At a chlorambucil concentration of 0.03 mg/ml, BLI
revealed approximately 90% cell death after 24 hours while trypan
blue revealed essentially no killing (FIG. 11C).
Visualization of HL-60luc in Mice
[0116] Mouse models for human leukemia utilize HL-60 cells that are
injected either i.p. (28) or i.v. (40,41). To determine if the
HL-60luc cells could be visualized in living mice, approximately
106 HL-60luc cells were injected i.p. or tail i.v. FIG. 12 shows
Swiss-Webster mice that were anesthesized with XXX and injected
with 106 HL-60luc cells intraperitoneally (i.p.; top) or
intravenously (i.v.; bottom) and followed by luciferin i.p. Mice
were imaged with the IVIS 50 instrument at different times
post-luciferin injection. The scale on the right of each image
indicates surface radiance (photons/second/cm2/steradian).
Luciferin was administered immediately following injection of cells
and the animals were imaged with the IVIS 50 instrument. The cells
could be detected with a 2-3 minute exposure when administered by
either route. The signal was greatest for i.p.-injected cells
immediately following injection while the signal for i.v.-injected
cells peaked approximately 35 minutes post luciferin (FIG. 12).
Interestingly, the signal observed for i.v. injection follows the
path of the tail vein and then dissipates as the cells become
diluted through other blood vessels. Thus, HL-60 cells can be
visualized in a living animal at concentrations normally used for
the SCID mouse model for human leukemia.
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[0162] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0163] While preferred embodiments of the invention have been shown
and described herein, it will be understood that such embodiments
are provided by way of example only. Numerous variations, changes
and substitutions will occur to those skilled in the art without
departing from the spirit of the invention. Accordingly, it is
intended that the appended claims cover all such variations as fall
within the spirit and scope of the invention.
* * * * *