U.S. patent application number 14/844882 was filed with the patent office on 2015-12-31 for method of therapy.
The applicant listed for this patent is Biotempus Limited. Invention is credited to Martin Leonard Ashdown.
Application Number | 20150377882 14/844882 |
Document ID | / |
Family ID | 34468632 |
Filed Date | 2015-12-31 |
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United States Patent
Application |
20150377882 |
Kind Code |
A1 |
Ashdown; Martin Leonard |
December 31, 2015 |
METHOD OF THERAPY
Abstract
Numerous diseases have been linked to the production of
regulator cells. The present invention relates to the observation
that the immune system is cycling in these diseases. Based on these
observations, the present invention provides methods for treating
diseases such as cancer and a HIV infection. The present invention
also relates to methods of determining when a therapy to treat a
disease characterized by the production of regulator cells should
be administered to a patient.
Inventors: |
Ashdown; Martin Leonard;
(Victoria, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biotempus Limited |
Victoria |
|
AU |
|
|
Family ID: |
34468632 |
Appl. No.: |
14/844882 |
Filed: |
September 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14063104 |
Oct 25, 2013 |
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14844882 |
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10576981 |
Mar 2, 2007 |
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PCT/AU04/01456 |
Oct 22, 2004 |
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14063104 |
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Current U.S.
Class: |
424/184.1 ;
424/173.1; 514/274; 514/44A; 514/492 |
Current CPC
Class: |
G01N 33/543 20130101;
A61K 31/282 20130101; G01N 33/57446 20130101; G01N 33/56988
20130101; A61P 35/00 20180101; A61P 31/00 20180101; A61P 31/18
20180101; G01N 33/57407 20130101; G01N 33/57449 20130101; A61K
31/513 20130101; G01N 33/574 20130101; G01N 33/56972 20130101; A61P
31/20 20180101; G01N 2333/4737 20130101; G01N 33/57484 20130101;
A61P 31/14 20180101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; A61K 31/513 20060101 A61K031/513; A61K 31/282 20060101
A61K031/282; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 24, 2003 |
AU |
2003905858 |
Claims
1-44. (canceled)
45. A method of treating a disease characterized by the production
of regulator T cells, wherein a patient suffering from the disease
has been analyzed for immune system cycling by monitoring the
patient for a regular oscillation of at least one of: a) number
and/or activity of regulator T cells, b) number and/or activity of
effector T cells, c) a marker molecule associated with the disease,
and/or d) an immune system marker, and comprising administering an
agent to treat the disease at a time selected such that the agent
exerts a proportionally greater effect against the regulator T
cells than the effector T cells, wherein the agent inhibits the
production of, limits the function of, and/or destroys, regulator T
cells, and wherein the agent is selected from the group consisting
of anti-proliferative drugs, radiation, dsRNA and antibodies which
inhibit the production and/or activity of regulator T cells, and
wherein the disease is cancer or an infection.
46. The method of claim 45, wherein the infection is a chronic
persistent infection characterized by the patient's immune system
not being able to eliminate the infection.
47. The method of claim 46, wherein the patient is infected with
HIV or Hepatitis C virus.
48. The method of claim 45, wherein the immune system marker
reflects the number and/or activity of regulator T cells, and/or
the number and/or activity of effector T cells.
49. The method of claim 45, wherein the immune system marker is an
acute phase inflammatory marker.
50. The method of claim 49, wherein the acute phase inflammatory
marker is selected from the group consisting of serum amyloid A,
serum amyloid P and c-reactive protein.
51. The method of claim 45, wherein the regulator T cells are
CD4+CD8- T cells.
52. The method of claim 45, wherein the agent is administered about
when CD4+CD8- T cells are detected.
53. The method of claim 45, wherein the effector T cells are
CD8+CD4- T cells.
54. The method of claim 45, wherein the agent is administered
approximately when CD8+CD4- T cell numbers have peaked.
55. The method of claim 45, wherein the marker molecule associated
with the cancer is an antigen produced by a cancer cell.
56. The method of claim 45, wherein the marker molecule associated
with the infection is an antigen produced by an infectious
agent.
57. The method of claim 45, wherein the agent is administered
approximately when levels of the marker molecule associated with
the cancer or infection begin to decrease.
58. The method of claim 45, wherein the patient is monitored for an
acute phase inflammatory marker, and a marker molecule associated
with the cancer or infection.
59. The method of claim 45, wherein the patient is monitored for a
period of at least 21 days.
60. The method of claim 45, the patient is monitored at least about
every 3 days.
61. The method of claim 45, wherein the anti-proliferative drug is
selected from the group consisting of taxol, vincristine,
vinblastine and anhydro vinblastine.
62. The method of claim 45, wherein the antibody is selected from
the group consisting of: anti-CD4+, anti-CTLA-4 (cytotoxic
lymphocyteassociated antigen-4), anti-GITR (glucocorticoid-induced
tumour necrosis factor receptor), anti-CD28 and anti-CD25.
63. The method of claim 45, wherein the patient has not been
exposed to a treatment for the cancer or infection for at least 21
days.
64. The method of claim 45, wherein the patient is a human.
65. A method of treating a disease characterized by the production
of regulator T cells, wherein a patient suffering from the disease
has been analyzed for immune system cycling by monitoring the
patient for a regular oscillation of at least one of: a) number
and/or activity of regulator T cells, b) number and/or activity of
effector T cells, c) a marker molecule associated with the disease,
and/or d) an immune system marker, and comprising administering a
vaccine to treat the disease at a time selected such that the
vaccine boosts the innate immune response, producing increased
numbers and/or activity of effector T cells, before the emergence
of regulator T cells, and wherein the disease is cancer or an
infection.
66. The method of claim 65, wherein the vaccine is administered
about when the levels of effector T cells are increasing.
67. The method of claim 65, wherein the vaccine is administered
about when the levels of a marker molecule associated with the
disease begin to decrease.
Description
FIELD OF THE INVENTION
[0001] Numerous diseases have been linked to the production of
regulator cells. The present invention relates to the observation
that the immune system is cycling in these diseases. Based on these
observations, the present invention provides methods for treating
diseases such as cancer and a HIV infection. The present invention
also relates to methods of determining when a therapy to treat a
disease characterized by the production of regulator cells should
be administered to a patient.
BACKGROUND OF THE INVENTION
[0002] In the past, attempts have been made to trigger the immune
system to mount an efficient response against malignant cells.
Despite significant and promising progress, such a response has yet
to be fully attained and many immune based therapies have proved
disappointing.
[0003] Numerous studies using in vitro cellular assays demonstrate
that cytotoxic lymphocytes have the ability to kill tumour cells.
Why this immune based destruction does not effectively control
tumour growth in vivo is a conundrum. The cancer patient also has
increased concentration of circulating immune complexes, indicating
the immune system is active, particularly against certain tumour
antigens. The level of these immune complexes can increase with
disease progression (Horvath et al, 1982; Aziz et al, 1998).
[0004] Regulatory cells (also referred to in the art as suppressor
cells) have been implicated in a subjects immune response to cancer
(North and Awwad, 1990; WO 03/068257). As most cancer antigens are
actually produced by the patient they are considered as "self" by
the immune system. Upon the presence, and/or increased quantity, of
tumour antigen the hosts immune system mounts a response
characterized by the production of effector cells which target
cells producing the tumour antigen. However, in many instances
these effector cells are recognized by the immune system as
targeting the hosts own cells, and hence a population of regulator
cells are produced to down-regulate the effector cell population.
Thus, the production of these regulator cells limits the ability of
the immune system to effectively remove cancer cells.
[0005] More recently, regulator cells have been shown to be
involved in a subjects immune response to a viral infection. WO
02/13828 describes the production of regulator cells during
retroviral infection, and methods of treating such infections by
down-regulating the regulator cell population whilst maintaining
the effector cell population. Furthermore, Peterson et al (2002)
observed that a population of CD4+ regulator cells were suppressing
the ability of CD8+ effector cells to control Friend murine
retrovirus infections in mice.
[0006] Measurements of certain acute-phase protein plasma
concentrations can be of diagnostic or prognostic value under
specific clinical conditions. The best known acute-phase protein is
C-reactive protein (CRP). CRP is a plasma protein that rises in the
blood with the inflammation from certain conditions. The level of
CRP in blood plasma can rise as high as 1000-fold with
inflammation. Conditions that commonly lead to marked changes in
CRP include bacterial and viral infection, trauma, surgery, burns,
inflammatory conditions, coronary and vascular disease and advanced
cancer.
[0007] Most acute phase proteins are synthesized by hepatocytes,
some are produced by other cell types, including monocytes,
endothelial cells, fibroblasts and adipocytes. Acute phase proteins
include serum amyloid A (SAA), CRP and serum amyloid P component
(SAP).
[0008] The immediate responsiveness of CRP and SAA to stimuli,
together with their wide concentration range and ease of automated
measurement, have led to plasma CRP and SAA levels being used to
monitor accurately the severity of inflammation and the efficacy of
disease management during certain disease conditions.
[0009] WO 03/070270 describes the use of acute phase inflammatory
markers in regimes for the effective treatment of HIV. These
methods rely on at least partially "resetting" the immune system by
a treatment such as HAART followed by the analysis of acute phase
inflammatory proteins as markers for effector/regulator cell
expansion. The emergence of acute phase inflammatory proteins
appears to be linked to effector cell expansion, which occurs
before regulator cell expansion, and thus the patient can be
treated with a suitable agent which allows the effector cell
population to be maintained whilst destroying, preventing the
production of, or reducing the activity of, regulator cells. In
essence, upon withdrawal of HAART treatment it was considered that
the patients immune system would treat the re-emerging HIV
particles as a new infection, and hence a new population of
effector cells would be produced.
[0010] Similar to WO 03/070270, WO 03/068257 relates to at least
partially resetting the immune system, however, in this instance in
the context of the treatment of cancer. Again, the treatment is
focussed on the initial re-emergence of effector cells following a
reduction in tumour load through techniques such as surgery or the
administration of anti-proliferative drugs.
[0011] Neither WO 02/13828, WO 03/070270 or WO 03/068257 appreciate
that the immune response is cycling in a cancer or HIV patient
regardless of the administration of treatment for these diseases.
The present invention is based on the realization of this cycling,
and thus provides methods for the treatment of diseases linked to
regulator cell production or activity.
SUMMARY OF THE INVENTION
[0012] The present inventor has surprisingly found that the immune
system is cycling during disease states characterized by the
presence of regulator cells. This cycling occurs on a regular basis
of approximately 14 to 15 days in humans.
[0013] Whilst not wishing to be limited by theory, it appears that
effector cell expansion against a target antigen is followed by the
expansion of regulator cells directed against the effectors. Upon
control of the effector cells by the regulator cells the numbers
and/or activity of both types of cells decrease, which in turn is
followed by the same cycle due to the continuous presence or
incomplete removal of antigen which results in an oscillating
persistent, but ineffective, immune response against the, for
example, tumour or virus.
[0014] Knowledge of this cycle can be used to treat diseases where
it is known that the emergence of regulator cells is detrimental to
the patient. Examples of such diseases include cancer and
persistent infections such as by the human immunodeficiency virus.
More specifically, treatment of a patient can be timed such that
effector cell numbers against a cellular or viral antigen are
maximized whilst regulator cell numbers are reduced or
abolished.
[0015] In fact, the present inventor has noted that the treatment
of a wide variety of cancers with anti-cancer drugs results, on
average, in a complete response rate in the range of 6.5 to 7%.
This range of 6.5 to 7% is consistent with an about 14 to 15 day
cycle of effector cell expansion followed by regulator cell
expansion. More specifically, when not taking into consideration
the cycling of effector and regulator cells, a medical practitioner
has an approximate 1 in 14.5 chance (6.8%) of administering an
anti-proliferative drug at a time where effector cells numbers are
high but regulator cell numbers have only begun to expand and hence
are vulnerable to treatments which target dividing cells. This
leaves high numbers of effector cells which target the cancer
cells, resulting in a complete response to the therapy.
[0016] Accordingly, in a first aspect the present invention
provides a method for determining when an agent should be
administered to a patient suffering from a disease characterized by
the production of regulator cells, the method comprising monitoring
the patient, or samples obtained therefrom, for at least one of: a)
effector cell numbers and/or activity, b) regulator cell numbers
and/or activity, c) a molecule associated with the disease, and/or
d) an immune system marker.
[0017] In another aspect, the present invention provides a method
of treating a disease characterized by the production of regulator
cells, the method comprising;
[0018] i) monitoring a patient suffering from the disease for at
least one of: [0019] a) number and/or activity of regulator cells,
[0020] b) number and/or activity of effector cells, [0021] c) a
molecule associated with the disease, and/or [0022] d) an immune
system marker, and
[0023] ii) exposing the patient to an agent to treat the
disease,
wherein the timing of administration of the agent is selected such
that the activity of effector cells is not significantly
reduced.
[0024] Preferably, the disease characterized by the production of
regulator cells is selected from, but not limited to, cancer and an
infection.
[0025] The infection can be caused by any type of infectious agent
such as, but not limited to, a virus, bacteria, protozoa, nematode,
prion, or fungus. Preferably, the infectious agent causes chronic
persistent infection characterized by the patient immune system not
being able to eliminate the infectious agent. Examples of
infectious agents which cause chronic persistent infection are
viruses such as HIV, the Hepatitis B virus and the Hepatitis C
virus.
[0026] Whilst not wishing to be limited by theory, it appears that
as antigen load, for example from increased tumour growth or viral
replication, increases following regulator cell activity the
patients immune system responds in a manner similar to a first time
exposure to the antigen. This immune response includes the
production of acute phase inflammatory markers such as serum
amyloid A and c-reactive protein.
[0027] An appropriate time to administer the agent is between when
the levels of acute phase inflammatory marker have peaked and
before the marker begins to rise in the next cycle. Accordingly, a
particularly preferred immune system marker is an acute phase
inflammatory marker. More preferably, the acute phase inflammatory
marker is selected from, but not limited to, the group consisting
of serum amyloid A, serum amyloid P and c-reactive protein.
[0028] Preferably, the immune system marker reflects the number
and/or activity of regulator cells, and/or the number and/or
activity of effector cells.
[0029] In one embodiment, the patient is monitored for an increase
in the number and/or activity of regulator cells by the analysis of
CD4+CD8- T cell levels. With regard to this embodiment, it is
preferred that the agent is administered about when CD4+CD8- T
cells are detected.
[0030] In another embodiment, the patient is monitored for an
increase in the number and/or activity of effector cells by the
analysis of CD8+CD4- T cell levels. With regard to this embodiment,
it is preferred that the agent is administered approximately when
CD8+CD4- T cell numbers have peaked.
[0031] In another embodiment, the molecule associated with the
disease is an antigen produced by a cancer cell or an infectious
agent. In this embodiment, the agent is administered approximately
when levels of the molecule associated with the disease begin to
decrease.
[0032] In a further embodiment, the disease is cancer and the
patient is monitored for fluctuations in the levels of tumour
antigen(s). With regard to this embodiment, it is preferred that
the agent is administered approximately when levels of tumour
antigen begin to decrease.
[0033] In yet a further embodiment, the disease is caused by an
infectious agent and the patient is monitored for fluctuations in
the levels of antigen(s) produced by the infectious agent. With
regard to this embodiment, it is preferred that the agent is
administered approximately when levels of antigen, or infectious
organisms or viruses (viral load), begin to decrease.
[0034] In another embodiment, the immune system marker is body
temperature. With respect to this embodiment, it is preferred that
the agent is administered when body temperature has peaked and
before body temperature begins to rise in the next cycle.
[0035] As outlined herein, the present inventor has noted that
fluctuations in numerous factors indicate that the immune system is
cycling in patients suffering from a disease characterized by the
production of regulator cells. These factors include acute phase
inflammatory markers, viral antigens, cancer antigens and body
temperature. These factors are linked, directly or indirectly, to
the general state of the immune system including, but not
necessarily limited to, effector cell production and/or activity,
regulator cell production and/or activity, and/or B cell production
and/or activity.
[0036] It will be appreciated by the skilled person that diseases
such are cancer and AIDS have a complex effect on the patient.
Furthermore, natural variations between individuals linked to
factors such as their genotype, nutrition, fitness, previous and
current disease status, all influence how a given individual
responds to a disease state. Thus, whilst in most cases the cycle
will be about 14 to 15 days, in some individuals this may be
slightly shorter or longer. In addition, like the menstrual cycle,
the length of the cycle may vary slightly within an individual due
to natural variation and/or environmental factors. Thus, individual
variation may at least be encountered with regard to, for example,
i) the length of the cycle, ii) the absolute numbers of effector or
regulator cells during the cycle, or iii) the levels of acute phase
inflammatory markers during the cycle. Such variation may be
exaggerated in patients with advanced cancer or infection, where
the patients immune system has been challenged for a considerable
length of time.
[0037] As result, it will most likely be desirable to monitor the
patient for a sufficient length of time to ensure that the dynamics
of the immune system cycling within a particular patient is
understood. Preferably, the patient is monitored for a period of at
least 7 days, more preferably at least 14 days, more preferably at
least 21 days, more preferably at least 28 days, more preferably at
least 35 days, more preferably at least 42 days, and even more
preferably at least 49 days.
[0038] Another complicating factor is that at least the levels of
some acute phase inflammatory markers have been found to cycle
about every 7 days (about half the length of a "full" immune system
cycle). Thus, it appears that relying on these types of markers
will improve the chance of successful treatment from about 6.8%
(based on random administration of the agent) to about 50% (based
on choosing the correct administration time by randomly choosing
which of the peaks is linked to the appropriate time to target
regulator cells). Whilst this is an improvement on current
techniques, it is preferred that such markers are monitored
inconjunction with other factors (for example, a molecule
associated with the disease, regulator cells and/or effector cells)
to optimize the chance of selecting the appropriate time to
administer the agent.
[0039] Thus, in another embodiment, the patient is monitored for an
acute phase inflammatory marker, and a molecule associated with the
disease. With regard to this embodiment, the agent is administered
between when the levels of the acute phase inflammatory marker have
peaked and before the marker begins to rise in the next cycle, and
when levels of the molecule associated with the disease begin to
decrease or would have been predicted to begin to decrease based
upon previous analysis of the molecule.
[0040] In general, it is preferred that numerous factors are
monitored at the same time. This is because, due to the factors
describe above, it is unlikely that each factor will have a perfect
cycle profile within a 14/15 day period, particularly over a number
of cycles, to routinely provide a clear indication of the
appropriate time to administer the agent. Whilst the analysis of
numerous factors of a long period may be costly, and may be of at
least some inconvenience to the patient, diseases such as cancer
and AIDS are life threatening. Hence it is worthwhile understanding
as much as possible regarding immune system cycling in a given
patient before the patient is treated.
[0041] In addition, although the analysis of different factors
cycling in some patients may result in complex profiles, given the
guidance provided herein it is well within the skill of the medical
practitioner to analyse the monitoring data to determine the
optimal time to administer the agent. An Example of the careful
analysis of multiple factors to determine the appropriate time to
effectively treat a disease characterized by the production of
regulator cells is provided herein.
[0042] A further complicating factor will be if the patient has
recently acquired a disease or trauma unrelated to that being
treated. For example, a patient being treated for a HIV infection
may also contract the common flu virus. The presence of the flu
virus will result in, for example, an increase in acute phase
inflammatory markers independent of the cycling of these markers
which is occurring due to the HIV infection. Other diseases which
may cause complications in monitoring effector/regulator cell
cycling for use in the methods of the present invention include,
rheumatoid arthritis, ulcers and chronic gum disease. Accordingly,
it is desirable to monitor the patient for any factors which may
result in elevated levels of, for example, acute phase inflammatory
markers to ensure that the factor being monitored truly reflects
effector/regulator cell cycling resulting from the disease being
treated.
[0043] Furthermore, it is preferred that the patient is monitored
as frequently as possible to ensure immune system cycling within a
given patient is suitably characterized. Naturally this will ensure
that the agent is administered at the appropriate time and that any
small variations in, for example, effector/regulator cell numbers
or activity, or markers thereof, is not misinterpreted. Preferably,
the patient is monitored at least every 3 days, more preferably at
least every 2 days, and most preferably at least every day.
Monitoring may occur more frequently, for instance every 12 hours,
when the cycling is reaching a stage where it is likely that the
timing would be appropriate to administer the agent.
[0044] Preferably, the agent inhibits the production of, limits the
function of, and/or destroys, regulator cells. More preferably, the
agent is selected from the group consisting of anti-cancer drugs
such as anti-proliferative drugs, radiation, dsRNA and antibodies
which inhibit the production and/or activity of regulator cells.
Preferably, the anti-proliferative drug is selected from the group
consisting of, but not limited to, taxol, vincristine, vinblastine
and anhydro vinblastine.
[0045] With regard to cancer, in contrast to typical anti-cancer
drug therapy which is administered to target tumour cells, the
method of treatment described herein actually targets regulator
cells. This leaves suitable numbers of effector cells to produce
the desired therapeutic effect.
[0046] Examples of preferred antibodies include, but are not
limited to, anti-CD4+, anti-CTLA-4 (cytotoxic lymphocyte-associated
antigen-4), anti-GITR (glucocorticoid-induced tumour necrosis
factor receptor), anti-CD28 and anti-CD25.
[0047] Preferably, the patient has not been exposed to a treatment
for the disease for at least 14 days, more preferably at least 21
days, and even more preferably at least 28 days.
[0048] The present inventor has also determined that treatment for
a disease characterized by the production of regulator cells can be
enhanced (or the chances of successful treatment can be increased)
when the vaccine is administered at the appropriate time. In these
instances, the vaccine boosts the innate immune response against
the disease. This will most likely be a result of increased numbers
and/or activity of effector cells. Although theoretically regulator
cells will still ultimately be produced, the boosting of the immune
system allows the patient to suitably control the disease before
the emergence of the regulator cells. This scenario would explain
why previous studies have shown that anti-HIV and anti-tumour
vaccines are only successful in a small number of patients. More
specifically, there is only a small chance the vaccine will be
administered at the same time the innate immune response to the
disease is occurring. Other times of administration in the prior
art occur when there are high numbers and/or activity of regulators
cells, or at times which uncouple the natural cycling of the immune
system.
[0049] Thus, in another aspect the present invention provides a
method for determining when a vaccine should be administered to a
patient suffering from a disease characterized by the production of
regulator cells, the method comprising monitoring the patient, or
samples obtained therefrom, for at least one of: a) effector cell
numbers and/or activity, b) regulator cell numbers and/or activity,
c) a molecule associated with the disease, and/or d) an immune
system marker.
[0050] In a further aspect, the present invention provides a method
of treating a disease characterized by the production of regulator
cells, the method comprising;
[0051] i) monitoring a patient suffering from the disease for at
least one of: [0052] a) number and/or activity of regulator cells,
[0053] b) number and/or activity of effector cells, [0054] c) a
molecule associated with the disease, and/or [0055] d) an immune
system marker, and
[0056] ii) exposing the patient to an vaccine to treat the
disease,
wherein the timing of administration of the vaccine is selected
such that the activity of effector cells is not significantly
reduced.
[0057] In one embodiment, the vaccine is administered about when
the levels of effector cells are increasing.
[0058] In another embodiment, the vaccine is administered about
when the levels of a molecule associated with the disease begin to
decrease.
[0059] In a further embodiment, the vaccine is administered about
when the levels of an acute phase inflammatory marker begin to
increase. As outlined above, at least some acute phase inflammatory
markers have been found to be cycling over about a seven day period
where only every second peak of acute phase inflammatory marker
levels is associated with effector cell numbers. Thus, in this
embodiment, the monitoring will most likely need to be combined
with the analysis of other factors described herein.
[0060] The observation that the immune system is cycling during
disease states characterized by the presence of regulator cells can
also be used as an indicator of the presence of such a disease.
These diagnosis procedures would be particularly useful for
analysing a patient for the recurrence of the disease state (such
as a tumour) following treatment, or for analysing a patient
determined to be susceptible to the disease (such as in cases where
the subject has previously been identified as possessing a cancer
susceptibility gene) for the emergence of the disease.
[0061] Thus, in a further aspect the present invention provides a
method of diagnosing a disease characterized by the production of
regulator cells, the method comprising monitoring the patient, or
samples obtained therefrom, for at least one of: a) effector cell
numbers and/or activity, b) regulator cell numbers and/or activity,
c) a molecule associated with the disease, and/or d) an immune
system marker, wherein cycling of any one of a) to d) indicates the
disease may be present.
[0062] Naturally, as outlined above, the patient will need to be
analysed for other disease states, such as minor infections such as
influenza etc, to ensure that any cycling observed (especially when
analysing acute phase inflammatory markers) is directly linked to a
disease characterized by the production of regulator cells.
[0063] Whilst ideally the monitoring should continue indefinitely,
this will most likely not be practical in a majority of situations.
Thus, the diagnosis procedure can be performed on an intermittent
basis based on assessed risk of the disease state emerging or
re-emerging. As the skilled addressee will appreciate from the
discussions herein, the term "intermittent basis" means that the
method will require a suitable number of samples be analysed over a
period of time to determine if immune cycling is occurring (for
example samples obtained at least every 3 days for a period of
about 14 days), however, if the test is negative this procedure may
not need to be repeated (for example) for another year.
[0064] In another aspect, the present invention provides for the
use of an assay which detects an immune system marker for
determining when an agent or vaccine should be administered to a
patient suffering from a disease characterized by the production of
regulator cells.
[0065] Preferably, the marker is an acute phase inflammatory
marker. More preferably, the marker is a positive acute phase
inflammatory marker. Even more preferably, the marker is selected
from the group consisting of, but not limited to, serum amyloid A
and c-reactive protein.
[0066] In another aspect, the present invention provides for the
use of an assay which detects effector cell numbers and/or activity
for determining when an agent or vaccine should be administered to
a patient suffering from a disease characterized by the production
of regulator cells.
[0067] Preferably, the assay detects the number of CD8+CD4- T
cells.
[0068] In another aspect, the present invention provides for the
use of an assay which detects regulator cell numbers and/or
activity for determining when an agent or vaccine should be
administered to a patient suffering from a disease characterized by
the production of regulator cells.
[0069] Preferably, the assay detects the number of CD4+CD8- T
cells.
[0070] In another aspect, the present invention provides for the
use of an assay which detects a molecule associated with a disease
characterized by the production of regulator cells for determining
when an agent or vaccine should be administered to treat the
disease.
[0071] Preferably, the assay detects an antigen produced by a
cancer cell or an infectious agent.
[0072] Preferably, the patient has not been exposed to a treatment
for the disease for at least 14 days, more preferably at least 21
days, and even more preferably at least 28 days.
[0073] In a further aspect, the present invention provides for the
use of an agent for the manufacture of a medicament for
administering to a patient suffering from a disease characterized
by the production of regulator cells, wherein the agent will be
administered at a time selected such that the activity of effector
cells is not significantly reduced, and wherein the patient has not
been exposed to a treatment for the disease for at least 14
days.
[0074] Preferably, the agent inhibits the production of, limits the
function of, and/or destroys, regulator cells.
[0075] As would be readily appreciated by those skilled in the art,
the methods of the present invention may be repeated to provide a
more complete treatment.
[0076] Preferably, the patient is a mammal. More preferably, the
mammal is a human.
[0077] In a further aspect, the present invention provides a kit
for determining when an agent or vaccine should be administered to
a patient suffering from a disease characterized by the production
of regulator cells, the kit comprising at least one reagent for
monitoring the patient, or samples obtained therefrom, for at least
one of: a) effector cell numbers and/or activity, b) regulator cell
numbers and/or activity, c) a molecule associated with the disease,
and/or d) an immune system marker.
[0078] Preferably, the kit comprises written instructions for
performing a method of the invention including reference to the
preferred number of samples to be analysed, and the timing between
sample analysis.
[0079] As will be apparent, preferred features and characteristics
of one aspect of the invention are applicable to many other aspects
of the invention.
[0080] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0081] The invention is hereinafter described by way of the
following non-limiting Examples and with reference to the
accompanying figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0082] FIG. 1. A) C-reactive protein and tumour marker CA125 levels
over a 14 day period in a patient with ovarian cancer. B) Serum
amyloid A levels in the same patient over the same period
(C-reactive protein levels from A) duplicated).
[0083] FIG. 2. C-reactive protein levels in response to taking a
first human HIV patient off HAART treatment.
[0084] FIG. 3. Viral load and CRP fluctuations in a second HIV
patient following the completion of HAART.
[0085] FIG. 4. CRP and C4 fluctuations in Mrs OM over 32 days shows
a distinct periodicity with an approximate repeating 7/14 day
oscillation. Measurements were taken every Monday, Wednesday and
Friday. In this case the C4 oscillation is more regular. Note the
rising trend in both parameters over the 32 day period.
[0086] FIG. 5. Serum Complement factors C4 and C3 fluctuations in
Mrs OM over 32 days show a near synchronous and regular periodicity
of approximately 7/14 days. Note the rising trend in both
parameters over the 32 day period.
[0087] FIG. 6. Serum Complement Factor C4 fluctuations and rising
CA125 levels with advancing disease in Mrs OM. Note the rising
trend in both parameters over the 32 day period.
[0088] FIG. 7. C-Reactive Protein versus Time in Mrs OM, (days)
Monitoring and Therapeutic events, 28 May 2004 (day 1)-9 Aug. 2004
(day 74). CRP monitoring began on the 28.sup.th of May (day 1) and
climbed steadily with advancing disease. The approx 14 day immune
response oscillation was derived from the combined interpretation
of serum CRP, C4 & CA125 collected data (see also FIG. 4).
Key:
[0089] A=Radiotherapy begins, day 38=5 Jul. 2004. B=Predicted CRP
peak, day 46, 47 & 48=13, 14, 15, Jul. 2004. C=Timing of first
chemotherapy application, day 49, 16 Jul. 2004. D=Predicted CRP
peak, day 63 &64=28, 29 Jul. 2004. Radiotherapy stops. E=Timing
of 2.sup.nd chemotherapy application, day 65=30 Jul. 2004. F=Fever,
day 66=31 Jul. 2004, Haemorrhage from Tumour, day 67=1 Aug. 2004.
G=CRP drops to 62.7 mg/l, day 69=4 Aug. 2004. H=Endoscopy reporting
no evidence of tumour, day 74=9 Aug. 2004.
[0090] FIG. 8. C-Reactive Protein and Serum Amyloid A versus time
in Mrs FO.
[0091] FIG. 9. C-Serum Amyloid A and IL-2 versus time in Mrs
FO.
[0092] FIG. 10. Serum Amyloid A and cancer marker CA125 versus time
in Mrs FO.
[0093] FIG. 11. C-Reactive Protein and C3 versus time in Mrs
FO.
[0094] FIG. 12. C-Reactive Protein versus time in Mr GA.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0095] As used herein the terms "treating", "treat" or "treatment"
include administering a therapeutically effective amount of an
agent sufficient to reduce or eliminate at least one symptom of the
disease.
[0096] As used herein, the term "tumour load" generally refers to
the number of cancerous cells in a subject at any given time.
Measuring the level of tumor antigen in the subject can be
considered as an indication of tumour load.
[0097] As used herein, the term "viral load" generally refers to
the number of viral particles in a subject at any given time.
Measuring the level of viral antigen in the subject can be
considered as an indication of viral load.
[0098] "Regulator cells" include, but are not necessarily limited
to, a subpopulation of CD4+ T cells. Such cells may also be
referred to in the art as "suppressor cells". Regulator cells may
either act directly on effector cells or may assert their affects
upon effector cells through other mechanisms.
[0099] CD4+ cells express the marker known in the art as CD4.
Typically, the term "CD4+ T cells" as used herein does not refer to
cells which also express CD8. However, this term can include T
cells which also express other antigenic markers such as CD25.
[0100] "Effector cells" include, but are not necessarily limited
to, the T cell population known as CD8+ cells.
[0101] As used herein, the term "limits the function of, and/or
destroys" when referring to the exposure of the "regulator cells"
to the agent means that the number, and/or activity, of regulator
cells is down-regulated by the agent. Most preferably, the number,
and/or activity, of regulator cells is completely eradicated by the
agent.
[0102] As used herein the term "disease characterized by the
production of regulator cells" refers to any condition wherein the
number or activity of regulator cells plays a role in prolonging
the disease state. Examples of such disease include, but are not
limited to, cancer and infections.
[0103] The term "immune system marker" generally refers to any
molecule or factor which provides an indication of the state and/or
activity of the immune system. These markers may be directly linked
to the activity and/or production of regulator and/or effector
cells, and/or may provide a more general indication of the overall
response of the immune system to an antigen. Examples of a suitable
immune system marker include acute phase inflammatory markers such
as c-reactive protein and serum amyloid A. Another example of an
immune system marker are indicators of cellular destruction such
as, but not limited to, cholesterol and beta-2-microglobulin in
serum. Cholesterol and beta-2-microglobulin are integral components
of cellular membranes. In particular, beta-2-microglobulin is the
accessory molecule to the Major Histocompatabilty Class I or MHC-I
receptor. Consequently, with the cycling of the anti-disease immune
response together with target cell destruction, the serum levels in
cancer patients of these two molecules is often elevated. Thus,
oscillations in indicators of cellular destruction, such as
cholesterol and beta-2-microglobulin, may also prove useful in
determining the beginning or end of the immune response cycle.
Naturally, upon the present discovery of the immune system cycling
in a disease characterized by the production of regulator cells,
the skilled addressee could readily identify further markers useful
in the methods of the invention.
[0104] As used herein, the term "a molecule associated with the
disease" refers to any molecule which is linked to the disease
state. In a preferred embodiment, the marker is a protein. Such
protein markers are well known in the art. Examples of suitable
tumour antigen markers are described herein. Suitable markers for,
if not all, infectious diseases are also well known, for example
the gag or env proteins of HIV.
[0105] As used herein the term "chronic persistent infection"
refers to the presence of an infectious agent in the patient which
is not readily controlled by the patients immune system or
available therapies. Examples include, but are not limited to,
infections with Mycobacterium tuberculosis (which causes
tuberculosis), HIV, the Hepatitis B virus or the Hepatitis C virus.
To be classified as a "chronic persistent infection" it is
preferred that the patient has at least had the infection for 3
months, more preferably at least 6 months.
[0106] For the purposes of this invention, the term "antibody",
unless specified to the contrary, includes fragments of whole
antibodies which retain their binding activity for a target
analyte. Such fragments include Fv, F(ab') and F(ab').sub.2
fragments, as well as single chain antibodies (scFv). Furthermore,
the antibodies and fragments thereof may be humanised antibodies,
for example as described in EP-A-239400.
[0107] As is known in the art, a cancer is generally considered as
uncontrolled cell growth. The methods of the present invention can
be used to treat any cancer including, but not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include breast cancer, prostate
cancer, colon cancer, squamous cell cancer, small-cell lung cancer,
non-small cell lung cancer, ovarian cancer, cervical cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, liver
cancer, bladder cancer, hepatoma, colorectal cancer, uterine
cervical cancer, endometrial carcinoma, salivary gland carcinoma,
mesothelioma, kidney cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma,
myeloma, various types of head and neck cancer, acute lymphoblastic
leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral
neuroepithelioma.
[0108] The "sample" refers to a material suspected of containing
regulator cells, effectors cells, immune system markers and/or a
molecule associated with the disease. The sample can be used as
obtained directly from the source or following at least one step of
(partial) purification. The sample can be prepared in any
convenient medium which does not interfere with the method of the
invention. Typically, the sample is an aqueous solution or
biological fluid as described in more detail below. The sample can
be derived from any source, such as a physiological fluid,
including blood, serum, plasma, saliva, sputum, ocular lens sweat,
faeces, urine, milk, ascites fluid, mucous, synovial fluid,
peritoneal fluid, transdermal exudates, pharyngeal exudates,
bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,
semen, cervical mucus, vaginal or urethral secretions, amniotic
fluid, and the like. Preferably, the sample is blood or a fraction
thereof. Pretreatment may involve, for example, preparing plasma
from blood, diluting viscous fluids, and the like. Methods of
treatment can involve filtration, distillation, separation,
concentration, inactivation of interfering components, and the
addition of reagents. The selection and pretreatment of biological
samples prior to testing is well known in the art and need not be
described further.
[0109] Unless otherwise indicated, the recombinant DNA and
immunological techniques utilized in the present invention are
standard procedures, well known to those skilled in the art. Such
techniques are described and explained throughout the literature in
sources such as, J. Perbal, A Practical Guide to Molecular Cloning,
John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.
A. Brown (editor), Essential Molecular Biology: A Practical
Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D.
Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4,
IRL Press (1995 and 1996), and F. M. Ausubel et al (editors),
Current Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-Interscience (1988, including all updates until present), Ed
Harlow and David Lane (editors) Antibodies: A Laboratory Manual,
Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al
(editors) Current Protocols in Immunology, John Wiley & Sons
(including all updates until present), and are incorporated herein
by reference.
Acute Phase Inflammatory Markers
[0110] Some acute phase inflammatory markers initially increase
during an immune response (referred to hereinafter as positive
acute phase inflammatory markers) whilst others initially decrease
during an immune response (referred to hereinafter as negative
acute phase inflammatory markers). Acute phase inflammatory markers
are also referred to in the art as acute phase reactants or acute
phase proteins. The skilled addressee will be aware of the many
assays which can be used to monitor acute phase inflammatory
markers.
[0111] Examples of positive acute phase inflammatory markers
include, but are not limited to, c-reactive protein, serum amyloid
A, serum amyloid P component, complement proteins such C2, C3, C4,
C5, C9, B, C1 inhibitor and C4 binding protein, fibrinogen, von
Willebrand factor, .alpha.1-antitrypsin, .alpha.1-antichymotrypsin,
.alpha.2-antiplasmin, heparin cofactor II, plasminogen activator
inhibitor I, haptoglobin, haemopexin, ceruloplasmin, manganese
superoxide dismutase, .alpha.1-acid glycoprotein, haeme oxygenase,
mannose-binding protein, leukocyte protein I, lipoporotein (a),
lipopolysaccharide-binding protein, and interleukins such as IL-1,
IL-2, IL-6, IL-10 and receptors thereof.
[0112] Example of negative acute phase inflammatory markers
include, but are not limited to, albumin, pre-albumin, transferrin,
apoAI, apoAII, .alpha.2 HS glycoprotein, inter-.alpha.-trypsin
inhibitor, histidine-rich glycoprotein.
[0113] Serum amyloid A (SAA) was discovered as a plasma component
that shares antigenicity with amyloid AA, the chief fibrillar
component in reactive AA amyloid deposits. SAA has been shown to be
an acute phase reactant whose level in blood is elevated to
1000-fold or higher as part of the body's responses to various
injuries including trauma, infection and inflammation.
[0114] SAA levels can be determined as known in the art, see for
example Weinstein et al (1984), Liuzzo et al (1994), O'Hara et al
(2000), Kimura et al (2001) and O'Hanlon et al (2002).
[0115] C-reactive protein (CRP) is an important positive acute
phase response protein, and its concentration in serum may increase
as much as 1,000-fold during the acute phase response. CRP is a
pentamer consisting of five identical subunits, each having a
molecular weight of about 23,500.
[0116] C-reactive protein levels can be determined using techniques
known in the art, these include, but are not limited to, those
disclosed in Senju et al (1983), Weinstein et al (1984), Price et
al (1987), Liuzzo et al (1994), Eda et al (1998), Kimura et al
(2001) and O'Hanlon et al (2002).
[0117] The complement proteins are a group of at least 20
immunologically distinct components. They normally circulate in the
blood in an inactive form. They are able to interact sequentially
with antigen-antibody complexes, with each other and with cell
membranes in a complex but adaptable way to destroy viruses and
bacteria and pathologically, even the hosts own cells. Abnormal
serum levels of complement proteins may be due to either inherited
or acquired diseases. At least circulating levels of C3 and C4
reflect a balance between complement consumption due to immune
complex formation and increased synthesis due to acute phase
response. Methods of measuring complement protein levels are well
known in the art.
[0118] Levels of different interleukins can also be determined
using procedures known in the art such as using the ProteoPlex.TM.
cytokine assay kit (EMD Biosciences Inc., CA, USA).
Agents
[0119] The agent can be any factor or treatment useful in treating
a disease characterized by the production of regulator cells.
Preferably, the agent selectively or non-selectively results in the
destruction, the inhibition of the production, or reduction of
activity, of regulator cells. For example, a CD4+ specific antibody
could be used to specifically target CD4+ T cells. However, in some
instances a non-selective agent could be used, such as an
anti-proliferative drug or radiation, both of which destroy
dividing cells. In particular, as with other cell types, regulator
cells are particularly vulnerable to destruction by anti-mitotic
(anti-proliferative) drugs or spindle poisons (e.g. Vinblastine or
paclitaxel) when dividing and specifically in mitosis.
[0120] The term "anti-proliferative drug" is a term well understood
in the art and refers to any compound that destroys dividing cells
or inhibits them from undergoing further proliferation.
Anti-proliferative drugs include, but are not limited to,
mechlorethamine, cyclophosphamide, ifosfamide, melphalan,
chlorambucil, hexamethyl-melamine, thiotepa, busulfan, carmustine,
lomustine, semustine, streptozocin, dacarbazine, methotrexate,
fluorouracil, floxuridine, cytarabine, mercaptopurine, thioguanine,
pentostatin, vinblastine, anhydro vinblastine, vincristine,
etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin,
bleomycin, plicamycin, mitomycin, L-asparaginase, cisplatin,
mitoxantrone, hydroxyurea, procarbazine, mitotane,
aminoglutethimide, prednisone, hydroxyprogesterone caproate,
medroprogesterone acetate, megestrol acetate, diethylstilbestrol,
ethinyl estradiol, tamoxifen, testosterone propionate, radioactive
isotopes, ricin A chain, taxol, diphtheria toxin, colchicine and
pseudomonas exotoxin A.
[0121] The agents are usually administered in the dosage forms that
are readily available to the skilled clinician, and are generally
administered in their normally prescribed amounts (as for example,
the amounts described in the Physician's Desk Reference, 55th
Edition, 2001, or the amounts described in the manufacture's
literature for the use of the agent).
[0122] In one embodiment, the agent is administered as a single
bolus injection. In another embodiment, the agent is administered
by infusion. The period of infusion can be, for example, at least 3
hours, at least 12 hours or at least 24 hours.
[0123] Recent studies have suggested that CD4+CD25+ T cells play an
important role in regulating immune cells directed against self
antigens (Salomon et al, 2000; Suri-Payer and Cantor, 2001).
Furthermore, targeting CD4+CD25+ T cells has been shown to enhance
the ability of an animal to control tumour growth (Onizuka et al,
1999; Shimizu et al, 1999; Sutmuller et al, 2001). Accordingly,
CD4+CD25+ T cells could be acting as regulator cells as used
herein. The activity of CD4+CD25+ T cells can be downregulated by
anti-GITR, anti-CD28 and/or anti-CTLA-4 (Read et al, 2000;
Takahashi et al, 2000; Shimizu et al, 2002). Thus, these antibodies
may be useful as agents for use in the methods of the present
invention.
[0124] Another example of an agent which can be administered in a
method of the invention is dsRNA. dsRNA is used in RNA interference
(RNAi) which is a phenomenon where upon introduction into a cell,
mRNA homologous to the dsRNA is specifically degraded so that
synthesis of gene products is suppressed. Examples of such an agent
causing RNAi include, but are not limited to, a sequence having at
least about 70% homology to the nucleic acid sequence of a target
gene or a sequence hybridizable under stringent conditions, RNA
containing a double-stranded portion having a length of at least 10
nucleotides or variants thereof. Examples of target genes include,
but are not limited to, a gene required for replication of a
regulator cell, a gene required for survival of a cancer cell, or a
gene required for growth and/or replication of an infectious
agent.
[0125] dsRNA having a length of about 20 bases (e.g.,
representatively about 21 to 23 bases) or less than about 20 bases,
which is called siRNA in the art, can be used. Expression of siRNA
in cells can suppress expression of a gene targeted by the siRNA.
In another embodiment, an agent capable of causing RNAi may have a
short hairpin structure having a sticky portion at the 3' terminus
(shRNA; short hairpin RNA). As used herein, the term "shRNA" refers
to a molecule of about 20 or more base pairs in which a
single-standed RNA partially contains a palindromic base sequence
and forms a double-strand structure therein (i.e., a hairpin
structure). shRNA can be artificially chemically synthesized.
Alternatively, shRNA can be produced by linking sense and antisense
strands of a DNA sequence in reverse directions and synthesizing
RNA in vitro with T7 RNA polymerase using the DNA as a template.
The length of the double-stranded portion is not particularly
limited, but is preferably about 10 or more nucleotides, and more
preferably about 20 or more nucleotides. The 3' protruding end may
be preferably DNA, more preferably DNA of at least 2 nucleotides in
length, and even more preferably DNA of 2-4 nucleotides in
length.
[0126] An agent capable of causing RNAi useful for the invention
may be artificially synthesized (chemically or biochemically) or
naturally occurring. There is substantially no difference
therebetween in terms of the effect of the present invention. A
chemically synthesized agent is preferably purified by liquid
chromatography or the like.
[0127] An agent capable of causing RNAi used in the present
invention can also be produced in vitro. In this synthesis system,
T7 RNA polymerase and T7 promoter can be used to synthesize
antisense and sense RNAs from template DNA. These RNAs are annealed
and thereafter are introduced into a cell.
[0128] dsRNA can be delivered to the patient using any means known
in the art. Examples of methods of delivering dsRNA to a patient
are described in, for example, US 20040180357, US 20040203024 and
20040192629.
Timing of Exposing the Subject to the Agent
[0129] For the investigator who randomly applies a single treatment
of anti-proliferative chemotherapy to a cancer patient there is an
approximate 1 in 14, to 1 in 15, chance of getting the timing
right. A one in fourteen chance equates to a 7% probability of
applying the therapy on the correct day, when the regulator cells
are vulnerable to inactivation. If this is done, the tumour should
regress mediated by immune destruction. More specifically, it is
our hypothesis that once the regulators cells have been removed by
therapeutic intervention, the immune response against the tumour or
virus can proceed unimpeded, ultimately leading to control of the
disease.
[0130] Whilst not wishing to be limited by theory, it is believed
that the relative number of effector cells expands in response to
an antigen before the regulator cells. Accordingly, as used herein,
the term "the activity of the effector cells is not significantly
reduced" means that the timing of the administration of the agent
is such that the agent exerts a proportionally greater effect
against the regulator cells than the effector cells. It is clearly
preferred that the agent is administered at a time when the ratio
of effect against the regulator cells to the effect against
effector cells is greatest.
[0131] As outlined above, the present invention relies on the
phenomenon that the immune system is cycling over an approximate 14
to 15 day period in a patient suffering from a disease
characterized by the production of regulator cells. In most
instances, the time point that the agent is to be administered will
need to be empirically determined in subjects at different stages
of disease as their immune response kinetics may vary. Other
factors such as the general health of the subject and/or the
genetic makeup of the subject will also impact upon when is the
appropriate time to administer the agent.
[0132] As will be appreciated by the skilled addressee, conditions
such as cancer and chronic persistent infectious are serious, often
life threatening, diseases. Due to many factors, not the least of
which is natural variations between individuals, it will be
typically be required that a patient be monitored for a reasonable
length of time to appreciate the nature of immune cycling in the
individual, and for monitoring to analyse a number of factors (such
as a combination of acute phase markers and disease antigens), to
ultimately determine the most appropriate time to administer the
agent to optimise the chances of an effective treatment.
[0133] Techniques known in the art can be used to monitor the
growing population of effector and/or regulator cells during the
"cycle".
[0134] Serial blood samples can be collected and quantitatively
screened for all CD4+ subsets by FACS analysis. This FACS
monitoring will need to be maintained until the regulator cells
begin clonally expanding in response to the disease state, whether
produced by the tumour or administered to the subject. Other
possible assays for monitoring the growing population of regulator
cells include lymphocyte proliferation/activation assays and
various cytokine level assays (for example an assay for IL-4, IL-6
or IL-10).
[0135] Also, serial blood samples can be collected and
quantitatively screened for all effector cell activity such as but
not limited to CD8+, CRP, SAA and various cytokines. Such effector
cell markers will precede the regulator cell markers.
[0136] When the disease is cancer another avenue of determining the
time point for administering the agent is to monitor the tumour
load. It is envisaged that the tumour load decreases due to the
activity of the effector cells, however, the subsequent increase in
regulator cells would down-regulate the effector cells resulting in
a slowing of the tumour load decrease. Accordingly, the agent could
be administered approximately prior to the slowing of the decrease
in tumour load. Techniques known in the art, for example RT-PCR or
antibody detection, of markers expressed by the tumour, could be
used to measure tumour load in these circumstances. Examples of
suitable tumour antigen marker assays include, but are not limited
to, for AFP (marker for hepatocellular carcinoma and germ-cell
tumours), CA 15-3 (marker for numerous cancers including breast
cancer), CA 19-9 (marker for numerous cancers including pancreatic
cancer and biliary tract tumours), CA 125 (marker for various
cancers including ovarian cancer), calcitonin (marker for various
tumours including thyroid medullary carcinoma), catecholamines and
metabolites (phaeochromoctoma), CEA (marker for various cancers
including colorectal cancers and other gastrointestinal cancers),
hCG/beta hCG (marker for various cancers including germ-cell
tumours and choriocarcinomas), 5HIAA in urine (carcinoid syndrome),
PSA (prostate cancer), sertonin (carcinoid syndrome) and
thyroglobulin (thyroid carcinoma).
[0137] Monitoring may need to be very frequent, for example as
often as every few hours, to ensure the correct time point is
selected for administration of the agent. Preferably, the
monitoring is conducted at least every 48 hours. More preferably,
the monitoring is conducted at least every 24 hours.
[0138] Optimally, the monitoring is continued to determine the
affect of the agent. Insufficient down-regulation, re-emergence of
the regulator cells or increases in, for example, tumour load will
mean that the method of the present invention should be repeated.
Such repeated cycles of treatment may generate immunological
memory. It is therefore possible that the present invention, used
in repetitive mode, may provide some prophylactic protective
effect.
Vaccines
[0139] As outlined above, the inventor has also noted after a
survey of the literature that the treatment of a variety of cancers
with therapeutic vaccines, on average yielded a complete response
rate of approximately 10% (see, for example, Trefzer et al, 2004;
Lotem et al., 2004; Smithers et al., 2003; Belli et al., 2002; Berd
et al., 2001; Wittig et al., 2001). This implies a window of
opportunity of therapeutic application of 1.5 days every 14 days
(10%). This is similar and well within the realms of probability of
the complete response rates of approximately 7% (1 day in 14) seen
in cancer chemotherapy reported herein. Thus a similar mechanism is
operating in the vaccine situation whereby the innoculation of a
cancer vaccine into the patient at the correct time is sufficient
to disturb the regulatory mechanisms/cells allowing the effectors
to kill the tumour resulting in a complete response.
[0140] Naturally, vaccines used in the present invention will
result in an immune against a disease characterized by the
production of regulator cells. Such vaccine will comprise at least
one antigen, or a polynucleotide encoding said antigen. The vaccine
can be provided as any form known in the art such as, but not
limited to, a DNA vaccine, ingestion of a transgenic organism
expressing the antigen, or composition comprising the antigen.
[0141] As used herein, an "antigen" is any polypeptide sequence
that contains an epitope which is capable of producing an immune
response against the disease.
[0142] Antigens which are capable of raising an immune response
against a cancer cell are well known in the art. Certain tumour
antigens can be recognized and targeted by the immune system. This
property may be due to overexpression by the tumour tissue. Some of
these antigens can be detected in normal tissue. The tumour
antigens targeted by T cells are generally proteins that are
processed intracellularly and presented as short peptide fragments
bound in the groove of the tumour MHC class I molecule to be
recognized by CD8.sup.+ cytotoxic T lymphocytes. The mere presence
of a tumour antigen is not always sufficient to trigger an immune
response. Co-stimulatory molecules such as B7.1 are sometimes
required. Once antigen-specific T cells are stimulated, they are
capable of recognizing and destroying the tumour. The conditions
needed for the activation of antigen-specific T cells are
stringent, but are open to genetic manipulation of target tumour
cells and T cells.
[0143] Antigens which can be used to treat infections, such as HIV,
are also well known in the art.
[0144] The antigen can be provided in any manner known in the art
which leads to an immune response. An antigen can be, for example,
native, recombinant or synthetic. Native antigens can be prepared,
for example, by providing cell lysates of a tumour cell.
[0145] Vaccines may be prepared from one or more antigens. The
preparation of vaccines which contain an antigen is known to one
skilled in the art. Typically, such vaccines are prepared as
injectables, or orals, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection or oral consumption may also be prepared. The
preparation may also be emulsified, or the protein encapsulated in
liposomes. The antigen is often mixed with carriers/excipients
which are pharmaceutically acceptable and compatible with the
active ingredient. Suitable carriers/excipients are, for example,
water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof.
[0146] In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine.
[0147] Typically, vaccines comprise an adjuvant. As used herein,
the term "adjuvant" means a substance that non-specifically
enhances the immune response to an antigen. Examples of adjuvants
which may be effective include but are not limited to:
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1-2-dipal-
mitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE), and RIBI, which contains three components
extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. Further examples of adjuvants include
aluminum hydroxide, aluminum, phosphate, aluminum potassium sulfate
(alum), bacterial endotoxin, lipid X, Corynebacterium parvum
(Propionobacterium acnes), Bordetella pertussis,
polyribonucleotides, sodium alginate, lanolin, lysolecithin,
vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked
copolymers or other synthetic adjuvants. Such adjuvants are
available commercially from various sources, for example, Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,
Detroit, Mich.).
[0148] The proportion of antigen and adjuvant can be varied over a
broad range so long as both are present in effective amounts. For
example, aluminium hydroxide can be present in an amount of about
0.5% of the vaccine mixture (Al.sub.2O.sub.3 basis). Conveniently,
the vaccines are formulated to contain a final concentration of
antigenic polypeptide in the range of from 0.2 to 200 .mu.g/ml,
preferably 5 to 50 .mu.g/ml, most preferably 15 .mu.g/ml.
[0149] After formulation, the vaccine may be incorporated into a
sterile container which is then sealed and stored at a low
temperature, for example 4.degree. C., or it may be freeze-dried.
Lyophilisation permits long-term storage in a stabilised form.
[0150] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1% to 2%. Oral formulations include such normally
employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10% to 95% of
active ingredient, preferably 25% to 70%. Where the vaccine
composition is lyophilised, the lyophilised material may be
reconstituted prior to administration, e.g. as a suspension.
Reconstitution is preferably effected in buffer.
[0151] Capsules, tablets and pills for oral administration to a
patient may be provided with an enteric coating comprising, for
example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose
acetate phthalate or hydroxypropylmethyl cellulose.
[0152] DNA vaccination involves the direct in vivo introduction of
DNA encoding an antigen into tissues of a subject for expression of
the antigen by the cells of the subject's tissue. Such vaccines are
termed herein "DNA vaccines" or "nucleic acid-based vaccines". DNA
vaccines are described in U.S. Pat. No. 5,939,400, U.S. Pat. No.
6,110,898, WO 95/20660 and WO 93/19183, the disclosures of which
are hereby incorporated by reference in their entireties.
[0153] To date, most DNA vaccines in mammalian systems have relied
upon viral promoters derived from cytomegalovirus (CMV). These have
had good efficiency in both muscle and skin inoculation in a number
of mammalian species. A factor known to affect the immune response
elicited by DNA immunization is the method of DNA delivery, for
example, parenteral routes can yield low rates of gene transfer and
produce considerable variability of gene expression. High-velocity
inoculation of plasmids, using a gene-gun, enhanced the immune
responses of mice, presumably because of a greater efficiency of
DNA transfection and more effective antigen presentation by
dendritic cells. Vectors containing the nucleic acid-based vaccine
of the invention may also be introduced into the desired host by
other methods known in the art, e.g., transfection,
electroporation, microinjection, transduction, cell fusion, DEAE
dextran, calcium phosphate precipitation, lipofection (lysosome
fusion), or a DNA vector transporter.
[0154] Transgenic plants producing a antigenic polypeptide can be
constructed using procedures well known in the art. A number of
plant-derived edible vaccines are currently being developed for
both animal and human pathogens. Immune responses have also
resulted from oral immunization with transgenic plants producing
virus-like particles (VLPs), or chimeric plant viruses displaying
antigenic epitopes. It has been suggested that the particulate form
of these VLPs or chimeric viruses may result in greater stability
of the antigen in the stomach, effectively increasing the amount of
antigen available for uptake in the gut.
EXAMPLES
Example 1
[0155] Provided below are examples of typical assays used to
monitor some acute phase inflammatory markers, as well as the
ovarian cancer marker CA125.
C-Reactive Protein
[0156] C-Reactive Protein was measured using a DADE Behring
Dimension RxL Chemistry Analyser, with reagents and calibrators
supplied by Dade Behring Diagnostics (Sydney, Australia)
(reagent-Cat No. DF-34; calibrators Cat. No. DC-34).
[0157] The CRP method is based on a particle enhanced turbidimetric
immunoassay technique. Latex particles coated with antibody to
C-Reactive Protein aggregate in the presence of C-Reactive Protein
in the sample. The increase in turbidity which accompanies
aggregation is proportional to the C-Reactive Protein
concentration.
TABLE-US-00001 INTRA-ASSAY INTER-ASSAY PRECISION PRECISION MEAN
MEAN mg/L CV N mg/L CV N 3.4 4.3% 20 4.6 5.6% 64 57.5 2.3% 20 37.0
3.0% 64 225.8 2.0% 20
REFERENCE RANGE: 0-5 mg/L ANALYTICAL RANGE: 0.5-500 mg/L
Cancer Antigen 125 (CA125)
[0158] AxSym CA 125 was based on Microparticle Enzyme Immunoassay
(MEIA) technology carried out on an Abbott Diagnostics AxSym with
reagents and calibrators supplied by Abbott Diagnostics (AxSym
Reagent pack-Cat No. 3B41-22; calibrators-Cat No. 9C22-01).
[0159] Sample, Anti CA 125 coated microparticles and specimen
diluent are pipetted in one well of the reaction vessel. The CA 125
binds to the Anti-CA 125 coated microparticles forming an Ab-Ag
complex. An aliquot of the reaction mixture containing the Ab-Ag
complex bound to the microparticles bind irreversibly to the glass
fiber matrix. The matrix cell is washed with the wash buffer to
remove the unbound materials. The anti-CA 125 subunit specific ALP
conjugate is dispersed onto the matrix cell and binds with the
Ab-Ag complex. The matrix cell is washed to remove unbound
material. The substrate, 4-methyl umbelliferyl phosphate, is added
to the matrix cell and the fluorescent product is measured by the
MEIA optical assembly.
[0160] Dilutions are made with Abbott CA 125 specimen diluent (No.
3B41-50).
[0161] The coefficient of Variation as assessed from routine
quality control sera at two levels (Abbott Tumour Marker Control
(9C22-10 levels 1, 2 & 3) is as follows:
TABLE-US-00002 MEAN SD CV % N LEVEL 1 U/mL 27 2.5 9.4 64 LEVEL 2
U/mL 78 5.5 7.1 64 LEVEL 3 U/mL 211 21.4 10.2 54
REFERENCE RANGES: 0-35 U/mL
ANALYTICAL RANGE: 2-600 U/mL
Interleukin 2 Receptor (IL2R)
[0162] The receptor of the cytokine interleukin 2 (IL2R) was
measured by a commercial automated chemiluminescent Enzyme Immuno
Assay (EIA) using an Immulite Analyser from Diagnostic Products
Corporation (Los Angeles, Calif., USA).
[0163] This is a competitive immunoassay using Alkaline Phosphatase
labelled IL2R as tracer and adamantyl dioxetane as luminescent
substrate for ALP enzyme.
[0164] All reagents and calibrators are supplied in kit form by
DPC--Cat No. LKIPZ.
[0165] Analytical Performance:
TABLE-US-00003 MEAN SD CV % LEVEL 1 213 U/mL 13 6.1 LEVEL 2 752
U/mL 49 6.5 LEVEL 3 2463 U/mL 189 7.7
ANALYTICAL RANGE: 5-7,500 U/mL
REFERENCE RANGE: 223-710 U/mL*
[0166] *Study performed on 87 apparently healthy adults.
Interleukin 6
[0167] The cytokine interleukin 6 was measured by a commercial
automated chemiluminescent Enzyme Immuno Assay (EIA) using an
Immulite Analyser from Diagnostic Products Corporation (Los
Angeles, Calif., USA).
[0168] This is a competitive immunoassay using Alkaline Phosphatase
labelled IL-6 as tracer and adamantyl dioxetane as luminescent
substrate for ALP enzyme.
[0169] All reagents and calibrators are supplied in kit form by
DPC--Cat No. LK6PZ.
[0170] Analytical Performance:
TABLE-US-00004 MEAN SD CV % LEVEL 1 88 pg/mL 4.5 5.1 LEVEL 2 230
pg/mL 12.2 5.3 LEVEL 3 638 pg/mL 46.6 7.3
ANALYTICAL RANGE: 2-1000 pg/mL REFERENCE RANGE: <4.1 pg/mL*
*Study performed on 60 apparently healthy laboratory
volunteers.
Interleukin 10
[0171] The cytokine interleukin 10 was measured by a commercial
automated chemiluminescent Enzyme Immuno Assay (EIA) using an
Immulite Analyser from Diagnostic Products Corporation, Los
Angeles, Ca USA.
[0172] This is a competitive immunoassay using Alkaline Phosphatase
labelled IL-10 as tracer and adamantyl dioxetane as luminescent
substrate for ALP enzyme.
[0173] All reagents and calibrators are supplied in kit form by
DPC--Cat No. LKXPZ.
[0174] Analytical Performance:
TABLE-US-00005 MEAN SD CV % LEVEL 1 18.2 pg/mL 1.8 9.9 LEVEL 2 46.0
pg/mL 2.2 4.8 LEVEL 3 177 pg/mL 8.0 4.5
ANALYTICAL RANGE: 5-1000 pg/mL REFERENCE RANGE: <9.1 pg/mL*
*Study performed on 55 apparently healthy adults.
Serum Amyloid A
[0175] Polystyrene particles coated with antibodies to human SAA
are agglutinated when mixed with samples containing SAA. The
intensity of the scattered light in the nephelometer depends on the
concentration of the analyte in the sample and consequently its
concentration can be determined by comparison with dilutions of a
standard of known concentration.
TABLE-US-00006 IMPRECISION: CV 4.7% @ 192 mg/L N = 404 CV 2.8% @
7.0 mg/L N = 40
REFERENCE RANGE: In a population with normal serum CRP levels
(95.sup.th percentile=5.0 mg/L N=483) the 95.sup.th percentile for
N Latex SAA was found to be at 6.4 mg/L ANALYTICAL RANGE: 3.0-200
mg/L
Complement C3
[0176] The automated method used to measure complement C3
concentration in serum samples by nephelometric analysis using a
Dade Behring ProSpect analyzer with reagents and calibrators
supplied by Dade Behring Diagnostics (Sydney, Australia).
[0177] Soluble antigen solution (sample) and specific antibodies
(antiserum Cat No. OSAP15) are mixed in the reaction cuvettes.
Insoluble antigen-antibody complexes form immediately, producing
turbidity in the mixture and increasing the amount of light
scattered by the solution. Following an incubation period the
absorbance of the solution is measured at the analytical
wavelength.
TABLE-US-00007 IMPRECISION: CV 5.5% @ 1.05 g/L N = 61 CV 3.2% @
2.70 g/L N = 61
REFERENCE RANGE: 0.81-1.85 g/L
ANALYTICAL RANGE: 0.10-3.50 g/L
Complement C4
[0178] The automated method used to measure complement C4
concentration in serum samples by nephelometric analysis using a
Dade Behring ProSpect analyzer with reagents and calibrators
supplied by Dade Behring Diagnostics (Sydney, Australia).
[0179] Soluble antigen solution (sample) and specific antibodies
(antiserum Cat No. OSAO15) are mixed in the reaction cuvettes.
Insoluble antigen-antibody complexes form immediately, producing
turbidity in the mixture and increasing the amount of light
scattered by the solution. Following an incubation period the
absorbance of the solution is measured at the analytical
wavelength.
TABLE-US-00008 IMPRECISION: CV 4.7% @ 0.20 g/L N = 61 CV 3.8% @
0.53 g/L N = 61
REFERENCE RANGE: 0.10-0.40 g/L
ANALYTICAL RANGE: 0.03-1.50 g/L
Example 2
[0180] An elderly female ovarian cancer patient was monitored for
about 12 days for fluctuations in the levels of c-reactive protein,
serum amyloid A and the tumour marker CA125. Monitoring was
performed using standard laboratory tests on blood samples
collected every other day. The patient had not recently been
exposed to any anti-cancer therapy. Furthermore, there was no
evidence that the patient was suffering from any diseases other
than cancer. The CA125 (an ovarian cancer marker) was monitored as
an indicator of disease burden.
[0181] As shown in FIG. 1A, c-reactive protein (CRP) levels peaked
at the beginning of the monitoring period. Furthermore, as shown in
FIG. 1B serum amyloid A levels were elevated at the same time of
the CRP peak.
[0182] These results indicate that;
[0183] i) the levels of acute phase inflammatory proteins are
fluctuating in a cancer patient in the absence of any other known
factors which might cause these fluctuations such as viral
infection or chemotherapy,
[0184] ii) elevated levels of acute phase inflammatory proteins was
associated with lower levels of tumour antigens suggesting the
presence of effector cells, and
[0185] iii) increased levels of tumour antigen is associated with
lower levels of acute phase inflammatory proteins suggesting that
regulator cells have counteracted the beneficial activity of the
effector cells such that these cells are no longer active against
the tumour cells.
Example 3
[0186] A human subject suffering from a HIV infection was subjected
to highly active antiretroviral therapy (HAART) for at least 6
months and then taken off the treatment. C-reactive protein levels
were determined using standard techniques on samples obtained
during and after the completion of HAART.
[0187] As can be seen in FIG. 2, the results show that upon
conclusion of HAART c-reactive protein levels began to cycle,
peaking approximately every 14 days.
Example 4
[0188] Serum CRP was used to monitor the immune response in HIV
patient who had stopped their anti-retroviral therapy (FIG. 3). In
this study CRP levels mimicked viral load fluctuations as the
immune response switched on and off (FIG. 3). It is interesting to
note that these CRP fluctuations have an approximate 14 day
cycle.
Example 5
[0189] The "Pubmed" database (http://www.ncbi.nlm.nih.gov/) was
searched for the abstracts of journal articles which described the
results of Phase II or Phase III clinical trials using anti-cancer
agents (such as vinblastine and taxol) for the treatment of cancer.
Other criteria that were used to select the "abstracts" were that
the cancer was at a late stage (stage III or stage IV) and the
disease had disseminated. Some studies used a single drug whereas
others used combinations. No other criteria were used and studies
with an atypical complete response rate were not disregarded.
[0190] The complete response rate (as indicated in the abstracts)
for each trial was used to determine the average complete response
rate of each type of cancer. The results are provided as Table 1.
Notably, the average complete response rate varied only a small
degree, namely between 5.1 to 8.2% for all cancers analysed. The
results provided in Table 1 were used to determine the overall
average complete response rate. This average complete response rate
was 6.6% over at least 10 different types of cancers when
considering the 144 trials analysed.
[0191] With specific regard to the data provided for ovarian cancer
it should be noted that one study (Adachi et al., 2001) observed a
complete response rate of 25% which was very large compared to the
other 143 trials. This study looked at eight patients, with two
patients providing a complete response rate. Whilst this is well
within the realms of possibility, if the study is ignored the
overall complete response rate for the remaining ovarian cancer
studies is 7.1%.
[0192] The complete response rates are remarkably consistent
between the different cancers, and treatment regimes thereof,
suggesting an underlying factor relevant to all cancers and
treatments thereof. As described herein, this factor is that the
immune system is cycling. Accordingly, it can be argued that the
complete response rates provided in Table 1 are the result of the
anti-cancer agent being administered at an appropriate time such
that effector cell numbers are maximized whilst regulator cell
numbers are reduced or removed, or activity is down-regulated or
compromised, by the anti-cancer agent sufficient to elicit a
complete response.
TABLE-US-00009 TABLE 1 Complete Response Rates Resulting from
Clinical Trails with Anti- Cancer Drugs against Various Cancers.
Complete Cancer Type Response Rate (%) Number of Trials
Mesothelioma.sup.a 5.1 10 Gastric.sup.b 7.33 15
Hepatocellular.sup.c 6.6 8 Pancreatic.sup.d 7.35 4 Melanoma.sup.e
7.5 15 Prostate.sup.f 5.15 7 NSC Lung.sup.g 5.85 6 Breast.sup.h
7.36 19 Ovarian.sup.i 8.2 15 Colorectal.sup.j 6.85 28
Miscellaneous.sup.k 6.0 17 .sup.aTsavaris et al (1997), Monnet et
al (2002), Pinto et al (2001), Kindler et al (1999), Yogelzang et
al (1997), Planting et al (1995), Chahinian et al (1993), Raghavan
et al (1990), Henss et al (1988) and Mbidde et al (1986).
.sup.bKollmannsberger et al (2000), Sugimachi et al (2000), Jeen et
al (2001), Yamada et al (2001), Aitini et al (2001), Cho et al
(2002), Kornek et al (2002), Hofheinz et al (2002), Constenla et al
(2002), Kim et al (2002), Louvet et al (2002), Kikuyama et al
(2002), Bar Sela et al (2002), Murad et al (1999) and Sakata et al
(1998). .sup.cPorta et al (1995), Pohl et al (2001), Oon et al
(1980), Choi et al (1984), Zeng et al (1998), Carr et al (1997),
Patt et al (2003) and Leung et al (1999). .sup.dMurad et al (2003),
Ashamalla et al (2003), Safran et al (2002) and Sherman et al
(2001). .sup.eRetsas et al (1996), Nathan et al (2000), Bafaloukos
et al (2002), Bafaloukos et al (2002), Buzaid et al (1998), Gibbs
et al (2000), Atkins et al (2002), Gundersen et al (1989), Johnson
et al (1985), Nystrom et al (2003), Einzig et al (1991), Bedikian
et al (1995), Einzig et al (1996), Nathan et al (2000) and Chapman
et al (2002). .sup.fHudes et al (1997), Kelly et al (2001),
Savarese et al (1999), Small et al (2001), Savarese et al (2001),
Trivedi et al (2000) and Picus et al (1999). .sup.gMariotta et al
(2002), Recchia et al (2002), Perng et al (2000), Ginopoulos et al
(1999), Paccagnella et al (1996) and Agelaki et al (2001).
.sup.hFreyer et al (2003), Morabito et al (2003), Kosmas et al
(2003), Gebbia et al (2003), Thomas et al (1994), Romero et al
(1994), Pectasides et al (2001), Frasci et al (2002), Stathopoulos
et al (2002), Gomez-Bernal et al (2003), Freyer et al (2003), Komek
et al (1998), Michelotti et al (1996), Kakolyris et al (1999),
Twelves et al (1994), Fumoleau et al (1993) and Ibrahim et al
(1999). .sup.iLi et al (2002), Sehouli et al (2002), Rose et al
(2003), Faivre et al (2002), Dieras et al (2002), Adachi et al
(2001), Sutton et al (1994), McClay et al (1995), Manetta et al
(1994), Guastalla et al (1994), Covens et al (1992), Einzig AI.
(1994), Kjorstad et al (1992), Ozols et al (1984), Planner et al
(1996) and Amadori et al (1997). .sup.jCassinello et al (2003),
Glimelius et al (2002), Calvo et al (2002), Scheithauer et al
(2002), Neri et al (2002), Falcone et al (2001), Kouroussis et al
(2001), Meropol et al (2001), Comella et al (2000), Cascinu et al
(1999), Sobrero et al (1995), Gamelin et al (1998), Romero et al
(1998), Beerblock et al (1997), Blanke et al (1997), Grem et al
(1993), Jeremic et al (1993), Posner et al (1992), Sinnige et al
(1990), LoRusso et al (1989), Petrelli et al (1989), Valdivieso et
al (1981), Cassinello et al (2003), Reina et al (2003), Comella et
al (1999), Neri et al (1998), Pyrhonen et al (1992) and Beck et al
(1984). .sup.kCancers included renal cell carcinoma,
adenocarcinoma, squamous cell carcinoma, uterine cervical cancer,
glioblastoma multiforme, metastatic osteosarcoma, urothelial cancer
and endometrial cancer. Described by Schornagel et al (1989), Liu
et al (2001), Forastiere et al (1987), Okuno et al (2002), Takasugi
et al (1984), Hurteloup et al (1986), Kakolyris et al (2002),
Morris et al (1998), Takeuchi et al (1991), Fountzilas et al
(1999), Rosenthal et al (2000), Goorin et al (2002),
Rodriguez-Galindo et al (2002), Ahmad et al (2002), DiPaola et al
(2003) and Lissoni et al (1996).
[0193] If the typical cycle of effector/regulator cell numbers is
considered as about 15 days, the data in Table 1 suggest a one day
window to administer the anti-cancer therapy to achieve a complete
response rate. Partial response rates in the order of 30% are
typically noted suggesting that if the agent is administered at a
24 to 36 hour period either side of this "one day window" a
beneficial effect can also be achieved.
Example 6
Patient
[0194] The patient was a 75 year old female designated herein "Mrs
OM".
History
[0195] Liver cirrhosis, ischaemic heart disease, insulin dependent
diabetic. Diagnosed with squamous cell carcinoma of lower
oesophagus by endoscopy and biopsy/histology May 2004. The cancer
resulted in the patient finding it difficulty to swallow.
Tumour Description
[0196] Five centimetre circumferential mass at the base of the
oesophagus, partially occluding the lumen. Unknown epithelial/mural
penetration.
Therapy Regimen
[0197] Radiotherapy approx 33 courses of 15 minutes duration every
week day over 6-8 weeks. Plus limited chemotherapy due to
underlying other medical conditions.
[0198] The oncologist agreed to give two application of
chemotherapy (.about.8 hr infusion of 5 Fluorouracil and
Carboplatin). The application would be coordinated with the
patient's immune response cycle/oscillation to attempt timed
down-regulation of cycling tumour specific regulator cells.
Monitoring and Therapeutic Intervention
[0199] To detect the immune response oscillation, monitoring of the
patient's immune response started on the 28 May 2004, day 1, using
the following assays; CRP, SAA, C3, C4 & CA125. CA125 was used
to monitor disease progression as this has been reported in the
literature in the case of squamous cell carcinoma of the
oesophagus.
[0200] During the initial stages of monitoring, the patient
reported increased difficulty in swallowing, most likely due to the
tumour growing. This was corroborated by a consistent rise in all
the measured parameters (see FIGS. 4 to 7).
[0201] Interestingly the climbing CA125 briefly plateaued over an
approximate 24 hr period, (FIG. 6 day 12-14) only to rise at a
steeper gradient beyond that point. This was interpreted as the
patient's immune response switching on and modulating the tumour
growth and marker (CA125), only to switch off due to immune
regulation at the end of the approximate 24 hr period.
[0202] This approximate 24 hr period established the end of one
about 14 day cycle and the beginning of the next, and therefore a
potential intervention point or a reference point for projecting
ahead to further intervention points.
[0203] Having defined the beginning and end of the .about.14 day
cycle it was now possible to anticipate and project forward a
number of days to best estimate two potential chemotherapeutic
intervention points approximately 2 weeks apart.
[0204] It was decided to take blood/measurements from the patient
on the Tuesday, Wednesday and Thursday (FIG. 7, 13, 14 & 15
July, days 46, 47 & 48, arrowed as B) to accurately define the
therapeutic intervention point or window. If the cycle had been
accurately determined, a peak followed by a down turn in the CRP
should be seen over those days on which analysis was carried out.
(FIG. 7). This pattern in the CRP should be repeated approximately
14 days later and in keeping with the persistent periodicity of the
immune response oscillation. This was found to be the case (FIG.
7).
[0205] Based on the CRP results, the inventor recommended to the
oncologist to administer the first application of chemotherapy
about Wednesday 14 Jul. 2004 or Thursday 15 Jul. 2004. However, Mrs
OM had already been booked for chemotherapy on Friday 16 Jul. 2004,
and the oncologist decided not to change this appointment. Since
this date was just after the peak in the CRP (FIG. 7, arrowed as C)
it was felt by the inventor that the window of opportunity may have
been missed because the application of therapy may be 24 hrs too
late. The inventor expected that at the time the therpay was
administered CRP would have begun to rise again. This prediction
proved correct as no effect was apparent on the tumour after
administration of the chemotherapy.
[0206] A second intervention point was determined/predicted and
blood was taken on the Wednesday and Thursday (FIG. 7, 28.sup.th
& 29.sup.th July, days 63 and 64 arrowed as D). The prediction
was confirmed by a peak in the CRP analysis indicating Friday 30
Jul. 2004, day 65 (FIG. 7, arrowed as E) as the optimal
intervention point for application of chemotherapy. Chemotherapy
was administered as an 8 hr infusion on the Friday. On this
occasion the inventor predicted that this would be appropriate time
to administer the therapy as the CRP would still be decreasing.
[0207] On the Saturday the patient developed a mild fever and felt
generally unwell. Early afternoon on the Sunday 1.sup.st August,
day 67, (FIG. 7, arrowed as F), the patient haemorrhaged from the
tumour site and consequently was admitted to hospital. The patient
lost about 150 mls of blood and received 2 units of blood that day
and intravenous fluids/nourishment for the next 9 days.
[0208] CRP was measured on 4 Aug. 2004, day 69 (FIG. 7, arrowed as
G), and was found to have dropped significantly.
[0209] On the last day of hospitalisation the patient's oesophagus
was examined endoscopically. No tumour was evident (FIG. 7, arrowed
as H).
Interpretation
[0210] The patient's oscillating antitumour immune response was
released from regulation by the timed targeting of tumour specific
regulator cells by the single administration of the
chemotherapeutic agents at the right designated time. This is when
immune regulatory cells are clonally active, in mitosis and thus
vulnerable to down-regulation. Once released from regulation the
anti-tumour immune response resulted in a febrile episode as
reported by the patient on day 66 and subsequent tumour
destruction. The immune mediated tumour destruction resulted in
haemorrhage due to the tumour's potential invasive involvement in
the epithelium/wall of the oesophagus.
[0211] The above actions and observations demonstrates the
following: [0212] It is possible to detect a persistent regular
oscillation in the cancer patient. [0213] This oscillation is
associated with the tumour burden. [0214] The oscillation has an
approximate 14 day periodicity with a 7 day sub cycle. [0215] The
beginning and end of the cycle can be determined by different
parameters such as but not limited to CRP, SAA, C3, C4 and tumour
antigen levels. [0216] The narrow window of opportunity for the
application of a single. administration of chemotherapy can be
determined. [0217] A single chemotherapeutic administration at the
correct time directed against the cancer patient's immune system
can lead to a successful therapeutic outcome.
Example 7
[0218] The patient was a 71 year old female designated herein "Mrs
FO". Previously Mrs FO was diagnosed with ovarian cancer, received
surgery and several rounds of standard chemotherapy. Patient
represented with elevated CA125 at 200 U/ml prior to
monitoring.
[0219] Patient was monitored (bled) every Monday, Wednesday &
Friday for 4 weeks. A well described near synchronous and regular
oscillation with a 7/14 day periodicity showing a close correlation
between CRP, SAA & IL-2 serum measurements (see FIGS. 8 and 9).
More interestingly, FIG. 10 which shows CRP & CA125 versus
time, the CRP and CA125 oscillations are out of phase, indicating
an inverse relationship between the immune system and the cancer
marker.
[0220] FIG. 11 shows the relationship over time between SAA and
complement factor C3. Note that the two major C3 peaks are
approximately 14 days apart and coincide with alternating SAA peaks
which are also approximately 14 days apart. This supports a
hypothesis that the 7 day peaks represent alternating T and B cell
clonal expansions and the major C3 peaks are B cell associated as
complement is associated with antibody mediated lysis. This
observation can assist in establishing the beginning and end of a
cycle and therefore can also assist in determining the therapeutic
intervention point.
Example 8
[0221] The patient was a 64 year old male designated herein "Mr
GA". Bowel cancer was first diagnosed 1997, following which the
patient was exposed to surgery, chemotherapy and radiotherapy. Lung
recurrence was diagnosed by needle biopsy in February 2004. The
patient was determined to possess multiple lesions and was
subjected to 12 rounds of chemotherapy. The last chemotherapy was
in September 2004. The most recent scan identified at least one 2
cm lesion upper left lung. Currently, relatively well/active (mid
October 2004).
[0222] Blood was taken every other day (Monday, Wednesday, Friday)
for 15 days. CRP was measured, with the resulting showing an
approximate and regular 7/14 day CRP oscillation.
Example 9
[0223] A post menopausal opherectomised patient (WB) with
re-emerging tumour and elevated CA125 levels was ask to record the
frequency of hot flushes or febrile episodes and grade them as
mild, moderate or severe. The intensity of these episodes were
matched to the immune response CRP oscillation. The more intense
episodes and their increased frequency were coincident with the
large peaks. Thus recording body temperature may be used as an
adjunct to define the beginning and or end of the immune response
oscillation for the purposes of timing the application of
therapy.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0224] The present application claims priority from Provisional
Patent Application No 2003905858 filed on 24 Oct. 2003, the
contents of which is incorporated herein by reference.
[0225] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
[0226] All publications discussed above are incorporated herein in
their entirety.
[0227] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date of each claim of
this application.
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References