Title: Hyperthermia and immunotherapy for leukemias
lymphomas, and solid tumors
United States Patent: 6,524,587
Issued: February 25, 2003
Inventors: Lyday; Bruce W. (12851 Haster St., #1-D, Garden
Grove, CA 92840)
Appl. No.: 499050
Filed: February 4, 2000
An improved method of inducing whole-body hyperthermia and enhanced
anti-tumor immune response through inoculation of a fever virus with nil
mortality and subsequent injection of irradiated tumor cells derived from
the patient. This therapy will safely reduce the tumor burden by 90-99.9% by
physical means (fever), before raising interferon levels to over 250 times
baseline. The Activated Lymphokine Killer cells produced by these high
interferon levels are capable of killing any cell expressing viral or tumor
antigens, even those which had previously escaped immune surveillance. As a
final step in the process, a specific class of Cytotoxic T Lymphocytes
programmed to destroy the patient's own cancer cells will be produced by
repeated inoculation of irradiated cancer cells harvested from the patient.
Through a combination of three methods of therapy never previously
integrated into a single regimen, it is logical to state that this therapy
has a high probability of completely eradicating cancer cells from the
patient. In addition, this therapy provides for life-long immunity to the
reoccurrence of the disease.
DESCRIPTION OF INVENTION
Dengue virus is an RNA virus of the Togavirus Family, subfamily
flavivirus. It has an icosahederal geometry, approximately 40-45 nanometers
in diameter with two major envelope proteins. The E1 protein or Hemagglutin
is very rich in the amino acid glycine, and has a molecular weight of
approximately 45,000 daltons. The E2 protein or Neuraminidase is rich in the
amino acids alanine, serine, and valine and has a molecular weight or
approximately 50,000 daltons. The E3 protein is a transmembrane structure
which anchors the E1 and E2 proteins to the viral core proteins.
Neutralizing antibodies are primarily directed against the E1 protein.
MATERIALS AND METHODS
Male or female subjects with stage I, II, III, or IV carcinomas, leukemias,
Dengue Virus (available at Walter Reed Army Hospital, Washington, D.C.)
passaged in African Green Monkey Kidney cells to less than 5 plaque-forming
units /ml. DBS-FRhL-2 roller flasks are then inoculated with seed virus at a
minimum of infection or MOI of 0.0005. After adsorption for 1.5 hrs. at 35
degrees Centigrade, the inoculum is removed and flasks are washed three
times with 100 ml of Hanks balanced salt solution (HBSS). Maintenance medium
(200 ml per roller) consisting of Eagle minimal essential medium with 0.25%
human serum albumin, 0.22% NaHCO3, streptomycin (50 micrograms/ml), and
neomycin (100 micrograms/ml). Medium on all flasks is changed by day four,
and supernatant fluids harvested on day six. Before centrifugation at
1,050.times.g for 20 min., human serum albumin is to be added, resulting in
a final concentration of 2.75%. Albumin pH is to be adjusted before addition
to the viral fluids. As a final step in clarification, fluid is to be
filtered through a 0.45 micrometer membrane filter (Nalge, Rochester, N.Y.)
Samples are then to be tested for adventitious microbial agents to be
performed as described in Public Health Service regulations for licensed,
live-attenuated viral vaccines (Code of Federal Regulations, Chapter 21,
subchapter F, Biologics).
After removal of samples for testing and plaque assay, remaining volume to
be held in ice baths in a 4 degree C. refrigerator for 7 days pending
results of safety testing and plaque assays. A final pool of virus to be
made from fluids from flasks containing less than 5 plaque-forming units/ml
at 39.3 degrees C., and no detectable large-plaque virus present. Average
titer for vials to be 850,000 PFU/ml, then freeze-dried for use after
neurovirulence testing. Intraspinal and intracerebral bihemispheric
inoculation of 0.5 ml virus fluid of male rhesus monkeys with 2 controls
receiving virus-free culture fluids. Monkeys to be observed daily for 20
days for evidence of CNS involvement or other physical abnormalities.
Following sacrifice, histological examination of lumbar and cervical cord,
lower and upper medulla oblongata, mesencephalon, and motor cortex to be
made for viral pathology.
Preparation of Dendritic Cells
Bone Marrow cells are depleted for lymphoctytes and MHC Class positive cells
by Fluorescent Activated Cell Sorting (FACS) with monoclonal antibodies for
CD3, CD4, and CD8. Remaining cells are cultured overnight in Iscove's
modified Dulbecco's medium (IMDM) supplemented with 10% Fetal Calf Serum (FCS),
2-ME, and 100 IU/ml penicillin, 1 mM sodium pyruvate, and 10 uM nonessential
amino acids at 37 C in a 5% CO2 atmosphere in 24-well plates with
approximately 1 million cells in 1 ml culture medium/well. After 24 hours,
the cells are replated and cultured in the presence of Granulocyte-Macrophage
Colony Stimulation Factor (GM-CSF), and recombinant IL-4 at 900 U/ml. After
3 to 4 days, media to be exchanged for fresh cytokine media.
Alternatively, dermal dendritic cells (DDC) can be prepared using the
following methods: Keratomes from healthy human volunteers are incubated in
a solution of the bacterial proteases Dispase type 2 at a final
concentration of 1.2 U/ml in RPMI 1640 for 1 hour at 37 C. After the
incubation period, epidermis and dermis can be easily separated. Epidermal
and dermal sheets are then cut into small (1-10 mm) pieces after several
washing with PBS, and placed in RPMI 1640 supplemented with 10% Fetal Bovine
Serum (FBS), and placed in 10-cm tissue culture plates. After 2-3 days,
pieces of tissue are removed, and the medium collected. Cells migrating out
of the tissue sections into the medium are spun down, resuspended in 1-2 ml
fresh medium and stained with trypan blue. Further enrichment can be
achieved by separation on a metrizamide gradient. Cells are layered onto
3-ml columns of hypertonic 14.5% metrizamide and sedimented at 650 g for 10
minutes at room temperature. Low density interphase cells are collected and
washed in two successively less hypertonic washes (RPMI 1640 with 10% FBS
and 40 mM NaCl ) to return cells to isotonicity.
Peptide Pulsing of Dendritic Cells
The previously described immunodominant peptides of the gene products can be
constructed using a variety of methods, but one is described here. An Abimed
422 multiple peptide synthesizer at 10 micromole scale can be employed to
construct the required peptides. Alternatively, genetic engineering
techniques using cloning and expression vectors to yield milligram amounts
of the desired peptides can be employed. After the desired peptides have
been isolated in sufficient quantities, they must be loaded onto the DC. On
day 8, DC can be pulsed for 2 hours at 37 C in 1 ml of IMDM supplemented
with 0.5% BSA and 10 micrograms of peptide. After several isotonic washings,
the peptides are ready for use in humans. The DC can be stored for up to two
years at -50 C in liquid nitrogen stasis until ready for use.
Peptide Synthesis Procedure
Although many methods of synthesizing short chains of amino acids may be
employed, the following method is described.
Step 1 (Synthesis)
Reagent grade methylene chloride to be distilled from anhydrous potassium
carbonate Reagent grade dimethylformamide to be treated with 0.4 nm
molecular sieves 48 hours prior to use. Additional materials and sources:
(All materials are Reagent-Grade unless otherwise specified).
Material Commercial Source
Isopropanol Fisher Chemical
Triflouroacetic acid Eastman Chemical
Dicyclohexylcarbodiimide Vega Biochemicals
Hydroxybenzotriazole Sigma Chemical
Protected amino acids Vega
Benzhdyrlamine resin 0.33 mmol/g Pierce Biochemical
Side chains of threonine and glutamic acid to be protected with O-benzyl
groups, tyrosine by ortho-bromobenzyl-oxycarbonyl, and cysteine with S-para-methoxybenzyl.
The inital amino acid (e.g., aspartic acid), converted to
butoxycarbonyl-B-benzylaspartic acid, and coupled to the bezhydralamine
resin. Coupling reactions to be checked at each step using ninhydrin and/or
picric acid at the end of the cycle. Double-coupling will be necessary
residues of aspartic acid, cysteine, proline, tyrosine, and phenylalanine.
An equimolar amount of butoxycarbonyl-asparaginine to be included to prevent
formation of cyanoalanine.
Following the final amino acid coupling, the N-terminal butoxycarbonyl group
to be removed and the resin vacuum dried overnight to yield the peptide
resin (95-97% pure). The peptide can be cleaved from the resin and the
side-chain protective groups removed by the treatment of 1.0 g of resin plus
1.0 ml anisole with 20 ml liquid HF for 1.0 hour at 4 C. After removal of HF
at 4 C with a stream of Nitrogen, the excess anisole can be removed by
extraction with anhydrous ether (100) ml, with the resulting mixture to be
exposed to high vacuum in the presence of NaOH pellets to remove any
volatile HF. The crude peptide resin can be extracted with Nitrogen-deareated
5% HOAc (100 ml), to remove any remaining reagents. The resulting mixture to
be diluted with water to 20% HOAc and put through a Sephadex G-10 column
equilibrated with 20% HOAc. Final product to be lyophilized to yield final
Injection of Patients
Patients to be injected by hypodermic needle into dermis with 2 ml of viral
sample fluids once in each limb. After 2-3 days, patients to be infused by
intralymphatic microcatheter with pulsed Dendritics, repeat injections until
patient is negative for disease.
Operation of Invention
a. Hyperthermia and Thermosensitivity of Cancer Cells
Cancer cells are more susceptible to heat than normal tissue cells due to
1. Tumor Type
2. Tumor Location
3. Blood Supply
4. Growth Rate
5. Intracellular pH
6. Cellular Metabolic Level
Generally speaking, tumors arising from connective tissue (bone, cartilage,
muscle) termed sarcomas are more susceptible to hyperthermia than tumors
arising from lining or epithelial tissue called carcinomas. This is
primarily due to the relative blood supplies of the tissue types. Epithelial
tissue has a much richer blood supply than connective tissue, so carcinomas
are more thermoresistant. Blood cancers such as leukemias and lymphomas are
not susceptible to heat damage. Malignant melanomas share both
characteristics as it arises from lining tissue, but has a poor blood
The location is also critical, as limb tumors are easier to subject to
selective heating than those in the body core. Most prior-art hyperthermic
successes have been with limb perfusion of sarcomas. Tumors near an
extensive network of arterioles are less sensitive than those far away from
these main blood vessels.
The relative growth rate of the tumor is also critical. An aggressive,
fast-growing tumor needs a richer blood supply than a dormant one.
Fast-growing tumors also build up large pools of acidic metabolites, and
this factor becomes critical during hyperthermia. At high temperatures, the
spindle apparatus which stabilizes chromosomes during their replication is
subject to denaturation and collapse, leading to cell death.
At moderate levels of hyperthermia (103.5-106 degrees F.), or (39.5-41
degrees C.) such as are induced by dengue fever, thermosensitivity is
primarily due to indirect factors such as blood supply, intracellular pH,
and cellular metabolism rates. Tumors have generally poor blood supply
because they arise from a single cell, and soon outstrip the local blood
supply. Secretion of a hormone called angiotensinogen encourages local blood
vessel proliferation, but demand outpaces perfusion rates. In moderate to
large tumors, the interior core is dead due to inability to obtain Oxygen,
glucose, and other nutrients. Only the outer tumor cells are able to carry
out sufficient gas and nutrient exchange for survival.
Tumors also have a lower intracellular pH than healthy tissue due to low
perfusion to carry away toxic, acidic by-products of metabolism. Cells have
two routes to generate the AdenosineTriPhosphate (ATP) currency molecules
used to carry out functional reactions: Aerobic Metabolism and Glycolysis.
Aerobic Metabolism uses oxygen and yields 36 ATP molecules per glucose
molecule, with little acidic waste products. Glycolysis, on the other hand,
is much faster, but yields only 2 ATP per glucose and produces lactic acid,
which lowers intracellular pH.
Tumors have higher glycolysis rates than healthy cells because of their
excessive growth. Thus, the tumor microenvironment is characterized by
hypoxia (low Oxygen), acidosis, and nutrient deprivation. The stage is set
for exploiting these weaknesses through hyperthermia.
When body temperature rises, so do cellular metabolism rates. Since
enzymatic reactions generally increase as a function of temperature, cells
must take in more nutrients and flush away their toxic wastes. Healthy
cells, with normal perfusion rates, can easily stand temperatures of up to
41 degrees C. Tumor cells, on the other hand, are presented with a lose-lose
scenario. If they do not increase their glycolytic rates, they will starve.
If they do increase their glycolysis rate, their intracellular pH will fall
until it reaches 6.7, almost a full logarithmic point below bloodstream pH.
At 6.0, the enzymes responsible for critical cell reactions begin to fail,
especially the Na-K ATPase pump system. This enzyme is responsible for
maintaining the relative gradient of sodium and potassium ions across the
cell membrane. When it fails, sodium ions flood in, and the cell dies.
Lysosomes and peroxisomes are cytoplasmic membrane-bound bodies containing
proteases. At pH of 6.0 and below, these bodies rupture, digesting the cell.
Tumor sensitivity is both time and temperature-dependent, according to the
reaction: s=Soe-kt where s is survival at any given time t and So is
survival at initial time and k is a constant representing inactivation rate
at given temperature and t is the duration of incubation.
As can be seen from the above equation, cell survival decreases
logarithmically. In simple terms, the higher the temperature, the shorter
the time period needed for a 1 log or 90% tumor kill rate, a 2 log or 99%
kill rate, or a 3 log, or 99.9% kill rate.
Dengue fever produces temperatures sufficient to produce a 1-2 log reduction
in viable tumor cells, but a comparatively small number will survive if they
are located near high-perfusion vessels. It is now up to the immune response
to identify and eliminate the cells.
b. Active non-specific immunotherapy operation of invention Dengue fever
virus infects and reproduces in two kinds of white blood cells: immature
monocytes (which mature into macrophages), and B-Lymphocytes, which mature
into antibody-producing Plasma Cells. In the first three days on infection,
dengue kills 60% of the circulating white blood cells, dropping WBC counts
from 5300/ml to 2200/ml. When ruptured, these cells liberate massive amounts
of interferon, interleukins, and lymphocyte structural proteins into the
bloodstream. These lymphokines stimulate NK cells to become LAK cells, which
are capable to killing viral-and tumor-antigen expressing cells without
regard to specificity. In previous in vitro experiments, dengue-activated
LAK cells killed cells of the human tumor line K562 to a high degree. LAK
cells are capable of killing tumor cells that are resistant to NK cells, and
this is a critical factor in the operation of the invention.
Dengue also induces mature macrophages to become tumorcidal through a
lymphokine called MAF or Macrophage Activation Factor. In this state,
macrophages become Tumor-Infiltrating Leukocytes capable of killing
cancerous cells. Even though this response, following a 1-2 log kill through
hyperthermia, may very well eradicate tumor cells from a patient, yielding a
cure, it will eventually subside. To be completely certain that no cancer
cell survives the heat and the LAK/TIL response, a third component, one that
provides for lifelong anti-tumor surveillance and killing capability, is
c. Specific Anti-tumor Response of Invention
LAK cells, while possessing formidable tumorcidal properties, have no
immunological memory. After interferon levels return to normal, these
nonspecific killer cells have no capacity for "remembering" the antigenic
structure that triggered them to destroy a cell. That function of the immune
system is delegated to the antibody arm of the humoral immunity, and to the
Cytotoxic T Lymphocytes.
Cytotoxic T Lymphocytes are derived from the Thymus, and migrate to lymph
tissue. Although T4 Helper lymphocytes can be cytotoxic to tumor cells,
these recognize longer peptides presented by Class II MHC on a limited
number of cell types. T8 CTL recognize shorter (8-11 mer) peptides presented
by Class I MHC, present on all cell types except in "privileged" sites: CNS,
retina, and gonads. T8 CTL use their T-Cell Receptor (TCR), to evaluate
peptides presented by MHC proteins coded for by the HLA A and B alleles.
Proteins manufactured in the endoplasmic reticulum are periodically cut by
proteases, and peptides fitting the HLA binding motif are loaded between the
MHC chains. Thus stabilized, the trimeric complex eventually is embedded in
the cell membrane, where the peptide can be "read" by the TCR. Cells
producing viral proteins, or tumor-related peptides, can thus be eliminated
if sufficient numbers of CTL can be generated. These CTL are
memory-competent, and can identify and kill cells expressing their target
antigens for the lifetime of the patient.
Many cancer researchers have attempted to generate tumor-specific CTL with
limited success. The major difficulties arise from a lack of prior debulking,
the seven tumor immune evasion methods, and T-cell tolerance to
Elimination of 99% (2-logs) of the existing tumor cells is a requirement for
optimum results from immunotherapy. Chemotherapy using cytotoxic drugs,
gamma-radiation, and major surgery can debulk the tumor, but depress
cellular immunity. Patients sometimes are "cured" of their cancer by these
methods only to die from infection due to their depressed immune system.
Hyperthemia is the only reliable debulking method which does not suppress
cellular immune responses.
The Seven Tumor Immune Evasion Methods
If a therapy achieves 2-log debulking, CTL and LAK cells are often
unsuccessful in eliminating tumor cells due to the seven tumor immune
1. Down-Regulation of Class I MHC expression.
2. Point Mutations in tumor epitopes recognized by CTL.
3. Expression of fetal trophoblast HLA-G protein to avoid NK recognition.
4. Tumor microvessels are inhibitory to LAK and CTL migration.
5. Tumor cells induce monocytes to secrete IL-10, which renders CTL anergic.
6. Tumor cells express Fas Ligand (FasL), which can kill Fas+CTL.
7. Tumor cells erect fibrous stromal barriers to impede CTL movement.
The described invention is engineered to defeat all seven of these
mechanisms through the release of cytokines induced by the dengue virus.
Tumor cells often decrease or eliminate Class I MHC expression by
restricting the beta-2-microglobin, which forms the floor of the MHC trimer.
Without B-2m, the MHC is unstable and cannot bind peptide. Tumor cells also
decrease the transporter proteins TAP-1 and TAP-2, which carry the MHC
complex to the membrane. Dysfunction of these proteins leads to absent Class
I expression, rendering the tumor cell invisible to the T-Cell Receptor of
Dengue virus infection induces large amounts of interferons alpha, beta, and
gamma. IFN-gamma can restore Class I expression in tumor cells by binding to
the promoter regions of the B-2m and TAP genes. IFN-alpha also increases
Class I expression, and acts in synergy with IFN-gamma to elevate Class I
levels to normal.
Tumor cells often suffer from high mutation rates as a result of faulty gene
regulation, and mutations in target peptides recognized by tumor-specific
CTL can help the tumor cell avoid lysis. If the mutation occurs in an amino
acid required for anchoring the peptide to the MHC chain, that peptide can
no longer bind an HLA molecule. If the mutation occurs in a residue required
for TCR recognition, the peptide will still bind MHC, but will no longer be
recognized by the CTL.
Dengue infection results in high levels of two important cytokines: IFN-beta,
and IL-2. When CTL are exposed to these cytokines together, they lose target
specificity and acquire LAK-like lytic ability. These non-fastidious CTL can
lyse tumor types from many cell lines without HLA-restriction, so mutations
in tumor-related peptides should not allow escape.
Tumor cells that have decreased MHC expression are vulnerable to NK lysis.
Natural Killers use a receptor that transmits a negative signal when bridged
by Class I MHC. If the cell lowers its HLA expression, it can be killed by
NK cells. Fetal trophoblast cells express a protein termed HLA-G, which
protects from maternal NK lysis. Tumor cells that activate the HLA-G gene
while decreasing their Class I MHC levels can avoid both arms of the
cellular immune system
Dengue infection induces very high levels of IL-2, which drives CTL
proliferation. IL-2 also transforms NK cells into LAK, which can kill NK-resistant
tumor lines like the Renal Cell Carcinoma line Cur.
Tumor blood vessels, especially the High Endothelial Venules (HEV), often
lack the P and E selecting, which ligate the CD11 integrins found on
activated killer cells. Together with high hydrodynamic shear rates, this
prevents LAK and CTL from exiting the bloodstream to engage the tumor cell
Dengue infection induces high levels of two proinflammatory cytokines, IL-1
and Tumro Necrosis Factor-alpha (TNF-a). IL-1 and TNF-a up-regulate the
selectins on tumor vessels, and widen the gap junctions between vessel
endothelial cells. This allows the killer cells created by the therapy to
exit the HEV and destroy tumor cells.
Tumor cells often secrete Transforming Growth Factor-Beta (TGF-b), which
causes monocytes in the vicinity to secrete IL10. IL-10 renders CTL anergic,
so that they are no longer capable of engaging tumor cells. Dengue virus
selectively reproduces in and kills monocytes, and the high levels of IL-2
induced will reverse the anergic state of the tumor-infiltrating lymphocytes
CTL cloned in response to a pathogen often express Fas, or CD95. When
bridged by the Fas ligand (FasL), the CTL dies by DNA fragmentation
(apoptosis). Cells in "privileged" sites often express FasL to kill any CTL
intruding into sensitive areas. Tumor cells that express FasL can kill
responding CTL, allowing for unchecked tumor growth.
Dengue infection induces high amounts of IL-6, which promotes expression of
FLIP (Fas-Ligand inhibitory Protein). FLIP acts as a "safety switch"
covering the Fas self-destruct trigger on CTL. As long as IL-2 levels are
high, FLIP will protect CTL from FasL. IL-2 levels in dengue infection stay
high for over 30 days.
Tumor cells secrete factors promoting growth of fibrovascular bundles called
stroma. This dense network of collagen bundles can impede CTL mobility. The
high levels of IFN-gamma induced by dengue infection activate macrophages to
express CD44, which digest stromal barriers with hyaluronidase enzymes. The
activated macrophages will be drawn to tumor areas by chemotactic signals
released by tumor-specific T4 and T8 CTL.
Overcoming T-cell Tolerance to Tumor-Related Peptides
The therapy described herein is unique due to its design to defeat each
tumor immune evasion method. A therapy achieving the first two goals,
debulking and defeating the tumor immune evasion mechanisms, has one other
barrier to overcome: T-cell Tolerance.
In the last months in utero, and for the first 18 months of life, the T-cell
immune system undergoes a cycle of clonal selection and deletion. T4 and T8
cells with receptors that have a high affinity for self-peptides presented
by the thymic epithelium are eliminated (deletion). Only T-cells with low
affinity for self-peptides are selected for, and leave the Thymus to reside
in peripheral lymph tissue. This is an important safeguard against
autoimmunity, but tumor cells take advantage of this system. If the tumor
arouses an immune response, the CTL created will have low affinity for the
peptide, and so exhibit low cytotoxicity against the tumor cells. Combined
with the immune evasion methods, this explains the low rates of success in
Clonal deletion is a less than perfect process, or autoimmune diseases would
be unknown. There is a growing body of evidence suggesting that tolerance
can be overcome, given the proper conditions. The first requirement is for
efficient presentation of antigen to the T-cells. Macrophages can function
as antigen-presenting cells (APC), but the most efficient APC are Dendritic
Cells (DC), derived from bone marrow. DC capture antigen, process it into
target peptides, then present the peptide to T-cells. The CTL then mold
their TCR to recognize the peptide, and become antigen-specific.
DC can prime CTL at rates of 1:1000, so 10 million DC pulsed with tumor
peptides can generate 10 billion CTL. Following a 2-log reduction through
hyperthermia from an initial tumor burden of 10 billion cells, this will
result in 100 CTL to each remaining tumor cell.
The second requirement for overcoming T-cell tolerance is high levels of
Th1-type cytokines: IFN-gamma, IL-2, IL-7, and IL-12. CTL expansion is
limited by two factors: APC and Th1 cytokine levels. Dengue infection
induces the high levels required.
Tumor-Associated Antigens (TAA)
Another major drawback to successful tumor immunotherapy is the scarcity of
TAA to serve as targets for CTL. Unlike the peptides captured by HLA in
virus-infected cells, TAA are generally synthesized in lower amounts. Each
cell has approximately 200,000 copies of a specific HLA type, HLA-A2, for
example. Each peptide fragment must compete with 10,000 others for HLA-binding,
and a minimum of 200 MHC complexes presenting the peptide are required for
Melanoma has the best-characterized TAA: Melan-A/MART-1, gp100, pmel7, and
the MAGE group antigens, which are expressed on other tumor types. The
following tumor types are listed, with specific TAA that can be loaded onto
allogenic DC to prime tumor-specific CTL:
Adenocarcinoma of the Breast
The most frequently expressed TAA by breast cancers are CarcinoEmbryonic
Antigen (CEA), and HER-2/neu, a proto-oncogene. CEA is a large fetal
oncoprotein expressed in large amounts in various adenocarcinomas, but in
only minor amounts in ordinary small intestinal epithelium tissue. An
example of a target peptide is YLSGANLNL (SEQ ID NO:1), restricted by
HLA-A2. HER-2/neu is overexpressed in many tumor types, and is a large (1255
amino acids) protein with many suitable targets for CTL. An example of a
target peptide is E75, containing residues 369-377, KIFGSLAFL (SEQ ID NO:2).
HER-2/neu is expressed up to 200-fold in tumor cells from breast, ovarian,
and lung tissue.
Cervical cancers are associated with the Human Pappiloma Virus (HPV) types
17 and 19. These DNA viruses are genetically stable, unlike the RNA viruses
Influenza A and HIV-1. HLA-restricted HPV peptides have been identified as
targets for CTL.
Colon cancer shares similar targets with breast cancer: CEA and HER-2-neu.
Stomach cancer cells have been found to over-express HER-2/neu.
Head and Neck Cancer
Head and Neck Squamous Epithelial cancers have been found to over-express
the melanoma-associated peptides from the MAGE-1 and MAGE-3 genes.
Leukemias and Lymphomas
Although unaffected by hyperthermia, as they are floating in an oxygen-rich
medium, leukemic and lymphoma cells are vulnerable to LAK cells. They also
express peptides from the oncogenes N-ras and K-ras, associated with Chronic
Myelogenous Leukemia and Acute Myeloid Leukemia. T-cell leukemias are
frequently caused by the Human T-Lymphotropic Leukemia viruses HTLV-1 and
II. The viral peptides presented by HLA in leukemic cells can serve as
targets for CTL.
The Epstein-Barr Virus is a large DNA virus associated with nasopharyngeal
carcinoma and Non-Hodgkin's Lymphoma. The peptides from this virus are
expressed by the cancer cells and can serve as targets for CTL.
Lung cancers are divided into squamous and adenocarcinomas. Target peptides
restricted by Aw68 have been isolated from squamous lung cancers, and
adenocarcinomas share peptides from the HER-2/neu, MAGE, GAGE, and related
Prostate-Specific Antigen has been used as a diagnostic marker for prostate
cancer, but it also contains CTL epitopes. Examples restricted by HLA-A2 are
PSA-1, and PSA-3.
Ovarian cancer cells have been found to express HER-2/neu and CEA.
Pancreas cancer cells have been found to express HER-2/neu and CEA.
Renal Cell Carcinoma
Renal Cell Carcinomas share many melanoma-related peptides, including MAGE
Uterine cancer cells have been found to express HER-2/neu and CEA.
Tumor-Related Peptides Expressed as Part of the Carcinogenesis Process p53
Tumor Suppressor Protein
The product of the p53 gene is a cornerstone of the carcinogenesis process.
Its product is a tetrameric protein that binds to DNA. This acts as a
barrier to RNA Polymerase II binding, preventing transcription of an
oncogene. Mutations arising from UV radiation, chemical carcinogens,
infection with a tumor-causing virus like SV40 can inactivate p53.
Inactivated p53 can no longer prevent transcription, and the oncogene
becomes activated, transforming the cell into an immortal, rapidly
proliferating tumor cell.
Mutated p53 genes are found in over 50% of all cancers, and peptides from
these genes are HLA-restricted. CTL specific for mutated p53, or even for
tumor cells over-expressing normal p53, can eradicate tumors in mice. For
this type of peptide to be used in cancer patients, the Class I MHC types
can be compared to cDNA libraries of p53, then the Polymerase Chain Reaction
(PCR), can be used to confirm the presence of mutated p53 in tumor tissue
The second general TAA is the enzyme telomerase. The ends of chromosomes are
capped by repeating units of telomeric DNA. This prevents unraveling of the
chromosome, as well as interchromosomal binding. Telomeric DNA is lost each
time a cell divides, and due to the semi-conservative nature of DNA
replication, they cannot be replaced by DNA Polymerase I. This appears to
serve as a cellular clock, limiting the number of times a cell can divide.
When the telomeres reach termination, the cell goes into arrest, unable to
Cancer cells, by definition, proliferate rapidly in defiance of restraints
against such activity. To add telomeres, they must synthesize large amounts
of the enzyme telomerase. Close to 90% of tumor cell lines show high
telomerase expression, and the catatlytic sub-unit, (hTERT) is an HLA-restricted
protein capable of serving as a target for CTL. Since telomerase is not
usually synthesized by normal cells, it represents a widely-expressed TAA
Although most TAA are present in some normal tissue types, especially
rapidly proliferating ones, they are made in minute amounts. Because the TAA
have to compete with 10,000 peptides derived from normal proteins, the HLA
presentation of these is low.
Conclusion, Ramifications, and Scope
Accordingly, the reader will see that the combined therapy previously
described provides a safe and effective means for eliminating cancer cells
from a human body. By combining three methods known to medicine as
beneficial to cancer patients, the therapy solves the dilemma posed by
current chemotherapy, radiation, and surgery. The above and various other
objects and advantages of the present invention are achieved without undue
risk to the patient, as full-strength wild-type dengue virus has a mortality
rate given by various Tropical Medicine texts as nil, nonexistent, and 1 in
61,000. No other reference to injecting volunteers with any other
full-strength virus could be found in any journal.
Although the description above contains many specifics, these should not be
construed as limiting the scope of the invention but as merely providing
illustrations of some of the presently preferred embodiments of this
Thus the scope of this invention should be determined by the appended claims
and their legal equivalents, rather than by the examples given.
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. Although any materials and methods
similar or equivalent to those described herein can be used in the practice
or in vitro and in vivo testing of the present invention, the preferred
methods and materials are described. All publications mentioned hereunder
are incorporated herein by reference. Unless mentioned otherwise, the
techniques employed herein are standard methodologies well known to one of
ordinary skill in the art.
The term "substantially pure" as used herein means as pure as can be
obtained by standard purification techniques.
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 2
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Target peptide for human CEA.
<400> SEQUENCE: 1
Tyr Leu Ser Gly Ala Asn Leu Asn Leu
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<223> OTHER INFORMATION: Target peptide E75 for human HER-2/neu.
<400> SEQUENCE: 2
Lys Ile Phe Gly Ser Leu Ala Phe Leu
Claim 1 of 30 Claims
1. A method of reducing tumor burden in a cancer patient with a human solid
tumor, comprising the steps of:
injecting said patient with substantially pure Dengue Virus; and
maintaining body temperature of said patient in excess of 103.5 degrees F.
for a sufficient period of time to reduce tumor burden, wherein said Dengue
virus injection maintains said body temperature in excess of 103.5 degrees.
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