U.S. patent application number 15/059586 was filed with the patent office on 2016-06-30 for individualized bacterial treatment of pancreatic cancer.
The applicant listed for this patent is AntiCancer, Inc.. Invention is credited to Fuminari Uehara, Ming Zhao.
Application Number | 20160184369 15/059586 |
Document ID | / |
Family ID | 50974895 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160184369 |
Kind Code |
A1 |
Zhao; Ming ; et al. |
June 30, 2016 |
INDIVIDUALIZED BACTERIAL TREATMENT OF PANCREATIC CANCER
Abstract
An individualized bacterial treatment of cancer is provided. The
treatment includes a strain of bacteria modified by in-vivo passage
through tumor grafts in experimental animals, where the modified
strain exhibits enhanced cancer cell-targeting of a specific
malignancy arising in a unique individual to the corresponding
parent strain of bacteria. The treatment uses this modified strain
for the treatment human solid-tumor malignancies by inoculating an
individual with a quantity of the strain; and repeating
inoculations at periodic intervals where repeated inoculations tend
to progressively eliminate the solid tumor malignancy in the
individual.
Inventors: |
Zhao; Ming; (San Diego,
CA) ; Uehara; Fuminari; (San Diego, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AntiCancer, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
50974895 |
Appl. No.: |
15/059586 |
Filed: |
March 3, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14140345 |
Dec 24, 2013 |
|
|
|
15059586 |
|
|
|
|
61745731 |
Dec 24, 2012 |
|
|
|
Current U.S.
Class: |
435/252.1 |
Current CPC
Class: |
C12N 1/20 20130101; C12N
1/36 20130101; A61K 35/74 20130101 |
International
Class: |
A61K 35/74 20060101
A61K035/74; C12N 1/36 20060101 C12N001/36; C12N 1/20 20060101
C12N001/20 |
Claims
1. A strain of enhanced cancer cell-targeting bacteria for
individualized cancer therapy comprising: A modified strain of
bacteria, wherein said modified strain is created by at least one
in-vivo passage through tumor grafts in experimental animals; and
said modified strain exhibits enhanced cancer cell targeting of a
specific malignancy arising in a unique individual compared to a
corresponding unmodified strain of bacteria.
2. The strain of claim 1 wherein said modified bacteria is a
modified Salmonella typhimurium.
3. The strain of claim 1, wherein said modified strain of bacteria
is created by at least one passage through cancer cells in
vitro.
4. The strain of claim 1, wherein said specific malignancy is a
pancreatic cancer.
5. The strain of claim 1, wherein said specific malignancy is a
sarcoma.
6. The strain of claim 1, wherein said specific malignancy is a
lung cancer.
7. The strain of claim 1, wherein said specific malignancy is a
breast cancer.
8. The strain of claim 1, wherein said specific malignancy is a
colon cancer.
9. The strain of claim 1, wherein said specific malignancy is a
prostate cancer.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S.
application Ser. No. 14/140,345 entitled "Individualized Bacterial
Treatment of Pancreatic Cancer," filed Dec. 24, 2013, which claims
priority from U.S. Patent Application No. 61/745,731, filed Dec.
24, 2012 and entitled "Individualized Bacterial Treatment of
Pancreatic Cancer," the disclosures of which are incorporated
entirely herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] This invention relates to bacterial therapy for the
treatment of cancer. In particular, the invention is highly
customized "designer" strains of enhanced cancer cell-targeting
bacteria for individualized cancer therapy, their method of
creation, and their use in the treatment of human patients with
cancer.
[0004] 2. State of the Art
[0005] For at least three hundred years, those tending to the sick
have occasionally observed regression of cancerous tumors in people
suffering from severe acute infectious illnesses. At times, when
the afflicted person survived an adequately severe and lengthy
infection, the cancer completely disappeared resulting in a cure.
In the late 19th and early 20th centuries, William B. Coley, a New
York oncologist, infected cancer patients with Streptococcus
pyrogenes or administered extracts of the bacteria deemed "Coley's
toxins, reportedly with remarkable results. (Hoffman, R. Bugging
tumors. Cancer Discovery Jul. 11, 2012; p. 588.)
[0006] This phenomenon was largely ignored after Cooley's death in
1936, but interest in this unique form of cancer treatment has
recently increased. The very conditions which make cancerous tumors
resistant to conventional chemotherapy--an acidic pH
microenvironment resulting from hypoxia and tumor necrosis--are
favorable for the growth of anaerobic bacteria. Various genera of
anaerobic bacteria, most notably Clostridium and Bifidobacterium,
selectively infect necrotic regions of tumors over healthy tissue.
But because these obligate anaerobes cannot establish infection in
a non-hypoxic microenvironment, therapy with anaerobic bacteria
must be combined with chemotherapy to kill viable solid tumors,
small metastatic deposits, and individual cancer cells.
Accordingly, Salmonella, a facultative anaerobe that exhibits
sustained growth in both viable and necrotic regions of a tumor,
has been shown to infect cancerous tumors, killing cancer cells in
all regions of the tumor. Tumoricidal activity has also been noted
in other genera of facultative anaerobes, such as Streptococcus and
Escherichia.
[0007] To be an effective cancer treatment in vivo, a bacterial
strain must not only be sufficiently cytotoxic to kill the cancer
cells, but also unable to sustain an infection in normal tissues
causing severe illness or death. Strategies employed to increase
safety center around inducing mutations in wild-type bacteria, and
then selecting for the mutated desired traits. Mutations decreasing
the virulence of bacteria by altering the organism's ability to
express cytotoxic characteristics, such as gene-based chemical
changes of the wild-type lipopolysaccharide ("LPS") resulting in an
attenuated host cytokine response are described. Also described are
creating mutated strains auxotrophic for one or more nutrients,
including purines and/or amino acids.
[0008] S. typhimuruim is a facultative anaerobe which can mount a
sustained infection in both healthy and necrotic tissue. A strain
of S. typhimurium auxotrophic for both arginine and leucine has
been developed. Mutagenesis of a wild population of Salmonella
typhimuruim is induced using nitrosoguanidine ("NTG"), and a
resulting dual auxotroph for the amino acids leucine and arginine
("Leu-Arg") is selected from the heterogeneous population of
auxotrophs, non-auxotroph mutations, and non-mutated wild bacteria.
This Leu-Arg dual auxotroph (S. typhimuruim A1, or "A1") is unable
to sustain an infection within somatic cells or normal tissue, but
grows actively in individual cancer cells and malignant tumors.
Otherwise, A1 has no other attenuating mutations limiting its
cytotoxicity in infected tissues. S. typhimurium, therefore, has
therapeutic potential. (Hoffman, R. Bugging tumors. Cancer
Discovery Jul. 11, 2012; p. 588.)
[0009] Ideally, a bacterial strain 100% specific for the cancer of
interest that is completely non-toxic to the host/patient is
needed, along with a simple reproducible method for creating and
using this strain in the treatment of human patients with cancer.
No such strain exists in the prior art that even approaches this
ideal, nor does a straightforward and reproducible method for
producing such a strain.
[0010] Citation of documents herein is not an admission by the
applicant that any is pertinent prior art. Stated dates or
representation of the contents of any document is based on the
information available to the applicant and does not constitute any
admission of the correctness of the dates or contents of any
document.
SUMMARY OF THE INVENTION
[0011] Embodiments of the present invention involve highly
customized "designer" strains of enhanced cancer cell-targeting
bacteria for individualized cancer therapy--sufficiently but not
over-attenuated as not to cause sustained infection in normal
tissues, their method of creation, and their use in the treatment
of human patients with cancer.
[0012] An embodiment includes a strain of enhanced cancer
cell-targeting bacteria for individualized cancer therapy
comprising a modified strain of bacteria, wherein said modified
strain is created by in-vivo passage through tumor grafts in
experimental animals; and said modified strain exhibits enhanced
cancer cell targeting of a specific malignancy arising in a unique
individual compared to a corresponding unmodified strain of
bacteria.
[0013] Another embodiment of the invention includes a method for
creating a strain of enhanced cancer cell-targeting bacteria for
individualized cancer therapy, said method comprises harvesting
cancer cells from an individual; transplanting a quantity of said
cancer cells into a quantity of experimental animals; incubating
the experimental animals to establish a cancer tissue graft within
the experimental animals; inoculating a quantity of bacteria.sub.A1
into a said experimental animal containing established said cancer
tissue graft; removing said cancer tissue graft from the said
experimental animal following a period of in-vivo incubation;
extracting bacteria.sub.R from said cancer tissue graft removed
from the experimental animal; incubating said bacteria.sub.R under
conditions suitable for bacterial multiplication; selecting said
bacteria.sub.R; incubating said bacteria.sub.R under conditions
suitable for bacterial multiplication; inoculating a quantity of
bacteria.sub.R into a second said experimental animal containing
established said cancer tissue graft; removing said cancer tissue
graft from the second said experimental animal following a period
of in-vivo incubation; extracting bacteria.sub.R1 from said cancer
tissue graft removed from the second said experimental animal;
incubating said bacteria.sub.R1 under conditions suitable for
bacterial multiplication; selecting said bacteria.sub.R1; and
incubating said bacteria.sub.R1 under conditions suitable for
bacterial multiplication.
[0014] Yet another embodiment includes a method for treatment of
human solid-tumor malignancies using a strain of enhanced cancer
cell-targeting bacteria for individualized cancer therapy. The
method comprises harvesting cancer cells from an individual with a
solid-tumor malignancy; creating tumor xenografts in
immunologically deficient experimental animals with harvested said
cancer cells; selecting a strain of enhanced cancer cell-targeting
bacteria by serial in-vivo passage of said bacteria through said
tumor xenografts within said experimental animals; performing
extraction, isolation, and propagation of said strain; performing
inoculation of said individual with a quantity of said strain; and
repeating inoculations at periodic intervals of a quantity of said
strain in said individual wherein repeated inoculations tend to
progressively eliminate said solid tumor malignancy in said
individual.
[0015] The foregoing and other features and advantages of the
present invention will be apparent from the following more detailed
description of the particular embodiments of the invention, as
illustrated in the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a flow chart of a method of individualized
bacteria treatment of cancer.
[0017] FIG. 2 is a flow chart of another method of individualized
bacteria treatment of cancer.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0018] Since the discovery of vaccines and antibiotics to prevent
and treat infectious disease, cancer has become a leading cause of
death in adults over the age of forty-five. Only heart disease is
responsible for more deaths annually. Despite the magnitude of this
disease, finding curative treatments for cancer has been difficult.
Cancer can arise from virtually any cell type. Cancers are
genetically unstable and often develop a phenotypically
heterogeneous cell population early in their course. This
heterogeneity leads to variable responses to treatment within the
same patient, including resistance. Through the process of
multiplication, local invasion, and metastasis, cancer cells become
interspersed within normal tissue, like weeds in a lawn. To
permanently rid the lawn of weeds, every individual weed must be
killed, lest even one resistant weed survive to multiply and
re-infest the entire lawn. Eliminating the weeds, however, cannot
be at the expense of destroying the lawn.
[0019] Current treatment strategies include surgical resection,
chemotherapy, radiotherapy, and various combinations of these
modalities. The choice of treatment depends on the cell type of the
tumor and the stage of the cancer at the time treatment is
initiated. All of these treatments have some degree of limited
efficacy and patient toxicity. Surgery is impractical when multiple
or widespread metastases are present. Where the disease is locally
advanced and invades surrounding structure vital for survival,
surgery is of limited or no value. Surgery for cancer is also
highly invasive and comes with all of the risks and side effects
associated with surgery for benign conditions. The well-known side
effects of chemotherapy are, at best, miserable; and at worst,
fatal. Chemotherapy kills asymptotically. With the exception of
small-cell lung cancer and certain lymphoid, hematogenous, and
germ-cell malignancies, chemotherapy always leaves a percentage of
viable cancer cells. Therefore, chemotherapy is typically not
curative. And because chemotherapy is unable to kill every cell,
chemotherapy selects for those cell types most resistant to
treatment. Like chemotherapy, radiotherapy also kills
asymptotically and selects for resistant cell lines. Depending on
the size of the radiated field, the type of surrounding tissue, and
the total dose of radiation, the side effects of radiotherapy may
be limited or severe.
[0020] Like conventional chemotherapy and radiotherapy, the use of
bacterial infectious agents to treat cancer would be similarly
limited by the current state of the art. Bacterial treatment of
cancer must temper cytotoxicity with specificity, restricting an
otherwise harmful infection to cancer cells and tumors.
[0021] Current strategies to maximize patient safety focus on
reducing the virulence of the bacterial strain used for treatment.
These include exposing a wild population of bacteria to mutagenic
agents, then selecting strains with attenuated pathogenicity.
Examples are strains that produce a fractional amount of a cytokine
or direct cytotoxin as those produced by the corresponding wild
strains. Reducing virulence, however, may lead to reduced tumor
targeting and decreased cytotoxicity. An attenuated strain of S.
typhimurium showed limited, if any, effectiveness in treating
metastatic malignant melanoma patients in a phase I clinical trial,
possibly because it was over-attenuated. (Toso, J. F. et al. Phase
I study of the intravenous administration of attenuated Salmonella
typhimurium to patients with metastatic melanoma. J. Clin Oncol.
20: 142-52 (2002).) In this study, the bacterial agent utilized was
a S. typhimuruim with two induced mutations; purI (creating an
auxotroph for the purine adenine), and msbB. The msbB mutation
altered the bacterial lipopolysaccharide ("LPS") such that upon
infection in mice, the normal host cytokine response was markedly
attenuated. Ten-fold lower levels of TNF.alpha. were measured in
mice infected with this mutant strain, versus mice infected with
the corresponding non-mutated S. typhimurium, which all died.
[0022] To be therapeutically efficacious, the "safe" bacterial
strain must also be able to infect cancer cells and tumors with the
highest degree of selectivity. Although the S. typhimurium
containing the purI and msbB mutations was demonstrated to
concentrate in tumors versus non-cancerous tissue at ratios greater
than 250:1, described as "tumor targeting," this "targeting" is
merely preferential replication of bacteria in a more favorable
tumor microenvironment. This is an endogenous characteristic of
wild strains of bacterial species, demonstrated by the historical
anecdotes of occasional cures of cancer patients surviving
life-threatening infections. Further, the S. typhimurium purI/msbB
strain (S. typhimurium VNP20009) only demonstrated minimal "tumor
targeting," selectively colonizing tumors in only some patients in
three published human clinical trials, deemed unsuccessful in
treating solid tumor cancers in human patients. (3 out of 25
patients: Toso, J. et al. Phase I study of the intravenous
administration of attenuated Salmonella typhimurium to patients
with metastatic melanoma. J. Clin. Oncol. 20, 142-52 (2002); 1 out
of 4 patients: Heimann, D. M. et al. Continuous intravenous
administration of live genetically modified Salmonella typhimurium
in patients with metastatic melanoma. J. Immunother. 26, 179-80
(2003); 2 out of 3 patients: Nemunatis, J. et al. Pilot trial of
genetically modified attenuated Salmonella expressing the E. coli
cytosine deaminase gene in refractory cancer patients. Cancer Gene
Ther. 10, 737-44 (2003).) Therefore, the prior art demonstrates
that S. typhimuruim VNP2009 is lacking in both virulence and
tumor-targeting for the treatment of human solid tumor cancers. A
different genetically-engineered, strain of bacteria is needed to
overcome these characteristic lacking in S. typhimurium VNP2009,
other strains of Salmonella, and other bacterial genera.
[0023] Additional examples of reducing virulence by selection of
mutated bacterial strains auxotrophic for one or more nutrients,
including purines and amino acids, are noted in the prior art. A
mutant strain of S. typhimurium auxotrophic for leucine and
arginine, "S. typhimurium A1", has no impairment of LPS, other
cytokine-inducing substances, or cytotoxins where the required
nutrients are present. This dual auxotroph, however, is unable to
sustain an infection in normal somatic tissues, making it
potentially safe for use as a therapeutic agent.
[0024] Thus, the A1 dual auxotroph variant is able to kill cancer
cells but not infect normal tissues. The strain can then be further
enhanced by selecting for tumor-targeting sub-strains in a nude
mouse model. Nude mice harboring human colon cancer orthotopic
tumorgrafts (xenografts) are infected with A1 and the bacteria are
subsequently extracted directly from the tumorgrafts following a
period of incubation. This "reselected" strain is denoted "A1-R."
A1-R was able to eradicate primary and metastatic tumors in
mono-therapy in the orthotopic-transplant nude mouse models of
human prostate (Zhao, et al. Mono-therapy with a tumor-targeting
mutant of Salmonella typhimuruim cures orthotopic metastatic mouse
models of human prostate cancer. Proc. Natl. Acad. Sci. USA 104:
10170-74 (2007)); breast (Zhao, M. et al. Targeted therapy with a
Salmonella typhimuruim leucine-arginine auxotroph cures orthotopic
human breast tumors in nude mice. Cancer Res. 66: 7647-52 (2006));
and pancreatic cancer (Nagakura, C. et al. Efficacy of a
genetically-modified Salmonella typhimurium in an orthotopic human
pancreatic cancer in nude mice. Anticancer Res. 29: 1873-78
(2009)). In the above studies, nude mice engrafted with human
tumors tolerated tail vein injection of 10.sup.7 colony forming
units ("CFUs") of the S. typhimurium A1-R dual auxotroph with
apparent systemic effects. All showed substantial tumor regression
and a high percentage were completely cured, depending on the
primary cancer cell type.
[0025] What is lacking in the prior art, however, is a strain of
bacteria that is custom-produced with the highest specificity to
target not merely an individual cancer cell type, but a genetically
and phenotypically unique malignancy arising within an individual
patient--a "designer bug" for each patient that, at a minimum,
preserves all of the cytotoxicity of the native strain of bacteria
while being unable to sustain an infection in non-cancerous somatic
cells and healthy tissues.
[0026] This invention addresses both of these fundamental
requirements--safety and specificity--by creating a unique,
modified strain of reselected auxotrophic bacteria using a novel
method. The result is a bacterial agent with a maximally-enhanced,
highest possible specific targeting of cancer cells wherever they
exist in the body while limiting host toxicity and not causing
significant illness, custom-tailored to the unique malignancy
present in a single individual patient. This invention discloses
such a strain, and further discloses a simple, reproducible method
for creating additional modified strains for enhanced cancer-cell
targeting of a specific malignancy arising in a unique individual.
Additionally, the invention discloses a method of using the strain
for the treatment of human patients with cancer.
[0027] Disclosed are strains of enhanced cancer cell-targeting
bacteria for individualized cancer therapy comprising a strain of
bacteria modified by in-vivo passage through tumor grafts in
experimental animals, wherein said modified strain exhibits
enhanced cancer cell-targeting of a specific malignancy arising in
a unique individual compared to the corresponding parent strain of
bacteria.
[0028] FIG. 1 shows a method 10 for treatment of human solid-tumor
malignancies using a strain of enhanced cancer cell-targeting
bacteria for individualized cancer therapy. The method 10 comprises
Step 11 of harvesting cancer cells from an individual with a
malignancy. Once the cancer cells are harvested, the method 10
includes Step 12 of creating tumor xenografts in immunologically
deficient experimental animals with said harvested cancer cells.
The method 10 then includes a Step 13 of selecting a strain of
enhanced cancer cell-targeting bacteria by serial in-vivo passage
of said bacteria through said tumor xenografts within said
experimental animals. Following Step 13, Step 14 is executed of
performing extraction, isolation, and propagation of said strain.
The method then includes Step 15 of performing inoculation of said
individual with a quantity of said strain. The method 10 then may
include repeating inoculations at periodic intervals of a quantity
of said strain in said individual wherein repeated inoculations
progressively eliminate said malignancy in said individual.
[0029] FIG. 2 depicts another method 20 for treatment of human
solid-tumor malignancies using a strain of enhanced cancer
cell-targeting bacteria for individualized cancer therapy. The
method 20 comprises Step 21 of harvesting cancer cells from an
individual. Step 21 is followed by Step 22 of transplanting a
quantity of said cancer cells into a quantity of experimental
animals. After transplanting a quantity of cancer cells into the
experimental animals, Step 23 includes incubating the experimental
animals to establish a cancer tissue graft within the experimental
animals. Step 23 leads to Step 24 of inoculating a bacterial
strain.sub.A1 into a said experimental animal containing
established said cancer tissue graft. The method 20 further
includes Step 25 of selecting a number of killed cancer cells
infected with the bacteria.sub.A1. The method 20 may then include
removing said cancer tissue graft from said experimental animal
inoculated with said bacterial strain following a period of in-vivo
incubation. The method 20 further comprises Step 26 of extracting
bacteria.sub.R from said cancer tissue graft removed from the
experimental animal inoculated with said bacterial strain; Step 27
of incubating said bacteria.sub.R under conditions suitable for
bacterial multiplication; Step 27 of inoculating a quantity of said
bacteria.sub.R into a second said experimental animal containing
established said cancer tissue graft; and Step 28 of selecting a
said bacteria.
[0030] Additional embodiments of the invention may continue with
steps 29-35 of Method 20. Step 29 of incubating the selected said
bacteria.sub.R under conditions suitable for bacterial
multiplication is followed by Step 30 inoculating a quantity of
selected said bacteria.sub.R into a second said experimental animal
containing established said cancer tissue graft; Step 31 removing
said cancer tissue graft from the said experimental animal
inovulated with a quantity of said bacteria.sub.R following a
period of incubation; Step 32 extracting bacteria.sub.R1 from said
cancer tissue graft removed from the experimental animal; Step 33
incubating said bacteria.sub.R1 under conditions suitable for
bacterial multiplication; and Step 34 selecting a said
bacteria.sub.R1.
[0031] Referring to FIGS. 1 and 2, the methods 10 and 20 may
further include creating a strain of enhanced cancer cell-targeting
bacteria for individualized cancer therapy, said method comprising
the steps of harvesting cancer cells from an individual
(experimental animal or human); transplanting a quantity of said
cancer cells into a quantity of experimental animals; incubating
the experimental animals to establish a cancer tissue graft within
the experimental animals; inoculating a bacterial strain.sub.A1
into a said experimental animal containing established said cancer
tissue graft; removing said cancer tissue graft from said
experimental animal inoculated with said bacterial strain.sub.A1
following a period of incubation of the inoculated said
experimental animal; extracting bacteria.sub.R from said cancer
tissue graft removed from the experimental animal inoculated with
said bacterial strain.sub.A1; incubating said bacteria.sub.R under
conditions suitable for bacterial multiplication; inoculating a
quantity of said bacteria.sub.R into a second said experimental
animal containing established said cancer tissue graft; removing
said cancer tissue graft from the second said experimental animal
following a period of in-vivo incubation; extracting
bacteria.sub.R1 from said cancer tissue graft removed from the
experimental animal; and incubating said bacteria.sub.R1 under
conditions suitable for bacterial multiplication.
[0032] In one embodiment of the methods 10 and 20, the bacteria are
a modified strain of Salmonella typhimuruim ("S. typhimurium").
This is not meant to be limiting, and strains of other facultative
anaerobes, for example Salmonella, Streptococcus, Escherichia, and
others may be used in various embodiments of the invention.
Mutations are induced in the wild strain and mutants are selected
for ability to grow in cancer cells and/or tumors without
sustaining an infection in non-cancerous host tissues and cells
using any variety of established techniques known to those skilled
in bacterial genetic engineering and related arts.
[0033] Further, intracellular replication and virulence of S.
typhimurium A1 or A1-R can be directly observed by transfecting S.
typhimuruim A1 with the green fluorescent protein ("GFP") gene pGFP
using electroporation. In other embodiments, the nuclei and
cytoplasm of the engrafted cancer cells can also be labeled through
stable transposition of retroviral red fluorescent protein ("RFP")
in the cytoplasm and retroviral GFP in the nucleus (by fusion of
GFP with histone H2B). Other labeling techniques may also be used.
This combination enhances the initial selection of bacteria that
are tumor targeting by allowing for the visualization of the
bacterial cancer cell interaction using dual color spatial-temporal
fluorescence microscopy. Apoptosis of the infected cancer cells is
readily determined by observing fragmentation of the GFP-expressing
nuclei. Cells with intact nuclei after a period of incubation not
containing cytotoxic bacteria are distinguished from apoptotic
cells specifically targeted and killed by intracellular cytotoxic
bacteria. In embodiments of the invention, only those killed cancer
cells harboring cytotoxic bacteria are selected for isolation and
propagation of the intracellular bacteria. This resulting
"reselected" strain is denoted A1-R.sub.n were "n" is zero or a
whole number representing the number of selection cycles, either
in-vitro or in-vivo, undergone by the original A1 strain.
[0034] According to the methods 10 and 20, to create orthotopic
tumorgrafts (xenografts) in the nude mouse model, human cancer
cells or biopsy samples of tumor tissue taken directly from the
cancer patient are used. The immunologically deficient athymic
"nude" mouse is used by way of example because this is the most
commonly used model today. The invention is intended to cover the
creation of human patient tumor xenografts using the family of
procedures established in the prior art with immunologically
deficient mice and other suitable experimental animals.
[0035] In various embodiments of the invention, and according to
methods 10 and 20, harvested cancer tissue may be homogenized with
the individual cells isolated, grown in tissue culture and
inoculated via the tail vein. The harvested cancer tissue may be
individual cancer cells harvested from a variety of sources,
including but not limited to patient blood (circulating tumor
cells), sputum (lung cancer), vaginal or endocervical swabs,
malignant ascites, and malignant pleural effusions. Alternatively,
the grafts may be surgically transferred directly to a favorable
host organ or tissue within the immunologically deficient mouse. In
various embodiments of the invention, this may be accomplished by
surgically implanting a 1 mm to 3 mm block of tissue beneath the
capsule of the host organ in the anesthetized animal, such as the
pancreas or prostate, into the mammary fat pad, or sub-serosally
into the cecal wall, depending on the cell type of the primary
tumor. This establishes a xenograft, whereby the human cancer grows
and metastasizes within the mouse or other experimental animal,
continuing to demonstrate the same phenotype--including human
histopathology and tumor-marker production--as the original human
tumor.
[0036] Embodiments of the invention may be practiced with an in
in-vitro inoculation of the parent strain of bacteria A1 into
harvested cancer cells grown in tissue culture, incubation,
selection of bacterial strain.sub.Rn from infected, killed cancer
cells in vitro, followed by any number of iterations passaging the
selected bacterial strain.sub.Rn through tumor xenografts in vitro.
In other embodiments if the invention, the in-vitro passage of the
selected bacterial strain.sub.Rn may be performed at any point
following step 23 or step 29 of the method 20. Embodiments of the
invention may be practiced using one, or greater than one in-vitro
passage of the selected bacterial strain.sub.Rn. Other embodiments
may omit this in-vitro passage cycle. Still other embodiments may
use in-vitro passage cycles only, with no passage through in-vivo
tumor xenografts. The foregoing embodiments utilizing at least one
passage of the bacterial strain through cancer cells in vitro is
particularly useful when solid tumor cancer specimens are not
available. This makes the invention useful where the cancer cells
are harvested from sources including but not limited to patient
blood (circulating tumor cells), sputum (lung cancer), vaginal or
endocervical swabs, malignant ascites, and malignant pleural
effusions.
[0037] It is understood that it is feasible to select
individualized bacteria for each person's cancer, the only
requirement being the availability of cancer cells. Individual
cells may be harvested according to the above and other
embodiments. If sufficient tumor material is available, such as
from a resection, in vivo growth of the tumor tissue is usually
feasible with modern techniques in immunodeficient mice or other
experimental animals wherein the "designer" bacteria can be
selected. With lesser amounts of material, down to individual
cells, the "designer" bacteria can be selected in vitro.
[0038] The invention provides for an individually-specific strain
of further modified attenuated cancer-specific cytotoxic bacteria;
derived from S. typhimuruim A1 in this particular embodiment. It is
appreciated that other species and strains may also be used. It is
further understood that throughout this application, where the
singular "strain" is written, other embodiments may be practiced
with any one of multiple strains of modified bacteria; similarly,
where the plural "strains" is used, an embodiment may use a single
strain of bacteria
[0039] The invention utilizes methods 10 and 20 to select the
modified strain by passage through experimental animal tumor
xenografts until the most tumor-specific tumoricidal variant strain
for that individual patient's cancer is created.
[0040] According to methods 10 and 20, a fresh portion of tumor
harvested by surgical resection or biopsy is transplanted
subcutaneously into a quantity of nude mice. Mice of the
NOOD.CB17-Prkd.sup.cscid/NcrCrl ("NOD/SCID") type may be used;
other experimental animals may otherwise be available. This cohort
of engrafted animals serves to preserve and propagate the human
cancer tissue for the single or multiple sequential passaging of
the sequential S. typhimuruim A strains. These "reselected" strains
are denoted A1-R, A1-R.sub.1, A1-R.sub.2, A1-R.sub.3, A1-R.sub.n,
A1-R.sub.n+1, etc. "A1" denotes the S. typhimurium dual auxotroph
strain for Leu-Arg. The invention anticipates other auxotrophs and
non-auxotrophic genetic mutations may also be used. The xenografts
are allowed to become established, which may occur after any number
of days depending on the cell type of the tumor and its individual
phenotype. Not all transplanted tumors will "take" in the
experimental animal, although the prior art describes in detail
methods creating optimal conditions for successful orthotopic
grafting.
[0041] For embodiments of the method 10 utilizing one or more than
one selection cycles in vitro, a portion of xenograft is resected,
homogenized in phosphate buffered saline solution ("PBS"), and a
suspension of individual cancer cells is prepared using standard
techniques known to persons of skill in the art. Certain
embodiments may use cancer cells from a non-solid tumor source
including but not limited to patient blood (circulating tumor
cells), sputum (lung cancer), vaginal or endocervical swabs,
malignant ascites, and malignant pleural effusions. In one
embodiment, the tumor tissue is homogenized and diluted with PBS.
Aliquots containing a quantity of cancer cells are placed in wells
containing buffered tissue culture media and grown using standard
tissue culture techniques to an approximate density of 10.sup.4 per
well. In other embodiments, any range of cancer cell densities from
a single cell may be used, although a preferred range is 10.sup.1
to 10.sup.6 cells per well. The wells are then inoculated with a
quantity of parent strain S. typhimurium A1 or A1-R.sub.n,
depending on whether the selection cycle is the initial selection
using the A1 strain, or an A1-R.sub.n strain from a subsequent
passage cycle.
[0042] In embodiments of methods 10 and 20 utilizing bacteria
labeled with GFP, the parent strain is labeled using established
techniques within the body of prior art. In one example from the
prior art, the parent strain is grown at 37.degree. C. to mid
logarithmic phase in Luria-Bertani ("LB") medium and harvested at
4.degree. C. A quantity of cells (2.0.times.10.sup.8) in 40 .mu.L
glycerol (10%) are mixed with 2 .mu.L of the pGFP vector and placed
on ice for 5 minutes before electroporation with a Gene Pulser
apparatus according to the manufacturer's instructions.
[0043] After a period of incubation, cancer cells containing
intracellular bacteria and exhibiting signs of apoptosis are
selected from the original inoculated aliquots. The selected strain
is denoted as A1-R.sub.n+1. Embodiments of the invention may use A1
or A1-R.sub.n transfected to express GFP to infect tumor xenografts
expressing RFP in the cytoplasm and GFP in the nucleus through
retroviral transfection, also using standard techniques previously
disclosed and known to those with skill in the art. In this manner,
only bacteria that are phenotypically 1) able to successfully
infect individual cancer cells; and 2) kill those infected cells
are selected from the larger population of A1 mutated bacteria.
Alternatively, one may choose to use other mutated strains in other
embodiments of the invention.
[0044] In still another embodiment of methods 10 and 20,
tumor-infecting bacteria present following any xenograft passage
cycle are selected using an adherence and invasion assay. In these
embodiments, the adherence and invasion assay is performed to
select the initial strain based upon enhanced tumor targeting. In
an example adherence and invasion assay from the prior art, A1 or
A1-R.sub.n bacteria are grown to late-log phase in LB broth. The
bacteria are diluted in cell culture medium to a concentration of
1.times.10.sup.6, added to the cancer cells in tissue culture wells
and placed in an incubator at 37.degree. C. After 60 minutes, the
cells are rinsed five times with 1 to 2 mL PBS. Adherent bacteria
are released by incubation with 0.2 mL 0.1% Triton X-100 for 10
minutes. LB broth (0.8 mL) is then added, and each sample is
vigorously mixed. Adherent bacteria are quantified by plating in
order to count CFUs on LB agar medium. To select based on invasion,
the bacterially-infected cancer cells are rinsed five times with 1
to 2 mL PBS and cultured in a medium containing gentamicin sulfate
(20 .mu.g/mL) to kill external but not internal bacteria. After
incubation with gentamicin for 12 hours, the cells are washed again
with PBS.
[0045] The cancer cells containing the A1-R.sub.n bacteria are
homogenized and the supernatant plated onto LB or other
nutrient-enriched media to accommodate the requirements of the
auxotroph, and incubated at 37.degree. C. until growth is observed.
This is usually apparent after overnight incubation. Embodiments of
the invention utilizing a GFP-expressing bacterial strain allow for
more reliable selection of the desired bacteria whereby the plates
are examined under an excitatory light source, such as a blue LED.
The colonies originating from the selected-tagged bacteria will
fluoresce brightly. Regardless of whether this technique is used,
the one colony appearing to be pure and growing the most vigorously
(or the GFP-expressing colony that fluoresces most brightly) is
further selected for propagation in appropriate media using
standard techniques. This strain, selected from the parent strain
of S. typhimurium for its ability to infect and kill cells from the
specific individual malignancy of interest, is denoted
A1-R.sub.n.
[0046] For each successive xenograft passage cycle, a quantity of
the strain A1-R.sub.n is then inoculated into an experimental
animal(s) containing an established cancer tissue graft
(xenograft). By way of example, 5.times.10.sup.7 CFU is used, but
this concentration may be varied in other embodiments of the
invention. In alternative embodiments of the invention, the
bacterial inoculation is intravenous (tail vein injection), or
directly intra-tumoral. Following a period of incubation, generally
one to five days, the xenograft or a portion thereof is excised and
the tissue is homogenized and diluted in PBS using established
techniques. The suspension of cellular debris is allowed to settle
and the supernatant is then plated onto LB agar or other media to
accommodate the nutrient requirements of the auxotroph. Following
an adequate period of incubation (overnight is typical), colonies
are again selected for apparent purity and vigor. Again, colony
fluorescence is a useful adjunct to selection if GFP or other
labeled bacteria are used. The single most vigorous, or most
brightly fluorescing where GFP is used, colony is selected. The
selected colony is then propagated in LB or other appropriate media
using standard techniques. This strain, further selected from the
S. typhimurium A1-R.sub.n for its ability to target and infect the
tumor of interest, is denoted A1-R.sub.n+1.
[0047] The modified strain of S. typhimurium A1-R.sub.n (S.
typhimuruim A1-R.sub.n+1) that results is more tumor-specific for
that individual from whom the original cancer was harvested than
the preceding strain A1-R.sub.n. The foregoing discussion also
discloses the method for creating the strain of enhanced cancer
cell-targeting bacteria for individualized cancer therapy. In this
invention, the enabling disclosure of the modified strain is by
disclosure of the method for its creation.
[0048] In still other embodiments of the invention according to
methods 10 and 20, the individual tumor-targeting capacity of
strain A1-R.sub.1 is enhanced by additional passage of sequential
strains (i.e. R.sub.2, R.sub.3, etc.) through tumor xenografts in
the established cohort of nude mice (or other experimental
animals). This is accomplished by serial repetition of the final
four steps: inoculation of a quantity of said bacteria.sub.R1 into
a said experimental animal containing established said cancer
tissue graft; removing said cancer tissue graft form the third said
experimental animal following a period of in-vivo incubation;
extracting bacteria.sub.R2 from said cancer tissue graft removed
from the experimental animal; and incubating said bacteria.sub.R2
under conditions suitable for bacterial multiplication.
[0049] In this way, the invention overcomes a lack of specificity
of attenuated strains of facultative anaerobic bacteria, such as S.
typhimuruim A1, for infecting a specific tumor arising in a unique
individual. The invention exploits the retention of individual
diversity and genetic heterogeneity demonstrated by orthotopic
tumorgrafts (xenografts) in nude mice (or other suitable
experimental animal) by selecting for those bacteria with the
highest infectivity for that tumor's genetically unique population
of malignant cells. The selection process may be facilitated by
multiple sequential passage of the chosen bacterial strain through
tumor xenografts until selection of the most tumoricidal variant
results.
[0050] It is understood that although the modified strain of
bacteria selected by passage through tumor grafts in experimental
animals and/or cancer cells in vitro exhibits enhanced cancer cell
targeting of a specific malignancy arising in a unique individual
compared to a corresponding unmodified strain of bacteria, this
enhanced cancer cell targeting is not necessarily limited to that
individual malignancy.
[0051] With regard to labeling of the bacteria, methods 10 and 20
may each include various types of labeling. For example, and
without limitation, the bacteria may be labeled with a fluorescent
label; the bacteria may be labeled with a fluorophore; the bacteria
is labeled with a fluorescent protein; and the bacteria is labeled
with a fluorescent antibody.
[0052] With regard to labeling the harvested cancer cells, methods
10 and 20 may each include labeling the nuclei of the harvested
cancer cells with a fluorescent label; labeling the nuclei of the
harvested cancer cells with a fluorophore; labeling the nuclei of
the harvested cancer cells with a fluorescent protein; and labeling
the nuclei of the harvested cancer cells with a fluorescent
antibody. Further, methods 10 and 20 may comprise labeling the
cytoplasm of the harvested cancer cells with a fluorescent label;
labeling the cytoplasm of the harvested cancer cells with a
fluorophore; labeling the cytoplasm of the harvested cancer cells
with a fluorescent protein; and labeling the cytoplasm of the
harvested cancer cells with a fluorescent antibody. Finally,
according to methods 10 and 20, labeling nucleus and the cytoplasm
of the harvested cancer cells with fluorescent labels.
[0053] While the present invention has been shown for the treatment
of cancer generally, particular embodiments are useful in the
treatment of specific malignancy, such as, a pancreatic cancer, a
sarcoma, a lung cancer, a breast cancer, a colon cancer or a
prostate cancer.
[0054] The embodiments and examples set forth herein were presented
in order to best explain the present invention and its practical
application, and to thereby enable those of ordinary skill in the
art to make and use the invention. However, those of ordinary skill
in the art will recognize that the foregoing description and
examples have been presented for the purposes of illustration and
example only. The description as set forth is not intended to be
exhaustive or to limit the invention to the precise form disclosed.
Many modifications and variations are possible in light of the
teachings above, and are intended to fall within the scope of the
appended claims.
* * * * *