U.S. patent application number 12/979105 was filed with the patent office on 2011-06-23 for treating cancer.
Invention is credited to Svetomir N. Markovic.
Application Number | 20110150902 12/979105 |
Document ID | / |
Family ID | 41466606 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110150902 |
Kind Code |
A1 |
Markovic; Svetomir N. |
June 23, 2011 |
TREATING CANCER
Abstract
This document relates to methods and materials involved in
treating cancer (e.g., melanoma). For example, methods and
materials involved in using an anti-chronic inflammation treatment
(e.g., chemotherapy) in combination with a cancer treatment agent
(e.g., a cancer vaccine) to treat cancer are provided.
Inventors: |
Markovic; Svetomir N.;
(Rochester, MN) |
Family ID: |
41466606 |
Appl. No.: |
12/979105 |
Filed: |
December 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2009/049511 |
Jul 2, 2009 |
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12979105 |
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61078203 |
Jul 3, 2008 |
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Current U.S.
Class: |
424/158.1 ;
424/184.1; 424/277.1; 514/171 |
Current CPC
Class: |
A61K 31/56 20130101;
A61K 2039/545 20130101; A61K 39/39533 20130101; A61K 39/001191
20180801; A61K 31/4188 20130101; C07K 16/30 20130101; A61K 45/06
20130101; A61K 39/0011 20130101; A61K 31/675 20130101; C07K 2317/76
20130101; A61K 39/001192 20180801; A61K 51/1069 20130101; A61K
39/00115 20180801; A61K 31/555 20130101; A61K 31/337 20130101; A61P
29/00 20180101; A61K 39/001156 20180801; A61P 35/00 20180101; C07K
16/22 20130101; A61K 2039/876 20180801; A61K 39/3955 20130101; A61K
31/337 20130101; A61K 2300/00 20130101; A61K 31/555 20130101; A61K
2300/00 20130101; A61K 31/4188 20130101; A61K 2300/00 20130101;
A61K 31/675 20130101; A61K 2300/00 20130101; A61K 31/337 20130101;
A61K 2300/00 20130101; A61K 39/0011 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/158.1 ;
514/171; 424/184.1; 424/277.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/56 20060101 A61K031/56; A61K 39/00 20060101
A61K039/00; A61P 29/00 20060101 A61P029/00; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method for treating a mammal having cancer, said method
comprising: (a) administering to said mammal an anti-chronic
inflammation treatment under conditions wherein the level of global
chronic inflammation in said mammal is reduced, and (b)
administering to said mammal a cancer treatment agent under
conditions wherein the presence of said cancer is reduced.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said cancer is melanoma.
4. The method of claim 1, wherein said cancer is stage IV
melanoma.
5. The method of claim 1, wherein said anti-chronic inflammation
treatment comprises chemotherapy, radiation, an anti-IL-4 agent, an
anti-IL-13 agent, or a steroid treatment.
6. The method of claim 1, wherein said cancer treatment agent is a
cancer vaccine.
7. The method of claim 1, wherein said cancer vaccine is a MART-1,
gp100, or survivin cancer vaccine.
8. The method of claim 1, wherein the period of time between the
last administration of said anti-chronic inflammation treatment and
the first administration of said cancer treatment agent is between
two weeks and six months.
9. A method for treating a mammal having cancer, said method
comprising: (a) administering to said mammal an anti-TGF.beta.
antibody under conditions wherein the level of global chronic
inflammation in said mammal is reduced, and (b) administering to
said mammal a cancer treatment agent under conditions wherein the
presence of said cancer is reduced.
10. The method of claim 9, wherein said mammal is a human.
11. The method of claim 9, wherein said cancer is melanoma.
12. The method of claim 9, wherein said cancer is stage IV
melanoma.
13. The method of claim 9, wherein said cancer treatment agent is a
cancer vaccine.
14. The method of claim 9, wherein said cancer vaccine is a MART-1,
gp100, or survivin cancer vaccine.
15. The method of claim 9, wherein the period of time between the
last administration of said anti-TGF.beta. antibody and the first
administration of said cancer treatment agent is between two weeks
and six months.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US2009/049511, filed Jul. 2, 2009, which claims
the benefit of priority from U.S. Provisional Application Ser. No.
61/078,203, filed on Jul. 3, 2008. The disclosures of the prior
applications are considered part of (and are incorporated by
reference in) the disclosure of this application.
BACKGROUND
[0002] 1. Technical Field
[0003] This document relates to methods and materials involved in
treating cancer (e.g., melanoma). For example, this document
relates to methods and materials involved in using an anti-chronic
inflammation treatment (e.g., chemotherapy) in combination with a
cancer treatment agent (e.g., a cancer vaccine) to treat
cancer.
[0004] 2. Background Information
[0005] Cancer is a serious illness that affects many people every
year. Melanoma is the most serious form of skin cancer. It is a
malignant tumor that originates in melanocytes, the cells which
produce the pigment melanin that colors skin, hair, and eyes and is
heavily concentrated in most moles. While it is not the most common
type of skin cancer, melanoma underlies the majority of skin
cancer-related deaths. About 48,000 deaths worldwide are registered
annually as being due to malignant melanoma. Worldwide, there are
about 160,000 new cases of melanoma each year. Melanoma is more
frequent in white men and is particularly common in white
populations living in sunny climates. Other risk factors for
developing melanoma include a history of sunburn, excessive sun
exposure, living in a sunny climate or at high altitude, having
many moles or large moles, and a family or personal history of skin
cancer.
SUMMARY
[0006] This document provides methods and materials related to
treating cancer. For example, this document provides methods and
materials for using an anti-chronic inflammation treatment (e.g.,
chemotherapy) in combination with a cancer treatment agent (e.g., a
cancer vaccine) to treat cancer. As described herein, cancer can
induce a global state of immune dysfunction and/or chronic
inflammation in the cancer patient. This global state of immune
dysfunction and/or chronic inflammation can prevent the patient
from mounting a successful response against the cancer. For
example, a cancer patient with a global state of chronic
inflammation can be in a state such that the patient is unable to
generate an anti-cancer immune response when given an anti-cancer
vaccine. The methods and materials provided herein can be used to
reduce the global state of immune dysfunction and/or chronic
inflammation present within a cancer patient such that the cancer
patient can better respond to a cancer treatment such as a cancer
vaccine. As described herein, chemotherapy, radiation, anti-IL-4
agents (e.g., anti-IL-4 antibodies), anti-IL-13 agents (e.g.,
soluble IL-13 receptor), steroids, and combinations thereof can be
used to reduce the global state of immune dysfunction and/or
chronic inflammation present within a cancer patient. Once the
global state of immune dysfunction and/or chronic inflammation
present within a cancer patient is reduced, the cancer patient can
be treated with an appropriate cancer treatment such as a cancer
vaccine or other immune stimulating agents (e.g., IL-2 or
IL-12).
[0007] In general, this document features a method for treating a
mammal having cancer. The method comprises, or consists essentially
of, (a) administering to the mammal an anti-chronic inflammation
treatment under conditions wherein the level of global chronic
inflammation in the mammal is reduced, and (b) administering to the
mammal a cancer treatment agent under conditions wherein the
presence of the cancer is reduced. The mammal can be a human. The
cancer can be melanoma. The cancer can be stage IV melanoma. The
anti-chronic inflammation treatment can comprise chemotherapy,
radiation, an anti-IL-4 agent, an anti-IL-13 agent, or a steroid
treatment. The cancer treatment agent can be a cancer vaccine. The
cancer vaccine can be a MART-1, gp100, or survivin cancer vaccine.
The period of time between the last administration of the
anti-chronic inflammation treatment and the first administration of
the cancer treatment agent can be between two weeks and six
months.
[0008] In another aspect, this document features a method for
treating a mammal having cancer. The method comprises, or consists
essentially of, (a) administering to the mammal an anti-TGF.beta.
antibody under conditions wherein the level of global chronic
inflammation in the mammal is reduced, and (b) administering to the
mammal a cancer treatment agent under conditions wherein the
presence of the cancer is reduced. The mammal can be a human. The
cancer can be melanoma. The cancer can be stage IV melanoma. The
cancer treatment agent can be a cancer vaccine. The cancer vaccine
can be a MART-1, gp100, or survivin cancer vaccine. The period of
time between the last administration of the anti-TGF.beta. antibody
and the first administration of the cancer treatment agent can be
between two weeks and six months.
[0009] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0010] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1: Effects of 1% patient plasma (vs. normal plasma) on
in vitro maturation of normal DC. Presented is a representative
experiment demonstrating the % co-stimulatory molecule positive
(CD80, 83, 86) DC.
[0012] FIG. 2. Allogeneic mixed lymphocyte reaction cultures
evaluating proliferation of T cells mixed at differing ratios with
mature or immature dendritic cells in the presence of normal
(control) plasma or plasma from a patient with metastatic melanoma
(MTB plasma). T cell proliferation was assessed by 3H-TdR
incorporation. Similar results were observed in two other
experiments.
[0013] FIG. 3. Three dimensional representation of the results of
Principal Component Analysis (PCA). The PCA was performed on 234
clinical samples for concentrations of 27 cytokines. Each sphere
represents one clinical sample. The dark spheres represent stage IV
melanoma patients, and the lighter spheres represent atypical nevi,
benign nevi, in situ melanoma, stage I melanoma, stage II melanoma,
or stage III melanoma. The axes are the main principal components.
Significant grouping is only evident for patients in the cohort of
stage IV (metastatic) melanoma (dark spheres).
[0014] FIG. 4: Mean plasma IL-4 levels (pg/mL).+-.SD across stages
of melanoma (melanoma in situ and stages: I, II, III, IV), patients
with atypical nevi (atypical) and benign nevi (normal
controls).
[0015] FIG. 5: Assessment of T-cell phenotype and function in
healthy donors and stage IV melanoma patients. The number of
T-cells exhibiting the FoxP3 (Treg) or PD-1 phenotype in peripheral
blood was determined in healthy donors and stage IV melanoma
patients (A). The frequency of FoxP3 positive cells were measured
by 3-color flow cytometry, CD4-PC5, CD25-PE and FoxP3-Alexa flour
488. The mean percent (+/-SD) of FoxP3 positive were determined
from the CD4 and CD25 double positive population. The mean
frequency (+/-SD) of PD-1+ cells was measured from the CD8+
population. The frequency (+/-SD) of tetramer positive (CMV or
MART-1) CD8+ T cells was compared among normal volunteers and
patients with stage IV melanoma (B).
[0016] FIG. 6: VEGF levels in patients with metastatic melanoma.
(A) RNA expression of cytokines in human metastatic melanoma
tissue. Twenty-four frozen biopsies of metastatic melanoma tumor
tissues was used to extract RNA for expression array analysis.
Illustrated are the RNA expression intensity profiles of 45 probes
for 24 cytokines. (B) Comparison of expression intensities between
genes coding for Th1 (IFN-.gamma. and IL-2), Th2 (IL-4, IL-5,
IL-10, and IL-13) cytokines and VEGF. There were no statistically
significant differences when comparing Th1 vs. Th2 cytokine
expression levels (p=0.04). There was a statistically significant
difference when comparing VEGF expression with Th1 or Th2 cytokines
(p<0.001). Levels of significance were determined using the
Wilcoxin signed-rank test. (C) ELISA (mean concentration +/-SD) for
VEGF-A was performed on plasma samples from healthy donors (n=30)
and stage IV melanoma patients (n=40).
[0017] FIG. 7. Healthy donor PBMCs were cultured in vitro for 48
hours in the presence of increasing concentrations of recombinant
human VEGFA. At the end of the incubation, cells were stimulated
with PMA and ionomycin, in the presence of brefeldin A and stained
for intracellular IFN.gamma., IL-4, or IL-13 and surface
immunophenotyped for CD294 or TIM-3. Cells were then analyzed for
the frequency of CD4 cells (% of CD4) expressing said phenotypes
using flow cytometry.
[0018] FIG. 8: Co-culture with recombinant human VEGF shifts
T-helper polarity from Th1 (IFN-.gamma.) to Th2 (IL-4)
predominance. PBMC (A) isolated from healthy donors were stimulated
with PMA and ionomycin in the presence of brefeldin-A,
permeabolized, and intracellularly stained for human IFN-.gamma.
(FITC) and human IL-4 (PE). PBMC were exposed to increasing
concentrations of VEGF (0-16 pg/mL) without/with IL-12. All cells
were immunostained with PC5 anti-human CD4. Purified CD4+ T-cells
(B) were negatively isolated using Miltenyi beads, cultured, and
stained in the same fashion as PBMC (A). Similar results were
observed in 5 different experiments.
[0019] FIG. 9. Changes in plasma VEGF levels (plasma VEGFA in
pg/mL; top left) at three time points in a single patient with
metastatic melanoma treated on protocol N047a correlate with
improved Th1/Th2 ratio as determined by intracellular staining of
CD4+ cells for IFN gamma or IL-4 (top right). These also correlate
with emergence of increased frequencies of tumor specific CTL
(bottom right) as determined by tetramer assay.
[0020] FIG. 10. Cellular interactions of acute and chronic
inflammation. MSC (myeloid suppressor cell); Th1 & Th2 (T
helper lymphocytes type 1 & 2); Treg: regulatory T cell; DC1
(dendritic cells, type 1); DC2 (dendritic cells type 2); CTL
(cytotoxic T lymphocyte); illustrated is central role of tumor
derived VEGF in polarizing immunity towards Th2 mediated "chronic
inflammation".
[0021] FIG. 11: Correlation of surface immunophenotyping for CD294
and TIM-3 with intracellular IL-4, IL-13 and IFN.gamma. for the
purposes of enumeration of Th2 and Th1 cells respectively.
[0022] FIG. 12: Changes in the ratio of human PBMC derived CD4 T
cell subsets (Th1 vs. Th2) following in vitro incubation with
varying concentrations of VEGFA or TGF.beta.. These results
indicate that both VEGFA and TGF.beta. have a similar effect on
Th1/Th2 polarity in human PBMC derived CD4 cells.
[0023] FIG. 13: Relative ratios of human PBMC derived CD4 T cells
subsets (Th1 vs. Th2) cultured in vitro with varying concentrations
of VEGFA in the absence or presence of increasing concentrations of
anti-TGF.beta. neutralizing antibody. Untreated (media) as well as
Th1 (Th1) and Th2 (Th2) favorable in vitro conditions are presented
as controls. These results indicate that presence of anti-TGF.beta.
antibodies reverses the Th1/Th2 modulation of VEGFA in vitro,
suggesting that the observed VEGF effect in these cells may be
TGF.beta. mediated.
DETAILED DESCRIPTION
[0024] This document provides methods and materials related to
treating cancer in mammals. For example, this document provides
methods and materials related to the use of an anti-chronic
inflammation treatment (e.g., chemotherapy) in combination with a
cancer treatment agent (e.g., a cancer vaccine) to treat
cancer.
[0025] The methods and materials provided herein can be used to
treat cancer in any type of mammal including, without limitation,
mice, rats, dogs, cats, horses, cows, pigs, monkeys, and humans.
Any type of cancer, such as skin cancer (e.g., melanoma), can be
treated. Examples of cancer that can be treated as described herein
include, without limitation, skin cancer, lung cancer, breast
cancer, prostate cancer, ovarian cancer, and colon cancer. In some
cases, stage I, stage II, stage III, or stage IV melanoma can be
treated using the methods and materials provided herein.
[0026] In general, cancer can be treated by administering an
anti-chronic inflammation treatment such that the global state of
immune dysfunction and/or chronic inflammation present within a
cancer patient is reduced. For example, chemotherapy, radiation,
anti-IL-4 agents (e.g., anti-IL-4 antibodies), anti-IL-13 agents
(e.g., soluble IL-13 receptor), steroids, and combinations thereof
can be used to reduce the global state of immune dysfunction and/or
chronic inflammation present within a cancer patient. In some
cases, chemotherapy such as paclitaxel, carboplatin, temozolomide,
or cyclophosphamide can be administered to a cancer patient to
reduce the global state of immune dysfunction and/or chronic
inflammation present within a cancer patient. In some cases, an
anti-chronic inflammation treatment can include, without
limitation, administering an anti-TGF.beta. antibody. For example,
anti-TGF.beta. antibodies can be administered to a cancer patient
to reduce the global state of immune dysfunction and/or chronic
inflammation present within a cancer patient. Examples of
anti-TGF.beta. antibodies include, without limitation, human
monoclonal anti-TGF-.beta.1 antibodies such as CAT-192 (Genzyme
Inc.).
[0027] Any appropriate method can be used to assess whether or not
the global state of immune dysfunction and/or chronic inflammation
was reduced following an anti-chronic inflammation treatment. For
example, cytokine profiles (e.g., IL-4, IL-13, IL-4, IL-13, IL-5,
IL-10, IL-2, and interferon gamma) present in blood can be assessed
before and after an anti-chronic inflammation treatment to
determine whether or not the global state of immune dysfunction
and/or chronic inflammation was reduced.
[0028] Once the global state of immune dysfunction and/or chronic
inflammation present within a cancer patient is reduced, the cancer
patient can be treated with an appropriate cancer treatment (e.g.,
an immune cancer treatment) such as a cancer vaccine. Examples of
appropriate cancer treatment agents include, without limitation,
immune stimulating cytokines (e.g., IL-2, IL-12, interferon alpha,
and interferon gamma), inhibitors of immune down-regulation (e.g.,
anti-CTLA4, anti-41bb, anti-PD-1, and anti-CD25), and cancer
vaccines (e.g., MART-1, gp100, survivin, and tyrosinase cancer
vaccines). It will be appreciated that paclitaxel, carboplatin,
bevacizumab, and anti-CTLA-4 can be used to treat (e.g., skin
cancer) upon administration either individually or in any
combination thereof (e.g., paclitaxel, carboplatin and
bevacizumab).
[0029] In some cases, the amount of time between administration of
an anti-chronic inflammation treatment and administration of a
cancer treatment can be between two weeks and twelve months (e.g.,
between two weeks and eleven months, between two weeks and ten
months, between two weeks and nine months, between two weeks and
eight months, between two weeks and seven months, between two weeks
and six months, between one month and twelve months, between one
month and six months, or between two months and six months). For
example, a chemotherapy agent (e.g., paclitaxel) can be
administered to a cancer patient to reduce the global state of
immune dysfunction and/or chronic inflammation present within the
cancer patient. Then, after one month without any type of
anti-chronic inflammation treatment, a cancer treatment (e.g., a
cancer vaccine) can be administered to the cancer patient.
[0030] Any appropriate method can be used to administer a cancer
treatment agent to a mammal For example, a cancer treatment agent
can be administered orally or via injection (e.g., subcutaneous
injection, intramuscular injection, intravenous injection, or
intrathecal injection). In some cases, cancer treatment agents can
be administered by different routes. For example, one cancer
treatment agent can be administered orally and a second cancer
treatment agent can be administered via injection.
[0031] In some cases, a cancer treatment agent can be administered
following resection of a tumor. Cancer treatment agent can be
administered to a mammal in any amount, at any frequency, and for
any duration effective to achieve a desired outcome (e.g., to
increase progression-free survival or to increase the time to
progression). In some cases, cancer treatment agents can be
administered to a mammal having skin cancer to reduce the
progression rate of melanoma by 5, 10, 25, 50, 75, 100, or more
percent. For example, the progression rate can be reduced such that
no additional cancer progression is detected. Any method can be
used to determine whether or not the progression rate of skin
cancer is reduced. For example, the progression rate of skin cancer
can be assessed by imaging tissue at different time points and
determining the amount of cancer cells present. The amounts of
cancer cells determined within tissue at different times can be
compared to determine the progression rate. After treatment as
described herein, the progression rate can be determined again over
another time interval. In some cases, the stage of skin cancer
after treatment can be determined and compared to the stage before
treatment to determine whether or not the progression rate was
reduced.
[0032] In some cases, a cancer treatment agent can be administered
to a mammal having cancer under conditions where progression-free
survival or time to progression is increased (e.g., by 5, 10, 25,
50, 75, 100, or more percent) as compared to the median
progression-free survival or time to progression, respectively, of
corresponding mammals having untreated cancer.
[0033] An effective amount of a cancer treatment agent can be any
amount that reduces the progression rate of cancer, increases the
progression-free survival rate, or increases the median time to
progression without producing significant toxicity to the mammal
Typically, an effective amount of a cancer treatment agent such as
bevacizumab can be from about 5 mg/kg/week to about 15 mg/kg/week
(e.g., about 10 mg/kg/week). If a particular mammal fails to
respond to a particular amount, then the amount of one or more of
the compounds can be increased by, for example, two fold. After
receiving this higher concentration, the mammal can be monitored
for both responsiveness to the treatment and toxicity symptoms, and
adjustments made accordingly. The effective amount can remain
constant or can be adjusted as a sliding scale or variable dose
depending on the mammal's response to treatment. Various factors
can influence the actual effective amount used for a particular
application. For example, the frequency of administration, duration
of treatment, use of multiple treatment agents, route of
administration, and severity of the cancer may require an increase
or decrease in the actual effective amount administered.
[0034] The frequency of administration can be any frequency that
reduces the progression rate of cancer, increases the
progression-free survival rate, or increases the median time to
progression without producing significant toxicity to the mammal
For example, the frequency of administration can be from about once
a month to about three times a month, or from about twice a month
to about six times a month, or from about once every two months to
about three times every two months. The frequency of administration
can remain constant or can be variable during the duration of
treatment. In addition, the frequency of administration of multiple
cancer treatment agents can be the same or can differ. For example,
one cancer treatment agent can be administered three times during a
28 day period, while a second cancer treatment agent can be
administered one time, and third cancer treatment agent can be
administered two times during the same period. A course of
treatment with a cancer treatment agent can include rest periods.
For example, a cancer treatment agent can be administered over a
two week period followed by a two week rest period, and such a
regimen can be repeated multiple times. As with the effective
amount, various factors can influence the actual frequency of
administration used for a particular application. For example, the
effective amount, duration of treatment, use of multiple treatment
agents, route of administration, and severity of the cancer may
require an increase or decrease in administration frequency.
[0035] An effective duration for administering a composition
provided herein can be any duration that reduces the progression
rate of cancer, increases the progression-free survival rate, or
increases the median time to progression without producing
significant toxicity to the mammal Thus, the effective duration can
vary from several days to several weeks, months, or years. In
general, the effective duration for the treatment of cancer can
range in duration from several weeks to several months. In some
cases, an effective duration can be for as long as an individual
mammal is alive. Multiple factors can influence the actual
effective duration used for a particular treatment. For example, an
effective duration can vary with the frequency of administration,
effective amount, use of multiple treatment agents, route of
administration, and severity of the cancer.
[0036] After administering a composition provided herein to a
mammal, the mammal can be monitored to determine whether or not the
cancer was treated. For example, a mammal can be assessed after
treatment to determine whether or not the progression rate of
cancer was reduced (e.g., stopped). As described herein, any
appropriate method can be used to assess progression and survival
rates.
[0037] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Patients, Methods, and Materials
Patients
[0038] Blood samples collected from patients with early stage
melanoma (melanoma in situ and melanoma stage I, II and III) and
benign nevi (atypical/dysplastic nevi) were newly diagnosed
patients with no previous treatment. All patients were tumor free
at the time of peripheral blood collection. Samples from patients
with metastatic melanoma (newly diagnosed, previously untreated) as
well as healthy volunteers/controls were collected under a separate
melanoma blood and tissue banking protocol. Both protocols were
reviewed and approved for use in these studies. All biospecimens
were collected, processed, and stored in uniform fashion following
established standard operating procedures. All patients signed an
informed consent document. The presented study describes data
obtained from 113 men and 96 women ranging in age from 21 to 85
(Table 1).
TABLE-US-00001 TABLE 1 Study patient population distributed by
clinical category, age, sex and assayed immune parameters. Assayed
immune parameters Age T-cell Total mean .+-. SD Cell Plasma
Function RNA Clinical category Patients (range) % female Subset
Cytokines Tetramer Assay array Benign Nevi 34 51 .+-. 12 68 26 34 7
2 0 (21-71) Atypical/Dysplastic 25 52 .+-. 16 44 22 16 11 1 0
(25-84) In situ melanoma 36 61 .+-. 16 36 30 35 16 3 0 (26-84)
Stage I 45 54 .+-. 17 44 36 44 16 4 0 (21-82) Stage II 16 55 .+-.
17 44 11 12 9 0 0 (22-81) Stage III 16 53 .+-. 19 44 14 16 6 1 0
(23-83) Stage IV 37 56 .+-. 14 43 32 30 27 16 24 (28-85)
Collection of Plasma and Peripheral Blood Mononuclear Cells
[0039] Peripheral venous blood (50-90 mL) was drawn into
heparinized Vacutainer tubes that were processed and separated into
plasma and peripheral blood mononuclear cells (PBMC) following
gradient centrifugation using Ficoll-Paque (GE Healthcare Uppsala,
Sweden). Plasma was collected and immediately frozen at -80.degree.
C. (1 mL aliquots). PBMC were collected, washed in phosphate
buffered saline (PBS), counted, diluted to 1.times.10.sup.7/mL and
viably frozen in 90% cosmic calf serum (Hyclone Inc. Logan, Utah)
and 10% DMSO (Sigma St. Louis, Mo.). All assays were batch-analyzed
at the end of the study.
Immunophenotyping
[0040] The following anti-human monoclonal antibodies were used in
PBMC immunophenotyping for flow cytometry: anti-CD3-APC, FITC and
PE, anti-CD4-FITC, anti-CD8-PE, anti-CD16 PE, anti-CD56 PE,
anti-CD62L APC, anti-CD69 FITC, anti-CD14 FITC, anti-CD16 FITC,
anti-CD19 FITC, anti-CD11c APC, anti-CD80 PE, anti-CD83 PE,
anti-CD86 PE, anti-CD40 APC, anti-HLA-DR PC5, anti-PD-1 (BD
Pharmingen San Jose, Calif.). The human monoclonal antibodies
anti-CD4 PC5 and anti-CD25 PE were purchased from Biolegend (San
Diego, Calif.) and used in conjunction with anti-human FoxP3 for
the enumeration of T.sub.reg cells. The following anti-human
monoclonal antibodies were used for intracellular staining for flow
cytometry: anti-IFN.beta. FITC, anti-IL-13 PE, anti-IL-4 PE (R and
D Systems Minneapolis, Minn.), and anti-FoxP3 Alexaflour 488
(Biolegend San Diego, Calif.).
[0041] Previously frozen PBMC (0.5-1.0 .times.10.sup.6 cells/mL)
were thawed and aliquoted into 96 well rounded bottom plates (100
.mu.L/well). The desired antibody or antibody pool was added at 5
.mu.L/well. The cells and antibodies were incubated for 30 minutes
at 4.degree. C. and washed twice with 1.times. PBS (Cellgro
Manassas, Va.), 0.1% BSA and 0.05% sodium azide (Sigma St. Louis,
Mo.). Four-color flow cytometry was performed on a LSRII flow
cytometer (Becton Dickenson San Jose, Calif.), and Cellquest
software (Becton Dickenson San Jose, Calif.) was utilized for data
analysis. For dendritic cells, a gate was set on cells, which were
HLA-DR.sup.+ and Lin.sup.- (CD3, CD14, CD16 and CD19). From this
population the percentage of cells, which were CD11c.sup.+ and
positive for costimulatory molecules (CD80, CD83 and CD86) was
determined as previously elsewhere (Fricke et al., Clin. Cancer
Res., 13:4840-8 (2007)). A panel of tumor associated antigen
tetramers, MART-1.sub.26-35, gp100.sub.264-272, gp100.sub.209-217,
and tyrosinase.sub.369-377 (Beckman Coulter San Jose, Calif.) were
used to enumerate the frequency of tumor antigen specific CD8
positive T-cells. Recall antigens, EBV.sub.280-288 and
CMV.sub.495-503 (Beckman Coulter San Jose, Calif.) were used as
positive controls. For tetramer frequencies, a gate was set on
lymphocytes, which were CD8.sup.+ and negative for CD4, CD14 and CD
19. Three-color flow cytometry was performed on a LSRII flow
cytometer (Becton Dickenson San Jose, Calif.) and Cellquest
software (Becton Dickenson San Jose, Calif.) was utilized for data
analysis.
[0042] Functional enumeration of tumor antigen specific CTL was
performed using an artificial antigen presenting cell method (aAPC)
as described elsewhere (Markovic et al., Clin. Exp. Immunol.,
145:438-47 (2006)). Briefly, frozen PBMC were thawed, labeled with
the desired tumor antigen peptide/class I tetramers (Beckman
Coulter Fullerton, Calif.) and stimulated for 6 hours with
streptavidin coated microbeads (Invitrogen Oslo, Norway) loaded
with HLA-A2 class I containing tumor antigen peptides of choice
(MART-1, gp100 or tyrosinase) and anti-human CD28 in the presence
of brefeldin A (Sigma, St. Louis, Mo.). After stimulation, the
cells were fixed with 2% paraformaldehyde (Sigma, St. Louis, Mo.)
and then permeabilized with 0.1% saponin (Sigma, St. Louis, Mo.) in
PBS. Cells were then immunophenotyped with anti-human CD4-PC5 or
CD8-APC and intracellular staining was done with anti-human
IFN.gamma. FITC or IL-4 PE. Four-color flow cytometry was performed
with a FACSCaliber and Cellquest software (Becton Dickenson San
Jose, Calif.) was utilized for data analysis.
Plasma Cytokine, Chemokines and Growth Factor Concentrations
[0043] Protein levels for 27 cytokines, chemokines, and growth
factors, including IL-1.beta., IL-1ra, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, Eotaxin,
FGF basic, G-CSF, GM-CSF, IFN-.gamma., IP-10, MCP-1, MIP-1.alpha.,
MIP-1.beta., PDGF, RANTES, TNF-.alpha., and VEGF, were measured
using the Bio-plex cytokine assay (Bio-rad, Hercules, Calif.) as
per manufacturer's instructions. Patient plasma was diluted 1:4 in
dilution buffer and 50 .mu.L was added to washed, fluorescently
dyed microspheres (beads) to which biomolecules of interest are
bound. The beads and diluted patient plasma were incubated for 30
minutes at room temperature with agitation. After the incubation
the beads were washed in Bio-plex wash buffer and placed in 25
.mu.L of detection antibody and incubated for 30 minutes as
described above. After washing, the beads were placed in
streptavidin-PE, incubated, and washed a final time. The bound
beads were resuspended in 125 .mu.L Bio-plex assay buffer and read
with the Luminex plate reader (Bio-rad, Hercules, Calif.). Protein
concentrations were determined using a standard curve generated
using the high PMT concentrations with sensitivity from 10-1000
pg/mL.
VEGF Mediated T.sub.h1/T.sub.h2 Polarity
[0044] To determine the effect of VEGF on T.sub.h1 and T.sub.h2
polarity, PBMC from healthy donors were stimulated for 3 days with
CD3/CD28 expander beads (Invitrogen Oslo, Norway) with and without
increasing doses of recombinant VEGF (1-16 pg/mL). Cells were also
cultured with 10 .mu.g/mL recombinant human IL-12 (R and D Systems,
Minneapolis, Minn.) or 8 .mu.g/mL of a monoclonal anti-human IL-12
(R and D Systems Minneapolis, Minn. clone #24910). After the
culture, the cells were harvested and restimulated with 50 ng/mL
PMA (Sigma, St. Louis, Mo.) and 1 .mu.g/mL ionomycin (Sigma, St.
Louis, Mo.) in the presence of 10 .mu.g/mL brefeldin A for 4 hours.
The cells were then stained with anti-human CD4, anti-human
IFN-.gamma. and anti-human IL-4 flow cytometry.
Tumor Tissue RNA Extraction and Microarray
[0045] Frozen tissue sections of melanoma biopsies, were examined,
regions of pure tumor with little/no evidence of necrosis or
stromal infiltration were outlined, scraped off the slides, and
used for RNA extraction. Total RNA was isolated from the excised
tumor tissue using the Qiagen RNA extraction kit (Qiagen Valencia,
Calif.). The quality of the RNA was evaluated by obtaining
electropherograms on Agilent 2100 Bioanalyzer and RNA integrity
number (RIN) using 2100 Expert software (Agilent Technologies, Inc.
Palo Alto, Calif.). cDNA was prepared from a total of 10 .mu.g of
RNA. Samples were quantified using standard spectrophotometry using
a Tecan spectrophotometer (Tecan US, Research Triangle Park, N.C.)
and considered acceptable if the A260/280 reading was >1.7. The
purified cDNA was used as a template for in vitro transcription
reaction for the synthesis of biotinylated cRNA using RNA
transcript labeling reagent (Affymetrix, Santa Clara, Calif.).
Labeled cRNA was then fragmented and hybridized onto the U133 Plus
2.0 array. Appropriate amounts of fragmented cRNA and control
oligonucleotide B2 were added along with control cRNA (BioB, BioC,
and BioD), herring sperm DNA, and bovine serum albumin to the
hybridization buffer. The hybridization mixture was heated at
99.degree. C. for 5 minutes followed by incubation at 45.degree. C.
for 5 minutes before injecting the sample into the microarray.
Then, the hybridization was carried out at 45.degree. C. for 16
hours with mixing on a rotisserie at 60 rpm. After hybridization,
the solutions were removed, and the arrays were washed and then
stained with streptavidin-phycoerythrin (Molecular Probes, Eugene,
Oreg.). After washes, arrays were scanned using the GeneChip
Scanner 3000 (Affymetrix, Santa Clara, Calif.). The quality of the
fragmented biotin labeled cRNA in each experiment was evaluated
before hybridizing onto the U133A expression array by both
obtaining electropherograms on Agilent 2100 Bioanalyzer and
hybridizing a fraction of the sample onto test-3 array as a measure
of quality control. GeneSpring GX 7.3 (Agilent Technologies, Inc.
Santa Clara, Calif.) data analysis software was used to analyze the
results of the microarray experiment. Gene expression values were
normalized by the GCRMA algorithm (Bolstad et al., Bioinformatics,
19:185-93 (2003)).
Statistical Analysis
[0046] The majority of samples analyzed in this report were
randomly assigned to batches for each laboratory assay due to the
fact that all samples were not collected/processed at the same
time. The randomization was stratified to assure an even
distribution across the stages of disease for each batch. The
distributions of the results of each run were examined, and those
that did not appear to be normally distributed were transformed
using either logarithmic or square root transformations. In order
to look at differences in various parameters between stages of
disease, analysis was performed utilizing analysis of covariance
(ANCOVA), adjusting for age, gender, and batch effects. Results of
this analysis were summarized by least square means and 95%
confidence intervals for each stage of disease. The p-values
presented are those from the overall ANCOVA, which compares the
means levels of each parameter across all stages of disease.
P-values <0.05 were considered to be statistically significant.
Due to the magnitude of the cytokine data from the multiplex assay
the data was processed using Partek 6.3 software (Partek Inc. St.
Louis Mo.) and analyzed using a principal component analysis (PCA)
approach. PCA was utilized in an effort to vector space transform a
multidimensional data set representing 27 variables for each
individual patient and group patients based on similar cytokine
concentrations revealing the internal relationships of cytokines
within patient groups (e.g., per stage of melanoma) in an unbiased
way.
Example 2
Systemic Immune Dysfunction
[0047] Preliminary results support the notion that systemic immune
dysfunction can lead to the observed induction of tolerance
following peptide vaccination in clinical trials. In this example,
normal donor myeloid DC were exposed to in vitro culture conditions
(GM-CSF, IL-4, and CD40L) that lead to their differentiation and
maturation (expression of co-stimulatory molecules) (FIG. 1). The
addition of patient plasma to these experimental conditions
resulted in a significant reduction in the number of DC expressing
key co-stimulatory molecules (maturation), suggesting the presence
of a soluble inhibitor(s) of DC maturation. Similar observations,
with varying degrees of "suppression" were made in another nine
experiments (nine other samples of patient plasma). Even when
normal DCs (mature or immature) were combined with other normal
donor lymphocytes in a mixed lymphocyte culture, the presence of
patient vs. normal plasma lead to significantly diminished
lymphocyte proliferation (FIG. 2). Thus, factors in the plasma of
patients with metastatic melanoma are interrupting normal immune
cell function.
Example 3
Evidence for Th2 Driven Systemic Chronic Inflammation in Patients
with Metastatic (Stage IV) Melanoma
[0048] The identity of the unknown factor(s) could be a known
cytokine. Thus, a screening study was performed to quantify the
plasma concentrations of 27 different cytokines (BioRad human
27-plex cytokine panel assaying for plasma concentrations of
IL-1.beta., IL-1r.alpha., IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-12 (p70), IL-13, IL-15, IL-17, basic FGF, eotaxin, G-CSF,
GM-CSF, IFN-.gamma., IP-10, MCP-1, MIP-1.alpha., MIP-1.beta., PDGF,
RANTES, TNF-.alpha., and VEGF) in plasma of over 200 patients with
all stages of melanoma (stage I thought IV), melanoma in situ,
atypical nevi (possible pre-malignant lesions) as well as normal
controls (patients with benign nevi). Due to the volume of data (27
assays for over 200 subjects), principal component analysis (PCA)
was used to identify patterns (groupings) of cytokine data based on
clinical subject classifications. The results suggested that the
most significant differences in plasma cytokine levels among the
different patient diagnostic categories was detected in the
category of patients with stage IV melanoma/metastatic melanoma
(circled brown spheres in FIG. 3). Closer analysis of the data
indicated that the most significant difference among the cytokines
was noted for IL-4 (FIG. 4, left), the key regulatory cytokine of
the Th2 immune response. Statistical comparisons (Student's t-test)
of the mean cytokine concentrations between patients with
metastatic melanoma (stage IV) versus all others depicted a pattern
of predominance of other Th2 cytokines in addition to IL-4 (IL-10,
IL-13, IL-5, eotaxin, IL-9) in patients with stage IV melanoma
(FIG. 4, right). For all of these cytokines, the pattern of plasma
concentrations across stages of melanoma was similar to that of
IL-4 (no significant changes among any of the patient cohorts
except stage IV melanoma). These results are complementary to
reports suggesting an increased frequency of Th2 cells in the blood
of patients with advanced cancer (as well as chronic infections)
relative to normal controls (Inagaki et al., Int. J. Cancer,
118(12):3054-61 (2006); Matsuda et al., Dis. Colon Rectum,
49(4):507-16 (2006); Agarwal et al., Cancer Immunol. Immunother.,
55(6):734-43 (2006); and Kumar et al., Oncol. Rep., 15(6):1513-6
(2006)).
[0049] Preliminary analysis of three random samples from patients
with stage IV melanoma also demonstrated an increased frequency of
Th2 cells. These data support the hypothesis of the presence of a
state of Th2 mediated chronic inflammation in patients with
metastatic melanoma and offer an explanation to the observed state
of systemic immune dysfunction (e.g., inability to generate
effective immunity following vaccination with cancer vaccines)
emulating other clinical conditions characterized by Th2 driven
systemic chronic inflammation.
[0050] PBMC isolated from patients with benign nevi, atypical
(including dysplastic) nevi, as well as patients with in situ,
stage I, II, III or IV melanoma were analyzed by flow cytometry to
determine the frequencies of T, NK, and dendritic (DC) cell
subsets. There were not significant differences in frequencies of
T-cell among stages of melanoma as determined by numbers of CD3,
CD4 or CD8 positive T-cells (Table 2). Similarly, no significant
differences were found in activated T-cells (CD3/CD69), total NK
cells (CD16/56.sup.+, CD3.sup.-), or most DC subset parameters. As
patients with stage IV melanoma appeared to differ significantly
from all others with regard to plasma cytokine profiles, the cell
subset analysis of patients with stage IV melanoma were compared
relative to all others. The analysis revealed no significant
differences among most parameters (Table 3) with the following
exceptions: (a) the frequency of naive T-cells (CD3/CD62L.sup.+) as
well as activated DC (CD11c/CD83.sup.+) were significantly less in
patients with stage IV melanoma; and (b) the frequency of tetramer
positive CTL for gp100 and tyrosinase (but not MART-1 or CMV and
EBV) were increased in patients with stage IV melanoma. Due to lack
of available biospecimens, T.sub.h1 and T.sub.h2 enumeration could
not be performed. These data suggested that there appeared to be
some level of "immune activation" in patients with metastatic
melanoma that was different from all other cohorts, and this was
consistent with a state of T.sub.h2 mediated "chronic
inflammation."
TABLE-US-00002 TABLE 2 Square root averages of cell subsets with
the 95% confidence interval (parenthesis). The p-value represents
the comparison across all stages of disease. Benign Atypical
In-Situ Stage I Stage II Stage III Stage IV Variable (N = 22) (N =
21) (N = 27) (N = 27) (N = 12) (N = 13) (N = 32) p-value Sqrt CD3
4.78 5.46 5.74 5.29 5.50 5.63 4.94 0.20 (4.20, 5.37) (4.88, 6.04)
(5.23, 6.25) (4.78, 5.80) (4.79, 6.20) (4.94, 6.32) (3.92, 5.96)
Sqrt CD3/4 3.97 4.54 4.57 4.40 4.51 4.64 4.00 0.59 (3.41, 4.53)
(3.98, 5.09) (4.08, 5.06) (3.91, 4.89) (3.84, 5.19) (3.98, 5.31)
(3.02, 4.97) Sqrt CD3/8 2.43 2.82 3.17 2.64 2.60 2.93 2.56 0.20
(1.96, 2.89) (2.36, 3.28) (2.77, 3.57) (2.23, 3.04) (2.04, 3.16)
(2.39, 3.48) (1.76, 3.37) Sqrt 2.92 2.77 2.91 2.28 2.67 3.15 1.13
0.17 CD3/62L (2.23, 3.60) (2.09, 3.45) (2.31, 3.50) (1.69, 2.88)
(1.84, 3.49) (2.35, 3.96) (0.00, 2.32) Sqrt CD3/69 0.40 0.54 0.56
0.50 0.50 0.48 0.49 0.95 (0.18, 0.61) (0.32, 0.75) (0.37, 0.75)
(0.31, 0.69) (0.24, 0.76) (0.23, 0.74) (0.12, 0.87) Sqrt 3.03 3.54
3.44 3.30 3.18 3.43 3.48 0.55 CD3/16 + 56 (2.62, 3.44) (3.13, 3.95)
(3.08, 3.80) (2.94, 3.66) (2.68, 3.68) (2.95, 3.92) (2.77, 4.20)
Sqrt 11c+/14- 1.88 2.15 1.89 1.89 2.00 1.98 1.64 0.66 (1.59, 2.17)
(1.86, 2.44) (1.64, 2.14) (1.64, 2.15) (1.65, 2.35) (1.64, 2.32)
(1.13, 2.14) Sqrt 11c+/14+ 2.74 3.07 2.60 3.11 2.86 3.50 2.80 0.33
(2.18, 3.3) (2.52, 3.63) (2.11, 3.09) (2.62, 3.60) (2.18, 3.54)
(2.84, 4.16) (1.82, 3.77) Sqrt 11c+/DR 0.80 0.91 0.74 0.87 0.74
0.86 0.78 0.48 (0.65, 0.95) (0.77, 1.06) (0.61, 0.87) (0.74, 1.00)
(0.56, 0.92) (0.69, 1.03) (0.53, 1.03) Sqrt 11c+/DR+ 1.06 1.21 1.06
1.05 1.11 1.01 1.33 0.45 (0.88, 1.23) (1.04, 1.39) (0.90, 1.21)
(0.90, 1.20) (0.90, 1.32) (0.81, 1.22) (1.03, 1.64) Sqrt 11c/80
0.22 0.26 0.24 0.24 0.21 0.25 0.18 0.46 (0.17, 0.26) (0.21, 0.30)
(0.20, 0.28) (0.20, 0.28) (0.15, 0.26) (0.20, 0.31) (0.10, 0.25)
Sqrt 11c/83 0.21 0.27 0.24 0.24 0.18 0.19 0.1 0.18 (0.15, 0.28)
(0.21, 0.33) (0.19, 0.30) (0.18, 0.29) (0.11, 0.26) (0.12, 0.26)
(0.00, 0.21) Sqrt 11c/86 0.70 0.82 0.71 0.76 0.70 0.75 0.74 0.84
(0.56, 0.83) (0.68, 0.95) (0.59, 0.82) (0.64, 0.87) (0.53, 0.86)
(0.59, 0.91) (0.50, 0.97) Sqrt DR/40 0.54 0.74 0.55 0.62 0.60 0.61
0.82 0.36 (0.37, 0.70) (0.58, 0.91) (0.40, 0.69) (0.47, 0.76)
(0.40, 0.80) (0.42, 0.80) (0.54, 1.11)
TABLE-US-00003 TABLE 3 p-value Cytokine (stage IV vs all other)
IL-4 1.73 .times. 10.sup.-12 RANTES (CCL5) 6.17 .times. 10.sup.-06
IL-10 5.29 .times. 10.sup.-05 Eotaxin (CCL11) 8.31 .times.
10.sup.-05 IP-10 (CXCL10) 0.0007 IL-13 0.002 IL-12p70 0.005 IL-7,
IL-9 0.009 VEGF, MIB-1b 0.02 (CCL4) GM-CSF 0.03 IL-5 0.05 IL-15,
TNFa, MIP-1a, >0.05 FGF, IL-2, G-CSF, IL- 8, IL-6, IFN-g, MCP-1,
IL-17, PDGF, IL-1ra, IL-1b
[0051] The emerging data seemed to suggest that patients with stage
IV melanoma, unlike all other patients with earlier stages of
melanoma (or healthy controls), existed in a state of systemic
T.sub.h2 dominance with some evidence of cellular immune activation
in peripheral blood (increased frequencies of tumor specific CTL
and decreased frequencies of naive T cells). This immune
homeostasis profile resembled a state of T.sub.h2 dominant "chronic
inflammation," similar to chronic viral infection (Sester et al.,
Am. J. Transplant, 5(6):1483-89 (2005)). A reflection of the
chronic inflammatory state of chronic viral infection as well as
metastatic melanoma is an increase in peripheral blood PD-1.sup.+
(exhausted) T-cells (Wong et al., Int. Immunol., 19:1223-34
(2007)). The same was found to be true in the patient cohort of
stage IV melanoma patients compared to healthy controls (FIG. 5a).
This was further supported by functional assessment of antigen
specific CTL, revealing a significant reduction in the frequency of
functional recall antigen (CMV.sub.495-503) specific CTL in
patients with stage IV melanoma versus healthy volunteers (FIG.
5b). Less than 5% of tumor antigen specific, PBMC derived, tetramer
positive CTL (MART-1) were capable of intracellular IFN-.gamma.
synthesis suggesting immune tolerance.
Example 4
Role of Malignant Melanoma Cells in Tumor-Associated Th.sub.2/IL-4
Mediated Systemic Chronic Inflammation
[0052] The plasma cytokine profiling data comparing patients across
all stages of melanoma suggested that the greatest differences in
the measured parameters occurred in the setting of metastatic
melanoma (stage 4 disease). Therefore, it was hypothesized, that
the presence of visible metastatic disease was in some way
responsible for the detected Th.sub.2 cytokine dominance in these
patients and was likely the result of molecules produced by the
tumor and/or its interaction with surrounding immune cells. To that
end, the mRNA expression profile of 24 biopsy specimens of human
metastatic melanoma was analyzed looking for up-regulation of
expression of known regulatory molecules of immunity (cytokines and
chemokines). The mRNA was extracted from frozen sections in areas
that by H&E staining appeared to contain pure tumor tissue
(devoid of necrotic tissue, stroma or lymphocytic infiltrates). The
RNA was analyzed using an Affymetrix U133 plus 2.0 array. In this
experiment, concurrent blood samples were not available from the
patients for whom tumor tissues existed. The expression of 23
cytokines (45 probes): IL1a and b, IL-1ra, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IFN-.gamma.,
CCL5, CCL11, CSF-2, MCP-1, TNF-.alpha., VEGF was analyzed. The
objective of the experiment was to determine whether or not the
malignancy itself was the source of T.sub.h2 cytokines that were
detected in plasma. The data revealed that many of the probed
cytokines, chemokines, and growth factors are up-regulated in tumor
tissue (FIG. 6a). However, there were no differences in expression
of T.sub.h1 vs. T.sub.h2 cytokines (IFN-.gamma. vs. IL-4, IL-5,
IL-10, and IL-13, FIG. 6b) in tumor tissues suggesting that the
observed T.sub.h2 cytokine predominance in plasma was not derived
from the tumor. However, of the tested cytokines, the most
highly/frequently up-regulated transcript in the tumor samples was
VEGF (FIG. 6b). Plasma VEGF levels were significantly higher in
metastatic melanoma patients relative to healthy donors, (FIG. 6c),
consistent with published reports (Tas et al., Melanoma Res.,
16:405-11 (2006)). Considering the described immune modulatory
(down-regulatory) properties of VEGF (Gabrilovich et al., Nat.
Med., 2:1096-103 (1996)), it was postulated that tumor derived VEGF
could be responsible for the T.sub.h2 polarization in patients with
stage IV melanoma (away from the normal state of T.sub.h1
dominance).
[0053] VEGF has been associated with DC polarization towards
DC.sub.2 leading to Th.sub.2 immune responses (e.g., asthma).
Likewise, Th.sub.2 cytokines (e.g., IL-4, IL-5, and IL-13) have
been associated with increased production of VEGF by a range of
different cell types including smooth muscle cells. Preliminary
data demonstrated that the addition of recombinant human VEGFA to a
2-day culture of normal blood-donor derived PBMC appeared to shift
Th polarity away from Th.sub.1 and towards Th.sub.2 in a dose
dependent fashion (FIG. 7). The addition of VEGF to the cell
culture favored a reduction of Th cells capable of IFN.gamma.
synthesis and TIM-3 expression (Th.sub.1) and increased the
frequency of Th cells capable of IL-4 synthesis and CD294 surface
expression (Th.sub.2). The effect on intracellular IL13 production
was less pronounced. Thus, it appeared that VEGFA had a direct
impact on human PBMC Th.sub.1/Th.sub.2 polarization in vitro.
[0054] In addition, healthy donor PBMC were stimulated with
CD3.sup.+/CD28.sup.+ expander micro-beads for 3 days with
increasing concentrations (1 pg/mL-16 pg/mL) of recombinant VEGF
and assessed intracellular cytokine production of IL-4 (T.sub.h2
cytokine) and IFN-.gamma. (T.sub.h1 cytokine) in CD4.sup.+ T-cells
at the end of in vitro culture (FIG. 8a). The data demonstrated
that increasing concentrations of VEGF resulted in a dose-dependent
reversal of the relative ratio of T.sub.h1 to T.sub.h2 cells in
favor of T.sub.h2. Increased concentrations of VEGF were associated
with a decrease in the number of T.sub.h1 cells
(CD4.sup.+/IFN.gamma..sup.+) with an associated reciprocal increase
in T.sub.h2 cells (CD4.sup.+/IL-4.sup.+). The polarizing effects of
VEGF were lost if the assay was performed with purified CD4 cells
only (FIG. 8b) suggesting that the observed T.sub.h polarization
effect of VEGF is indirect, likely mediated by other PBMC. The
addition of 10 .mu.g/mL of IL-12 to the culture containing 16 pg/mL
of VEGF prevented the shift in T-helper polarity from T.sub.h1 to
T.sub.h2; addition of anti-human IL-12 antibody to the stimulated
PBMC mimicked the effect of VEGF (FIG. 8a). These data suggest a
possible role for monocyte/macrophages in the PBMC preparation as
the mediators of the VEGF induced T.sub.h polarization.
[0055] To gain further insight into the potential role of tumor
produced VEGF on systemic immune homeostasis in vivo in humans with
metastatic melanoma, frozen peripheral blood specimens were
randomly selected from a recently completed clinical trial (N047a)
where patients with metastatic melanoma were treated with
chemotherapy (paclitaxel+carboplatin) and a specific anti-VEGFA
antibody (bevacizumab). They were analyzed for changes in plasma
VEGFA levels and Th.sub.1/Th.sub.2 polarity as well as frequency of
tumor specific CTL (tetramer assay). If VEGF was responsible for
Th.sub.2 polarization, its suppression using chemotherapy/anti-VEGF
therapy would revert the Th.sub.1/Th.sub.2 balance back to normal
(normal ratio is 1:1) and perhaps result in emergence of naturally
processed anti-tumor specific CTL. Also, this particular clinical
trial was chosen because of: (1) available, appropriately stored
biospecimens; and (2) this single arm phase II clinical trial
yielded favorable clinical outcomes for patients with metastatic
melanoma, suggesting possible clinical relevance of this
therapeutic strategy. As illustrated in this example from a single
patient (FIG. 9), coincident with the decrease of plasma VEGFA
concentrations (as a result of therapy, FIG. 9, top left), there
was an increase in Th.sub.1:Th.sub.2 ratio away from Th.sub.2 and
towards Th.sub.1 (FIG. 9, top right) at the same time as the
increase in frequency of tumor specific CTL in peripheral blood
(reactive against melanoma differentiation antigens: MART-1, gp100
or tyrosinase, FIG. 9, bottom right). Similar observations were
made in two other patient samples. In all three cases, the
increases in tumor specific CTL tetramer frequency coincided with a
reduction in plasma VEGFA levels to normal levels and a shift in Th
polarity away from Th.sub.2 and towards Th.sub.1. Of note, such an
increase in CTL frequency was not observed when analyzing the same
immunological parameters from patients with metastatic melanoma
treated with chemotherapy alone (nab-paclitaxel+carboplatin)
without the anti-VEGF antibody (three patients analyzed from
protocol N057e). Of note, the presented data were consistent with a
published anecdotal observation from a prior clinical trials
demonstrating increase in tumor specific CTL tetramer frequency
coincident with reduction of plasma VEGFC levels in patients with
metastatic melanoma treated with a thrombospondin-1 analog, ABT-510
(Markovic et al., Am. J. Clin. Oncol., 30(3):303-9 (2007)).
Therefore, viewed in the context of the current hypothesis, in
addition to the originally postulated anti-tumor and
anti-angiogenic goals of paclitaxel/carboplatin/bevacizumab
therapy, the effect of chemotherapy (paclitaxel and carboplatin)
may also have depleted (lymphodepleted) the pre-existing state of
"chronic inflammation"; and the VEGF inhibitor (bevacizumab) may
have allowed reconstitution of tumor-specific immunity in a
Th.sub.1 (not Th.sub.2) dominant systemic environment. Thus, it is
possible that the additional, unanticipated, immunomodulatory
effect of this therapy may have added to the observed therapeutic
clinical result. Repeated treatments with lymphodepleting
chemotherapy may have also inadvertently lead to ultimate depletion
of the beneficial anti-tumor immune response as well, allowing
tumor progression. Perhaps, this explains in part the clinical
outcomes of protocol N047a demonstrating a dramatic improvement in
median progression free survival (from 6 weeks to 6 months) with a
not nearly as significant an improvement in overall survival (from
8 months to 12 months).
[0056] In summary, preliminary data suggests that patients with
advanced (metastatic) melanoma exhibit systemic features of
Th.sub.2-mediated chronic inflammation that appears at least in
part mediated by tumor-secreted VEGF (FIG. 10). This state of
chronic inflammation effectively dampens spontaneously developed
anti-tumor CTL immune responses and significantly reduces the
efficacy of de novo immunization efforts with cancer
vaccines/immune modulation in this patient population. Evidence
exists that the observed Th.sub.2/VEGF pathway could be self
sustaining and exists in both physiologic (pregnancy) as well as
other pathologic states (e.g., asthma) in humans. Disruption of the
Th.sub.2 driven systemic chronic inflammation in patients with
advanced melanoma (and possibly other malignancies) and
reconstitution of effective immunity (Th.sub.1 dominance) could
potentially translate into effective therapy with clinically
meaningful results. Therefore, an improved understanding of this
mechanism of tumor mediated immune dysfunction/tumor progression as
a function of Th.sub.2-mediated chronic inflammation could yield
therapeutic targets for cancer therapy with agents already in
clinical development for Th.sub.2 mediated disorders (e.g.,
anti-IL-4 antibody).
Example 5
Confirm the Mechanism of Tumor-Induced (VEGF Mediated), Th.sub.2
Driven Chronic Inflammation Across Stages of Melanoma Focusing on
the Functional State of cellular Anti-Tumor Immunity
[0057] The identified cytokine profiles of plasma suggests the
existence of a Th.sub.2 dominant systemic immune environment in the
blood of patients with metastatic melanoma. The analysis of the
cellular/functional counterpart of the immune response in these
patients across all stages of melanoma remains unknown. To confirm
the hypothesis of Th.sub.2 dominant systemic immunity, the
existence of reciprocal, Th.sub.2 polarized, changes in the
cellular immune response in these patients across stages of
melanoma that will correlate with the described changes in plasma
cytokine and VEGF concentrations is determined To address this, one
can (a) enumerate Th.sub.1, Th.sub.2 and T.sub.reg cells across
stages of melanoma; (b); analyze the numbers and
functional/differentiation state of circulating DC
(DC.sub.1/DC.sub.2) across stages of melanoma (DC.sub.2 driven
Th.sub.2 polarization) and (c) analyze the functional status
(active vs. tolerant) of both tumor-specific (e.g. MART-1, gp100,
tyrosinase) and recall antigen specific (EBV, CMV) CTL in the
HLA-A2.sup.+ subset of patients, across stages of melanoma. These
data can be combined with the existing data on plasma cytokine
levels and correlated looking for patterns of Th.sub.1 vs. Th.sub.2
cytokine/cellular profiles across patients and in relation to
plasma VEGF levels.
[0058] Enumeration of Th.sub.1, Th.sub.2 and T.sub.reg cells across
stages of melanoma. In order to assess whether the Th.sub.2
cytokine predominance in plasma of patients with metastatic
melanoma is truly a reflection of a systemic immune polarization
towards Th.sub.2 driven chronic inflammation, one can ascertain the
expected corresponding changes in the frequencies of circulating Th
cell subsets across disease stage using frozen PMBC samples
corresponding to plasma cytokine samples described above. The
available frozen PBMC can be thawed and analyzed for the relative
numbers of Th.sub.1, Th.sub.2 and T.sub.reg. CD4 cells can be
isolated from thawed PBMC specimens using paramagnetic beads coated
with anti-CD4 (Dynal, Oslo, Norway), and they can be incubated with
mouse-anti-human CD3/CD28 coated "stimulator" micro-beads (R and D
Systems Minneapolis, Minn.) for 6 hours in the presence of 1 ug/ml
brefeldin A, (Sigma Aldrich, St Louis, Mo.). After stimulation, the
cells can be fixed, permeablized, and stained with APC conjugated
mouse anti-human CD4 (Becton Dickinson, San Jose, Calif.) and FITC
conjugated mouse anti-human IFN.gamma. and anti-IL-13 (R and D
Systems Minneapolis, Minn.). The stained samples can be analyzed by
flow cytometry (FACScan and Cellquest software (Becton-Dickinson,
San Jose, Calif.). The results can indicate the percentage of
IFN.gamma. positive/IL-13 (or IL-4) negative (Th.sub.1) and
IFN.gamma. negative/IL-13 (or IL-4) positive (Th.sub.2) helper T
cells.
[0059] GLP validation of anti-CD294 and anti-TIM-3 cell-surface
immunostaining can be completed for the distinction of Th.sub.2 vs.
Th.sub.1 cells in ex vivo (unstimulated) frozen PBMC, respectively.
Preliminary data suggested that CD4/CD294 positive Th.sub.2 cells
exclusively produce IL-4 and IL-13 and not IFN.gamma. upon CD3/CD28
stimulation. Conversely, CD4/TIM-3 positive Th.sub.1 cells
exclusively produced IFN.gamma. and not IL-4 and IL-13 following
the same in vitro stimulation (FIG. 11). Once validated, the assay
can be standardized and applied to the battery of tests described
herein.
[0060] Enumeration of T.sub.reg can be performed using
intracellular staining for FoxP3 of CD4/25 positive lymphocytes
Immunophenotyping can be conducted using commercially available
monoclonal antibodies (Biolegend; San Diego, Calif.). Samples can
be analyzed by flow-cytometry by FACScan.RTM. and data processed
using Cellquest.RTM. software (Becton-Dickinson, Franklin Lakes,
N.J.).
[0061] Assessment of peripheral blood DC subset (DC.sub.1/DC.sub.2)
and activation/differentiation status. In order to ascertain
whether or not the state of Th.sub.2 driven systemic chronic
inflammation is primarily a function of systemic DC polarization
towards DC.sub.2 (from DC.sub.1) leading to HTL polarization from
Th.sub.1 to Th.sub.2 in patients with advanced melanoma, one can
quantify the relative numbers and functional states of DC.sub.1 and
DC.sub.2 in patients with melanoma across stages of disease. These
data can be analyzed in conjunction with corresponding plasma
cytokine/VEGF and Th subset data (above). Therefore, the available,
frozen PBMC corresponding to the plasma cytokine samples described
above can be thawed and analyzed for the relative numbers of DC
subsets defined by expression of CD11c.sup.+/CD123-(DC.sub.1), and
CD11c-/CD123.sup.+ (DC.sub.2). Each subset can be analyzed for
surface expression of co-stimulatory molecules (CD80, 83, 86)
Immunophenotyping can be conducted using commercially available
monoclonal antibodies (BD Pharmingen; San Jose, Calif.). Samples
can be analyzed by flow-cytometry by FACScan.RTM. and data
processed using Cellquest.RTM. software (Becton-Dickinson, Franklin
Lakes, N.J.).
[0062] Analysis of the functional status (active/tolerant) of tumor
specific (MART-1, gp100, tyrosinase) and recall antigen specific
(EBV, CMV) CTL in the HLA-A2.sup.+ subset of patients across stages
of melanoma. Available frozen PBMC for the same cell repository as
described herein can be analyzed for the frequency and functional
capacity (exhausted, tolerant vs. non-tolerant subsets) of tumor
specific and recall antigen specific CTL. If the hypothesis of
tumor mediated, Th.sub.2-driven chronic inflammation is correct,
the predominant phenotype of the tumor (and recall) antigen
specific CTL can be one of tolerance (inability to synthesize
intracellular IFN.gamma. upon congnant stimulation with
tumor-specific peptides) and exhaustion (expression of PD-1). The
latter has already been suggested to be true (Rosenberg et al., J.
Immunol., 175(9):6169-76 (2005)) Immunophenotyping of PBMC can be
performed using tetramers for melanoma differentiation antigen
specific, HLA-A2 congnant peptides (MART-1.sub.27-35,
gp100.sub.209-217 and tyrosinase.sub.368-376) as well as A2 cognant
peptides of EBV and CMV (Beckman Coulter, San Diego, Calif.). For
tetramer analysis, thawed PBMC can be stained with FITC conjugated
anti-CD8, PC5 conjugated anti-human CD4, CD14 and CD19, and
conjugated HLA-A2 tetramers containing peptides from CMV, EBV
(controls), MART-1.sub.27-35, gp100.sub.209-217 and
tyrosinase.sub.368-376. Samples can be analyzed by flow-cytometry
and data processed using Cellquest.RTM. software (Becton-Dickinson,
Franklin Lakes, N.J.). Gates can be set on lymphocytes that were
CD4, CD14, and CD19 (PC5) negative and CD8 (APC) positive. Ongoing
quality assurance (QA) data suggests an inter-assay variability
with a coefficient of variation (CV) below 5%. Standard control
samples can be run alongside all experiments. If the standard
control samples generate results beyond .+-.2SD of the mean, all
assay results can be rejected and the experiment repeated.
[0063] Functional analysis of tetramer positive CTL can be
performed in patient samples demonstrating tetramer frequencies of
at least 0.1% to melanoma differentiation or recall antigens. One
can proceed to ascertain the ability of tetramer positive CTL to
synthesize interferon-y (IFN.gamma.) upon stimulation with cognate
peptide presented in the context of HLA-A2 and anti-CD28
co-stimulation using artificial antigen presenting cell (aAPC)
stimulation. The details of this method are described elsewhere
(Markovic et al., Clin. Exp. Immunol., 145(3):438-47 (2006)). In
brief, previously frozen patient PBMC can be thawed in batches,
labeled with PE conjugated tetramers (Beckman Coulter, San Diego,
Calif.), and stimulated for 6 hours, in the presence of 1 mg/mL
brefeldin A, (Sigma Aldrich, St Louis, Mo.) with pararamagnetic
beads (Dynal, Oslo, Norway) coated with peptide loaded HLA-A2
(Beckman Coulter San Diego, Calif.) and mouse anti-human CD28 (R
and D Systems Minneapolis, Minn.). After stimulation, the cells can
be fixed, permeablized, and stained with APC conjugated mouse
anti-human CD8 (Becton Dickinson, San Jose, Calif.) and FITC
conjugated mouse anti-human IFN-gamma (R and D Systems Minneapolis,
Minn.). The stained samples can be analyzed by flow cytometry
(FACScan and Cellquest software (Becton-Dickinson, San Jose,
Calif.). The results can indicate the percentage of tetramer
positive CTL able vs. unable to synthesize intracellular
IFN.gamma.. Ongoing QA data suggests an inter-assay variability
with a CV of below 9%. Standard control samples can be run
alongside all experiments. If the standard control samples generate
results beyond .+-.2SD of the mean, all assay results can be
rejected, and the experiment repeated.
[0064] Laboratory data summary and statistical analysis. All blood
samples were registered, collected, processed and annotated. Sample
break down per diagnostic category is described in Table 4.
TABLE-US-00004 TABLE 4 Summary of available biospecimens (frozen
PBMC and plasma) Clinical diagnostic category Benign Atypical In
Situ Stage I Stage II Stage III Stage IV nevi nevi melanoma
melanoma melanoma melanoma melanoma Available frozen 26 16 35 36 12
16 30 PBMC with already available corresponding plasma cytokine
data Additional available frozen PBMC and frozen plasma as of May
1, 2008 Therapeutic 0 0 0 0 0 44 198 clinical trials Melanoma blood
10 0 22 43 38 209 421 and tissue bank (MC997 g)
Example 6
Assess the Impact of Systemic Therapeutics on the Hypothesized
Tumor-Driven Th.sub.2 Mediated State of Systemic Chronic
Inflammation With Special Emphasis on the Role of VEGF
[0065] The effects of melanoma-specific therapeutic interventions
on the VEGF/Th.sub.2 state of tumor-induced chronic inflammation
can be examined using peripheral blood biospecimens from patients
with stage IV malignant melanoma enrolled on ongoing or completed
clinical trials for changes in a range of immune parameters with a
primary focus on Th.sub.1/Th.sub.2 balance and functional tumor
specific CTL immunity. The trials are listed in the Table 5. The
patients enrolled into these trials had a blood specimen collected
before initiation of therapy and after one cycle of treatment. This
can provide data on the immediate impact of therapy on our
VEGF/Th2/immune parameters of interest.
TABLE-US-00005 TABLE 5 Summary of available biospecimens from
therapeutic clinical trials for stage IV melanoma Study Number of
patients enrolled number Treatment regimen (accrual as of May 1,
2008) N0377 RAD001.sup.2 53 (completed) N047a Paclitaxel +
Carboplatin + 53 (completed) Bevacizumab.sup.1 N057e Abraxane +
Carboplatin 74 (completed) MC057f Temozolomide 86 (12) Paclitaxel +
Carboplatin 86 (0) N0675 RAD001.sup.2 + Temozolomide 43 (6) N0775
Abraxane + Carboplatin + 43 (0) Bevacizumab.sup.1 Temozolomide +
Bevacizumab 43 (0) .sup.1Humanized anti-VEGFA antibody;
.sup.2rapamycin analog, inhibitor of mTOR (down-regulation of VEGF
synthesis)
[0066] For each individual patient, the biospecimens collected
before initiation of therapy and after one cycle of treatment can
be used. This can provide data on the immediate impact of therapy
on VEGF/Th2/immune parameters of interest. Additional testing for
later time-points can be pursued only if justified by the initial
analysis suggesting beneficial changes in the studied immune
parameters. This can allow one to gain insight into the effects of
a broad range of clinical interventions on immune homeostasis using
an available (but limited) resource of biospecimens and only pursue
further analysis if justified by the generated data.
[0067] Laboratory analyses of the stored PBMC can include the same
assays described above and can also include: (a) PBMC
immunophenotyping for immune cell subset analysis; and (b) plasma
cytokine profiling.
[0068] PBMC immunophenotyping for immune cell subset analysis. Pre
and post-treatment frozen PBMC biospecimens can be analyzed for the
"global" impact of therapy on immune cell subsets. One can analyze
the relative numbers of T, B, NK cells, monocytes and DC, and their
activation status using commercially available monoclonal
antibodies directed at the following antigens: CD3, CD4, CD8,
CD11c, CD14, C16, CD19, CD20, CD25, CD45RA/RO, CD56, CD69, CD63L,
CD80, CD83, CD86, CD123, DR, foxP3 (BD Pharmingen; San Jose,
Calif.) Immunophenotyping can be performed using manufacturer's
instruction in batch samples of the same patients analyzed on the
same day. The stained samples can be analyzed by flow cytometry
(FACScan and Cellquest software (Becton-Dickinson, San Jose,
Calif.). PBMC isolated from patients prior to B7-DC XAb and 15 days
after antibody treatment can be assayed. Changes in the numbers of
cells bearing the lymphocyte markers in pared comparisons for
patients prior to B7-DC XAb treatment can be used to ascertain
antibody treatment effects.
[0069] Plasma cytokine profiling. In order to complement the PBMC
derived cellular immunity analyses, one can add plasma cytokine
measurements in the same samples (paired PBMC and plasma testing).
To that end, one can profile the serum cytokine changes as a result
of specific therapy for all available specimens before and after
treatment. The BioRad human 27-plex cytokine panel can be used (Cat
# 171-A11127, Bio-Rad, San Diego Calif.) for the measurements of
plasma concentrations of IL-1.beta., IL-1r.alpha., IL-2, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15,
IL-17, basic FGF, Eotaxin, G-CSF, GM-CSF, IFN-.gamma., IP-10,
MCP-1, MIP-1.alpha., MIP-1.beta., PDGF, RANTES, TNF-.alpha., and
VEGF. The assay can be performed as per the manufacturer's
directions. Briefly, 100 .mu.L of Bio-Plex assay buffer can be
added to each well of a MultiScreen MABVN 1.2 .mu.m microfiltration
plate followed by the addition of 50 .mu.L of the multiplex bead
preparation. Following washing of the beads with the addition of
100 .mu.L of wash buffer, 50 .mu.L the samples or the standards can
be added to each well and incubated with shaking for 30 minutes at
room temperature. The plasma (1:3 dilution) and standards can be
diluted using the Bio-Plex human serum diluent kit and plated in
duplicate. Standard curves can be generated with a mixture of 27
cytokine standards and eight serial dilutions ranging from 0-32,000
pg/mL. The plate can then be washed 3 times followed by incubation
of each well in 25 .mu.L of pre-mixed detection antibodies for 30
minutes with shaking. The plate can further be washed and 50 .mu.L
of streptavidin solution were added to each well and incubated for
10 minutes at room temperature with shaking. The beads can be given
a final washing and resuspension in 125 .mu.L of Bio-Plex assay
buffer. Cytokine levels in the sera can be quantified by analyzing
100 .mu.L of each well on a Bio-Plex using Bio-Plex Manager
software version 4.0. Normal values for plasma cytokine
concentrations were generated by analyzing 30 plasma samples from
healthy donors (blood donors at the Mayo Clinic Dept. of
Transfusion Medicine). A set of five normal plasma samples
(standards) can be run along side all batches of plasma analysis.
If the cytokine concentrations of the "standard" samples differ by
more than 20%, results can be rejected, and the plasma samples
re-analyzed.
Example 7
Prospectively Follow Changes of Immune Homeostasis (Evolution of
Systemic Chronic Inflammation) in High-Risk Patients After Complete
Resection of Advanced Melanoma Until Subsequent Tumor Relapse
[0070] To better understand the clinical relevance of these
differences in immune homeostasis and study the kinetics of their
evolution in humans as they develop clinically detectable
metastatic cancer (melanoma), one can perform a prospective
clinical trial in which patients with surgically resected
metastatic melanoma (and in a state of chronic inflammation)
undergo complete resection of their tumors as part of their
clinical care and are subsequently followed at regular
time-intervals until tumor relapse. It is hypothesize that
following surgical resection, the state of systemic "chronic
inflammation" will resolve. These patients can then be followed at
regular intervals (every 2 months), and their blood analyzed for
emergence of Th.sub.2-mediated chronic inflammation, until clinical
tumor relapse/recurrence of metastatic melanoma (approximately 50%
of patients will relapse within 18 months of surgery). This study
will depict the time-sequence and thresholds of systemic changes in
immune homeostasis (chronic inflammation) as they evolve towards
the development of relapsed metastatic melanoma. Sufficient blood
specimens (100 mL every 2 months) can be collected to allow
complete analysis as well as provide some additional material for
further testing (if necessary). The clinical trial can be powered
based on the inter-patient variability of the most prominent immune
abnormality in patients with metastatic melanoma (e.g. variability
of plasma IL-4 concentrations) determined herein. If successful,
these data can clinically validate the changes in immune
homeostasis as they impact the natural history of metastatic
melanoma and describe potential targets for future therapy.
Additionally, as all patients on this study will undergo surgical
resection of metastatic melanoma as well as undergo concurrent
comprehensive immunological testing, their surgical tissues can be
processed and preserved for analysis addressing the influence of
the tumor on the observed immunological profile (multiple frozen
blocks for future immunohistochemical study and mRNA extraction).
All tumor tissue can undergo genomic mRNA expression profiling as
well as IHC analysis for infiltrating immune cell subsets (funded
under separate, existing instruments). These data can correlate the
relationship of immunity in the tumor microenvironment with that of
systemic immunity in metastatic cancer.
[0071] Study design. The clinical trail can be conducted in the
context of the clinical trials program of the Melanoma Study Group
of the Mayo Clinic Cancer Center. All patients with the diagnosis
of metastatic melanoma that are planned to undergo complete
surgical resection of their malignancy can be offered participation
in this study. The objective of the study can be to profile the
changes in immune homeostasis from pre-surgery, post-surgery and
all through the time of clinically detectable tumor relapse. It is
hypothesized that the state of VEGF/Th.sub.2 driven chronic
inflammation can be established pre-surgery, resolved soon after
surgery and slowly re-develop in the months prior to clinical tumor
relapse.
[0072] For the purposes of this study, patients can be clinically
followed in accordance to clinical practice (every 2 months).
Patients can be asked to donate 100 mL of blood at each follow-up
time point. The blood can be collected, processed, and stored in
accordance to existing procedures for immunological testing Immune
homeostasis analysis can be conducted in batches to limit
inter-assay variability. Specific focus/priority can be given to
parameters reflecting Th.sub.1/Th.sub.2 balance, frequency of
functional/tolerant tumor (or recall) antigen specific CTL as well
as plasma cytokine and VEGF levels. At the time of clinical
relapse, patients can be re-tested, and the tumor biopsied for
histologic confirmation. Available tumor tissues can be analyzed
for expression of tumor associated antigens (immunohistochemistry
for MART-1, gp100 and tyrosinase) as well as tumor infiltrating
lymphocytes and compared to the original surgical specimen for each
individual patient. In the rare events where patients can undergo
another curative surgical resection at the time of relapse, they
may continue on study following the outlined follow-up/testing
schedule until such a time when a tumor relapse is no longer
surgically resectable. See, e.g., Table 6.
[0073] Eligibility Criteria.
[0074] Required Characteristics/Inclusion Criteria:
1. HLA-A2.sup.+ adult patients (age 18 years) with metastatic
malignant melanoma who are planned to undergo complete resection
for metastatic disease as part of their regular medical care. 2.
The following laboratory values obtained 14 days prior to
registration: hemoglobin .gtoreq.9.0 g/dL; platelet count
.gtoreq.75,000/.mu.L; and AST.ltoreq.3.times.ULN. 3. Ability to
provide informed consent. 4. Willingness to return to clinic for
follow-up. 5. ECOG performance status 0, 1 or 2. 6. Willingness to
participate in the mandatory translational research component of
the study.
[0075] Contraindications/Exclusion Criteria:
1. Uncontrolled or current infection. 2. Known standard therapy for
the patient's disease that is potentially curative or proven
capable of extending life expectancy. 3. Any of the following prior
therapies with interval since most recent treatment: (a)
chemotherapy .ltoreq.4 weeks prior to registration; or (b) biologic
therapy weeks prior to registration. 4. Any of the following as
this regimen may be harmful to a developing fetus or nursing child:
(a) pregnant women; (b) nursing women; or (c) women of childbearing
potential or their sexual partners who are unwilling to employ
adequate contraception (condoms, diaphragm, birth control pills,
injections, intrauterine device (IUD), surgical sterilization,
subcutaneous implants, or abstinence, etc). 5. Known immune
deficiency or ongoing immunosuppressive therapy.
TABLE-US-00006 TABLE 6 Test schedule Every 2 months after
.ltoreq.14 days prior <7 days prior to surgery until surgically
At time of Tests and procedures to registration scheduled surgery
unresectable relapse.sup.2 tumor relapse.sup.2 History and exam, X
X X weight, performance status Vital signs X X Disease evaluation X
X X (clinical/imaging) Hematology group X X X WBC, ALC, ANC, Hgb,
platelets Chemistry group X X X AST, LDH, Alk Phos, Creat, K, Na,
LDH Immunology studies X.sup.R X.sup.R X.sup.R HLA typing X.sup.R,
Tumor typing for X.sup.R X.sup.R MART1, gp100 and tyrosinase;
profile of infiltrating lymphocytes Serum pregnancy test.sup.1
X.sup.R .sup.1Only for women of child-bearing age; .sup.2if a
patient has a melanoma relapse that is surgically completely
resectable, they may continue on study with the same
follow-up/testing schedule until relapse is no longer surgically
resectable; .sup.Rresearch funded
Example 8
TGF.beta. Alters Th1/Th2 Ratios
[0076] CD4 T cells (Th1 and Th2 CD4 T cells) derived from human
PBMC were incubated in vitro with varying concentrations of VEGFA
(rhVEGFA; 10 ng/mL, 50 ng/mL, and 200 ng/mL) or TGF.beta.
(rhTGF.beta.; 10 ng/mL, 50 ng/mL, and 200 ng/mL). After six hours
of incubation at 37.degree. C., the ratio of Th1 vs. Th2 (Th1/Th2)
was determined Both VEGFA and TGF.beta. exhibited a similar effect
on Th1/Th2 polarity in human PBMC derived CD4 cells (FIG. 12).
[0077] CD4 T cells (Th1 and Th2 CD4 T cells) derived from human
PBMC were incubated in vitro with VEGFA alone (1 ng/mL, 5 ng/mL, 10
ng/mL, 100 ng/mL, or 1000 ng/mL) or VEGFA (100 ng/mL) plus an
anti-TGF.beta. antibody (1 ng/mL, 10 ng/mL, 100 ng/mL, 1 .mu.g/mL,
5 .mu.g/mL, or 10 .mu.g/mL). The anti-TGF.beta. antibody was
obtained from Genzyme Corp. (Cambridge, Mass.). Untreated cells
(media only) as well as cells exposed to Th1 or Th2 favorable in
vitro conditions were used as controls. After six hours of
incubation at 37.degree. C., the ratio of Th1 vs. Th2 (Th1/Th2) was
determined. The presence of anti-TGF.beta. antibodies reversed the
Th1/Th2 modulation of VEGFA in vitro, suggesting that the observed
VEGF effect in these cells may be TGF.beta. mediated (FIG. 13).
OTHER EMBODIMENTS
[0078] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *