Cancer
Cancer immunotherapy attempts to stimulate the immune
system to reject and destroy tumors. Immuno cell therapy
for cancer was first introduced by Rosenberg and his
colleagues of National Institute of Health USA. In the
late 80s, they published an article in which they reported
a low tumor regression rate (2.6-3.3%) in 1205 patients
with metastatic cancer who underwent different types of
active specific immunotherapy (ASI), and suggested that
Immuno Cell Therapy with specific chemotherapy is the
future of cancer Immunotherapy . In the beginning
Immunotherapy treatments involved administration of
cytokines such as Interleukin with an aim of inducing the
lymphocytes which will carry their activity of destroying
the tumor cells. Thereafter the adverse effects of such
intravenously administered cytokines lead to the
extraction of the lymphocytes from the blood and
culture-expand them in the lab and then to inject the
cells alone enable them destroy the cancer cells .
Though the concept of this treatment started in the US in
80s, fully fledged clinical treatments on a routine basis
have been in practice in Japan since 1990. Randomized
controlled studies in different cancers with significant
increase in survival and disease free period have been
reported and its efficacy is enhanced by 20 - 30% when
cell based immunotherapy is combined with other
conventional treatment methods.
BCG immunotherapy for early stage (non-invasive) bladder
cancer utilizes instillation of attenuated live bacteria
into the bladder, and is effective in preventing
recurrence in up to two thirds of cases. Topical
immunotherapy utilizes an immune enhancement cream
(imiquimod) which is an interferon producer causing the
patients own killer T cells to destroy warts,, actinic
keratoses, basal cell cancer, vaginal intraepithelial
neoplasia., squamous cell cancer, cutaneous lymphoma, and
superficial malignant melanoma. Injection immunotherapy
uses mumps, candida the HPV vaccine, or trichophytin
antigen injections to treat warts (HPV induced tumors).
Lung cancer has been demonstrated to potentially respond
to immunotherapy.
In many parts of Asia, Medicinal mushrooms are thought to
be able to boost the immune system naturally. Cellular and
animal research has shown that Agaricus blazei may
stimulate immune system cells and the production of
interferons and interleukins (reviewed by G. Hetland).
Mushroom isolates like PSK also are used to increase
immune system parameters (reviewed by Kobayashi). Used in
conjunction with chemotherapy, PSK has increased the
survival time of cancer patients in randomized, control
studies, with a variety of cancer types.
Autologous Immune Enhancement Therapy (AIET)
In the multipronged approach to treat CANCER, one very
useful latest weapon would be AIET which is in clinical
practice in Japan since 90s for more than 15 years with
good effectiveness and several clinical trials and
researches are reported from institutions all over the
world including from the US. AIET is a treatment method in
which some immune cells are taken out of a patient's body
which are cultured and processed to be activated or to
acquire additional functions until their resistance to
cancer is strengthened, then the cells are put back in the
body. Researchers have found that the thus activated
immune system might also be able to determine the
difference between healthy cells and cancer cells to
eliminate the cancer cells from the body. In AIET,
specific type of cells mainly the NK cells and T
lymphocytes are isolated from the peripheral blood of the
cancer patients (during remission in patients who undergo
chemotherapy) by proven methods, expanded to 25 - 30 fold
and activated and then reinfused back into the patient’s
body. These cells act against the cancer cells effectively
and recharge the immune system. Upon encountering a tumor
cell, the activated NK cell attaches to the membrane of
the cancer cell and injects toxic granules which dissolve
the target cell. In less than five minutes, the cancer
cell dies and the NK cell moves on to its next target
cancer cell. A single NK cell can destroy up to 27 cancer
cells before its lifespan. This is the mechanism by which
AIET is effective in Cancer therapy.
Dendritic cell based immunotherapy
This utilizes dendritic cells to activate a cytotoxic
response towards an antigen. Dendritic cells, a type of
antigen presenting cell, are harvested from a patient.
These cells are then either pulsed with an antigen or
transfected with a viral vector. The activated dendritic
cells are then placed back into the patient; these cells
then present the antigens to effector lymphocytes (CD4+ T
cells, CD8+ T cells, and in specialized dendritic cells, B
cells also). This initiates a cytotoxic response to occur
against these antigens and anything that may present these
antigens. One use for this therapy is in cancer
immunotherapy. Tumor Antigens are presented to dendritic
cells, which cause the immune system to target these
antigens, which are often expressed on cancerous cells.
The Dendreon product candidate Provenge is one example of
this approach. T cell based adoptive immunotherapy
Adoptive cell therapy (ACT) using autologous
tumor-infiltrating lymphocytes is an effective treatment
for patients with metastatic melanoma; this is based on
adoptive immunity.
Adoptive cell transfer, or "ACT," uses T cell-based
cytotoxic responses to attack cancer. T cells that have a
natural or genetically engineered reactivity to a
patient's cancer are expanded, made more effective, in
vitro using a variety of means and then adoptively
transferred into a cancer patient.
For example, T cells with a naturally occurring reactivity
to a patient’s cancer can be found infiltrated in the
patient's own tumors. The tumor can be harvested, and
these tumor-infiltrating lymphocytes (TIL) can then be
expanded, or made more effective, in vitro using high
concentrations of interluekin-2 (IL-2), anti-CD3 and
allo-reactive feeders. These T cells can then be
transferred back into the patient along with exogenous
administration of IL-2 to further boost their activity.
Thus far, a 51% objective response rate has been observed;
and in some patients, tumors shrank to undetectable size.
The initial studies of adoptive cell transfer using TIL,
however, revealed that persistence of the transferred
cells in vivo was too short. Before reinfusion,
lymphodepletion of the recipient is required to eliminate
regulatory T cells as well as normal endogenous
lymphocytes that compete with the transferred cells for
homeostatic cytokines. Prior lymphodepletion to transfer
of the expanded TIL was made by total body irradiation.
The trend for increasing survival as a function of
increasing lymphodepletion was highly significant
(P=0.007). Transferred cells expanded in vivo and
persisted in the peripheral blood in many patients,
sometimes achieving levels of 75% of all CD8+ T cells at
6-12 months after infusion.
Morgan et al. (2006) demonstrated that the adoptive cell
transfer of lymphocytes transduced with retrovirus
encoding T cell receptors (TCRs) that recognize a cancer
antigen can mediate anti-tumor responses in patients with
metastatic melanomas.
In such T cell genetic engineering, TCRs that have been
identified to have reactivity against tumor-associated
antigens are cloned into a replication-incompetent virus
that is capable of genomic integration. A patient's own
lymphocytes are exposed to these viruses and then expanded
non-specifically or stimulated using the engineered TCR.
The cells are then transferred back into the patient. This
therapy has been demonstrated to result in objective
clinical responses in patients with refractory stage IV
cancer. The Surgery Branch of the National Cancer
Institute (Bethesda, Maryland) is actively investigating
this form of cancer treatment for patients suffering
aggressive melanomas.
Combination of ACT with such genetic engineering of T
cells has opened possibilities for the extension of ACT
immunotherapy to patients with a wide variety of cancer
types and is a promising new approach to cancer treatment.
In June 2008, it was announced that US doctors from the
Clinical Research Division led by Dr. Cassian Yee at Fred
Hutchinson Cancer Research Center in Seattle had
successfully treated a patient with advanced skin cancer
by injecting the patient with immune cells cloned from his
own immune system. The patient was free from tumours
within eight weeks of treatment. Dr. Cassian Yee described
the research findings at The Cancer Research Institute
International 2008 Symposia Series. . Responses, however,
were not seen in other patients in this clinical trial.
Larger trials are now under way.
Monday, April 26, 2010
18 patients treated with vaccines for RENAL CANCER
Renal cell carcinoma treated by vaccines for active
specific immunotherapy: correlation of survival with skin
testing by autologous tumor cells.
McCune CS, O'Donnell RW, Marquis DM, Sahasrabudhe DM.
University of Rochester Cancer Center, University of
Rochester School of Medicine and Dentistry, New York
14642. Abstract
Eighteen patients with metastatic renal cell carcinoma,
who were treated by vaccines for active specific
immunotherapy, also completed skin testing with autologous
tumor cells, both prior to and following vaccine
treatment. All patients have now been followed for more
than 5 years. Ten patients who remained skin-test-negative
following treatment had no clinical responses, and all had
expired by 22 months. Eight patients became
skin-test-positive; three of these had clinical
regressions and three remain alive after more than 69
months. The survival times of the skin-test-positive group
were significantly superior to those of the
skin-test-negative group. The results suggest that skin
testing with autologous tumor cells may accurately
identify those patients who have acquired antigen-specific
cell-mediated antitumor immunity.
http://www.ncbi.nlm.nih.gov/pubmed/2289200
specific immunotherapy: correlation of survival with skin
testing by autologous tumor cells.
McCune CS, O'Donnell RW, Marquis DM, Sahasrabudhe DM.
University of Rochester Cancer Center, University of
Rochester School of Medicine and Dentistry, New York
14642. Abstract
Eighteen patients with metastatic renal cell carcinoma,
who were treated by vaccines for active specific
immunotherapy, also completed skin testing with autologous
tumor cells, both prior to and following vaccine
treatment. All patients have now been followed for more
than 5 years. Ten patients who remained skin-test-negative
following treatment had no clinical responses, and all had
expired by 22 months. Eight patients became
skin-test-positive; three of these had clinical
regressions and three remain alive after more than 69
months. The survival times of the skin-test-positive group
were significantly superior to those of the
skin-test-negative group. The results suggest that skin
testing with autologous tumor cells may accurately
identify those patients who have acquired antigen-specific
cell-mediated antitumor immunity.
http://www.ncbi.nlm.nih.gov/pubmed/2289200
Cancer immunotherapy ASI - active specific immunotherapy
Cancer immunotherapy attempts to stimulate the immune
system to reject and destroy tumors. Immuno cell therapy
for cancer was first introduced by Rosenberg and his
colleagues of National Institute of Health USA. In the
late 80s, they published an article in which they reported
a low tumor regression rate (2.6-3.3%) in 1205 patients
with metastatic cancer who underwent different types of
active specific immunotherapy (ASI), and suggested that
Immuno Cell Therapy with specific chemotherapy is the
future of cancer Immunotherapy. In the beginning
Immunotherapy treatments involved administration of
cytokines such as Interleukin with an aim of inducing
the lymphocytes which will carry their activity of
destroying the tumor cells. Thereafter the adverse effects
of such intravenously administered cytokines lead to
the extraction of the lymphocytes from the blood and
culture-expand them in the lab and then to inject the
cells alone enable them destroy the cancer cells .
Though the concept of this treatment started in the US in
80s, fully fledged clinical treatments on a routine basis
have been in practice in Japan since 1990. Randomized
controlled studies in different cancers with significant
increase in survival and disease free period have been
reported and its efficacy is enhanced by
20 -- 30% when cell based immunotherapy is combined with
other conventional treatment methods.
BCG immunotherapy for early stage (non-invasive)
bladder cancer utilizes instillation of attenuated live
bacteria into the bladder, and is effective in preventing
recurrence in up to two thirds of cases. Topical
immunotherapy utilizes an immune enhancement cream
(imiquimod) which is an interferon producer causing the
patients own killer T cells to destroy warts,, actinic
keratoses, basal cell cancer, vaginal intraepithelial
neoplasia., squamous cell cancer, cutaneous
lymphoma, and superficial malignant melanoma.
Injection immunotherapy uses mumps, candida the HPV
vaccine, or trichophytin antigen injections to
treat warts (HPV induced tumors). Lung cancer has been
demonstrated to potentially respond to immunotherapy.
In many parts of Asia, Medicinal mushrooms are thought to
be able to boost the immune system naturally. Cellular and
animal research has shown that Agaricus blazei may
stimulate immune system cells and the production of
interferons and interleukins (reviewed by G. Hetland).
Mushroom isolates like PSK also are used to increase
immune system parameters (reviewed by Kobayashi). Used
in conjunction with chemotherapy, PSK has increased the
survival time of cancer patients in randomized, control
studies, with a variety of cancer types.
Autologous Immune Enhancement Therapy (AIET)
In the multipronged approach to treat CANCER, one very
useful latest weapon would be AIET which is in clinical
practice in Japan since 90s for more than 15 years with
good effectiveness and several clinical trials and
researches are reported from institutions all over the
world including from the US. AIET is a treatment method in
which some immune cells are taken out of a patient's body
which are cultured and processed to be activated or to
acquire additional functions until their resistance to
cancer is strengthened, then the cells are put back in the
body. Researchers have found that the thus activated
immune system might also be able to determine the
difference between healthy cells and cancer cells to
eliminate the cancer cells from the body. In AIET,
specific type of cells mainly the NK cells and T
lymphocytes are isolated from the peripheral blood of the
cancer patients (during remission in patients who undergo
chemotherapy) by proven methods, expanded to 25 – 30 fold
and activated and then reinfused back into the patient’s
body. These cells act against the cancer cells effectively
and recharge the immune system. Upon encountering a tumor
cell, the activated NK cell attaches to the membrane of
the cancer cell and injects toxic granules which dissolve
the target cell. In less than five minutes, the cancer
cell dies and the NK cell moves on to its next target
cancer cell. A single NK cell can destroy up to 27 cancer
cells before its lifespan. This is the mechanism by which
AIET is effective in Cancer therapy.
This utilizes dendritic cells to activate a cytotoxic
response towards an antigen. Dendritic cells, a type of
antigen presenting cell, are harvested from a patient.
These cells are then either pulsed with an antigen or
transfected with a viral vector. The activated dendritic
cells are then placed back into the patient; these cells
then present the antigens to effector lymphocytes (CD4+ T
cells, CD8+ T cells, and in specialized dendritic cells, B
cells also). This initiates a cytotoxic response to occur
against these antigens and anything that may present these
antigens. One use for this therapy is in cancer
immunotherapy. Tumor Antigens are presented to dendritic
cells, which cause the immune system to target these
antigens, which are often expressed on cancerous
cells. The Dendreon product candidate Provenge is one
example of this approach.
Adoptive cell therapy (ACT) using autologous
tumor-infiltrating lymphocytes is an effective treatment
for patients with metastatic melanoma; this is based
on adoptive immunity.
Adoptive cell transfer, or "ACT," uses T cell-based
cytotoxic responses to attack cancer. T cells that have a
natural or genetically engineered reactivity to a
patient's cancer are expanded, made more effective, in
vitro using a variety of means and then adoptively
transferred into a cancer patient.
For example, T cells with a naturally occurring reactivity
to a patient’s cancer can be found infiltrated in the
patient's own tumors. The tumor can be harvested, and
these tumor-infiltrating lymphocytes (TIL) can then be
expanded, or made more effective, in vitro using high
concentrations of interluekin-2 (IL-2), anti-CD3 and
allo-reactive feeders. These T cells can then be
transferred back into the patient along with exogenous
administration of IL-2 to further boost their activity.
Thus far, a 51% objective response rate has been observed;
and in some patients, tumors shrank to undetectable
size.
The initial studies of adoptive cell transfer using TIL,
however, revealed that persistence of the transferred
cells in vivo was too short. Before reinfusion,
lymphodepletion of the recipient is required to eliminate
regulatory T cells as well as normal endogenous
lymphocytes that compete with the transferred cells for
homeostatic cytokines. Prior
lymphodepletion to transfer of the expanded TIL was made
by total body irradiation. The trend for increasing
survival as a function of increasing lymphodepletion was
highly significant (P=0.007). Transferred cells
expanded in vivo and persisted in the peripheral blood in
many patients, sometimes achieving levels of 75% of all
CD8+ T cells at 6-12 months after infusion.
Morgan et al. (2006) demonstrated that the adoptive
cell transfer of lymphocytes transduced with retrovirus
encoding T cell receptors (TCRs) that recognize a cancer
antigen can mediate anti-tumor responses in patients with
metastatic melanomas.
In such T cell genetic engineering, TCRs that have been
identified to have reactivity against tumor-associated
antigens are cloned into a replication-incompetent virus
that is capable of genomic integration. A patient's own
lymphocytes are exposed to these viruses and then expanded
non-specifically or stimulated using the engineered TCR.
The cells are then transferred back into the patient. This
therapy has been demonstrated to result in objective
clinical responses in patients with refractory stage IV
cancer. The Surgery Branch of the National Cancer
Institute (Bethesda, Maryland) is actively investigating
this form of cancer treatment for patients suffering
aggressive melanomas.
Combination of ACT with such genetic engineering of T
cells has opened possibilities for the extension of ACT
immunotherapy to patients with a wide variety of cancer
types and is a promising new approach to cancer
treatment.
In June 2008, it was announced that US doctors from the
Clinical Research Division led by Dr. Cassian Yee at Fred
Hutchinson Cancer Research Center in Seattle had
successfully treated a patient with advanced skin cancer
by injecting the patient with immune cells cloned from his
own immune system. The patient was free from tumours
within eight weeks of treatment. Dr. Cassian Yee described
the research findings at The Cancer Research Institute
International 2008 Symposia Series. . Responses,
however, were not seen in other patients in this clinical
trial.
Larger trials are now under way.
system to reject and destroy tumors. Immuno cell therapy
for cancer was first introduced by Rosenberg and his
colleagues of National Institute of Health USA. In the
late 80s, they published an article in which they reported
a low tumor regression rate (2.6-3.3%) in 1205 patients
with metastatic cancer who underwent different types of
active specific immunotherapy (ASI), and suggested that
Immuno Cell Therapy with specific chemotherapy is the
future of cancer Immunotherapy. In the beginning
Immunotherapy treatments involved administration of
cytokines such as Interleukin with an aim of inducing
the lymphocytes which will carry their activity of
destroying the tumor cells. Thereafter the adverse effects
of such intravenously administered cytokines lead to
the extraction of the lymphocytes from the blood and
culture-expand them in the lab and then to inject the
cells alone enable them destroy the cancer cells .
Though the concept of this treatment started in the US in
80s, fully fledged clinical treatments on a routine basis
have been in practice in Japan since 1990. Randomized
controlled studies in different cancers with significant
increase in survival and disease free period have been
reported and its efficacy is enhanced by
20 -- 30% when cell based immunotherapy is combined with
other conventional treatment methods.
BCG immunotherapy for early stage (non-invasive)
bladder cancer utilizes instillation of attenuated live
bacteria into the bladder, and is effective in preventing
recurrence in up to two thirds of cases. Topical
immunotherapy utilizes an immune enhancement cream
(imiquimod) which is an interferon producer causing the
patients own killer T cells to destroy warts,, actinic
keratoses, basal cell cancer, vaginal intraepithelial
neoplasia., squamous cell cancer, cutaneous
lymphoma, and superficial malignant melanoma.
Injection immunotherapy uses mumps, candida the HPV
vaccine, or trichophytin antigen injections to
treat warts (HPV induced tumors). Lung cancer has been
demonstrated to potentially respond to immunotherapy.
In many parts of Asia, Medicinal mushrooms are thought to
be able to boost the immune system naturally. Cellular and
animal research has shown that Agaricus blazei may
stimulate immune system cells and the production of
interferons and interleukins (reviewed by G. Hetland).
Mushroom isolates like PSK also are used to increase
immune system parameters (reviewed by Kobayashi). Used
in conjunction with chemotherapy, PSK has increased the
survival time of cancer patients in randomized, control
studies, with a variety of cancer types.
Autologous Immune Enhancement Therapy (AIET)
In the multipronged approach to treat CANCER, one very
useful latest weapon would be AIET which is in clinical
practice in Japan since 90s for more than 15 years with
good effectiveness and several clinical trials and
researches are reported from institutions all over the
world including from the US. AIET is a treatment method in
which some immune cells are taken out of a patient's body
which are cultured and processed to be activated or to
acquire additional functions until their resistance to
cancer is strengthened, then the cells are put back in the
body. Researchers have found that the thus activated
immune system might also be able to determine the
difference between healthy cells and cancer cells to
eliminate the cancer cells from the body. In AIET,
specific type of cells mainly the NK cells and T
lymphocytes are isolated from the peripheral blood of the
cancer patients (during remission in patients who undergo
chemotherapy) by proven methods, expanded to 25 – 30 fold
and activated and then reinfused back into the patient’s
body. These cells act against the cancer cells effectively
and recharge the immune system. Upon encountering a tumor
cell, the activated NK cell attaches to the membrane of
the cancer cell and injects toxic granules which dissolve
the target cell. In less than five minutes, the cancer
cell dies and the NK cell moves on to its next target
cancer cell. A single NK cell can destroy up to 27 cancer
cells before its lifespan. This is the mechanism by which
AIET is effective in Cancer therapy.
This utilizes dendritic cells to activate a cytotoxic
response towards an antigen. Dendritic cells, a type of
antigen presenting cell, are harvested from a patient.
These cells are then either pulsed with an antigen or
transfected with a viral vector. The activated dendritic
cells are then placed back into the patient; these cells
then present the antigens to effector lymphocytes (CD4+ T
cells, CD8+ T cells, and in specialized dendritic cells, B
cells also). This initiates a cytotoxic response to occur
against these antigens and anything that may present these
antigens. One use for this therapy is in cancer
immunotherapy. Tumor Antigens are presented to dendritic
cells, which cause the immune system to target these
antigens, which are often expressed on cancerous
cells. The Dendreon product candidate Provenge is one
example of this approach.
Adoptive cell therapy (ACT) using autologous
tumor-infiltrating lymphocytes is an effective treatment
for patients with metastatic melanoma; this is based
on adoptive immunity.
Adoptive cell transfer, or "ACT," uses T cell-based
cytotoxic responses to attack cancer. T cells that have a
natural or genetically engineered reactivity to a
patient's cancer are expanded, made more effective, in
vitro using a variety of means and then adoptively
transferred into a cancer patient.
For example, T cells with a naturally occurring reactivity
to a patient’s cancer can be found infiltrated in the
patient's own tumors. The tumor can be harvested, and
these tumor-infiltrating lymphocytes (TIL) can then be
expanded, or made more effective, in vitro using high
concentrations of interluekin-2 (IL-2), anti-CD3 and
allo-reactive feeders. These T cells can then be
transferred back into the patient along with exogenous
administration of IL-2 to further boost their activity.
Thus far, a 51% objective response rate has been observed;
and in some patients, tumors shrank to undetectable
size.
The initial studies of adoptive cell transfer using TIL,
however, revealed that persistence of the transferred
cells in vivo was too short. Before reinfusion,
lymphodepletion of the recipient is required to eliminate
regulatory T cells as well as normal endogenous
lymphocytes that compete with the transferred cells for
homeostatic cytokines. Prior
lymphodepletion to transfer of the expanded TIL was made
by total body irradiation. The trend for increasing
survival as a function of increasing lymphodepletion was
highly significant (P=0.007). Transferred cells
expanded in vivo and persisted in the peripheral blood in
many patients, sometimes achieving levels of 75% of all
CD8+ T cells at 6-12 months after infusion.
Morgan et al. (2006) demonstrated that the adoptive
cell transfer of lymphocytes transduced with retrovirus
encoding T cell receptors (TCRs) that recognize a cancer
antigen can mediate anti-tumor responses in patients with
metastatic melanomas.
In such T cell genetic engineering, TCRs that have been
identified to have reactivity against tumor-associated
antigens are cloned into a replication-incompetent virus
that is capable of genomic integration. A patient's own
lymphocytes are exposed to these viruses and then expanded
non-specifically or stimulated using the engineered TCR.
The cells are then transferred back into the patient. This
therapy has been demonstrated to result in objective
clinical responses in patients with refractory stage IV
cancer. The Surgery Branch of the National Cancer
Institute (Bethesda, Maryland) is actively investigating
this form of cancer treatment for patients suffering
aggressive melanomas.
Combination of ACT with such genetic engineering of T
cells has opened possibilities for the extension of ACT
immunotherapy to patients with a wide variety of cancer
types and is a promising new approach to cancer
treatment.
In June 2008, it was announced that US doctors from the
Clinical Research Division led by Dr. Cassian Yee at Fred
Hutchinson Cancer Research Center in Seattle had
successfully treated a patient with advanced skin cancer
by injecting the patient with immune cells cloned from his
own immune system. The patient was free from tumours
within eight weeks of treatment. Dr. Cassian Yee described
the research findings at The Cancer Research Institute
International 2008 Symposia Series. . Responses,
however, were not seen in other patients in this clinical
trial.
Larger trials are now under way.
Sunday, April 25, 2010
Dedication to William Bradley Coley
Dedicated to William Bradley Coley
(1862-1936)
William Bradley Coley pioneered the study of immunotherapy at the turn of the 20th century.
A young surgeon at what is now Memorial Sloan-Kettering Cancer Center in New York, Coley
observed that patients with sarcoma who developed superficial erysipelas streptococcal skin in-
fection after surgery had better outcomes than those patients who did not develop infections.
Coley later combined a gram-positive, heat-killed Streptococcus with the gram-negative, heat-
killed Serratia bacteria to treat patients with cancer. During his career he successfully treated
hundreds of patients with this bacterial vaccine.
Participants at the Walker’s Cay Colloquium advance the field of immunotherapy that Coley’s
observations began.
Cancer Immunol Immunother (2003) 52(Suppl1):S1-S38TABLE OF CONTENTS
About the Albert B. Sabin Vaccine Institute
Introduction - H.R. Shepherd, DSc
Session I: Translational Research
Quick Look S5
DNA Vaccines and Immunocytokines for Cancer Therapy
- Ralph Reisfeld, PhD S5
Recombinant DNA Vaccines - Jeffrey Schlom, PhD S5
Immunization Against Ubiquitous Tumor-Associated Anti-
gens - Malcolm S. Mitchell, MD S6
Session II: Tumor Antigens
Quick Look S7
Identification and Optimization of TCR Specific Ligands
for Vaccine Design from Combinatorial Peptide Libraries -
Darcy Wilson, PhD S7
Probing Degeneracy of Recognition by Epitope-Specific
Cytotoxic T cells (CTLs) for Superagonists for Vaccine
Design - June Kan-Mitchell, PhD S7
Prostate Cancer Antigens and Vaccines - Michael L. Sal-
galler, PhD S8
Human Her-2/neu - Wei-Zen Wei, PhD S9
Helper Epitopes - Jay Berzofsky, MD, PhD S9
Session III: Involvement of HLA-Class II Molecules in
Tumor Rejection
Quick Look S10
Activating Tumor-Specific CD4+
T Cells: Tumor Cells as
Antigen Presenting Cells and Vaccines - Suzanne
Ostrand-Rosenberg, PhD S10
The Role of Epitopes for Tumor-Reactive T cells - Esteban
Celis, MD, PhD S11
CD4+
T cell Responses in Melanoma/RCC and DC-Based
Repolarization - Walter Storkus, PhD S11
Session IV: Discussion Session: Regulatory Perspectives,
Including FDA
Quick Look S13
Developing Cancer Immunotherapies for the Clinic: Regu-
latory Perspectives - Lucio Miele, MD PhD S13
Session V: Strategies for Immunization
Quick Look S15
PLGA Nanosphere Delivery of Peptides and Lipopeptides
to Dendritic Cells - John Samuel, PhD S15
Large Multivalent Immunogen (LMI) Immunotherapy -
Matt Mescher, PhD S15
The Role of the Effector in Antitumor Response Dick Dut-
ton, PhD S16
Venezuelan Equine Encephalitis Virus for Treatment of
Cervical Cancer - W. Martin Kast, PhD S17
IRX-2: A Natural Cytokine Stimulant for Cancer Vaccines
- Harvey Brandwein, PhD S17
Gene Vaccination Against HER-2/neu - Lawrence B. Lach-
man, PhD S18
Magnitude and Duration of Antigen Specific Immune
Response May Affect Clinical Response - H. Kim Lyerly,
MD S18
Session VI: Costimulatory Molecules
Quick Look S19
CD137(4-1BB), More than a Costimulator - Lieping Chen,
MD, PhD S19
Immune Gene Therapy Using Recombinant Proteins of the
Tumor Necrosis Factor Family - Thomas J. Kipps, MD,
PhD S20
T help for CTL - Stephen P. Schoenberger, PhD S21
Session VII: Tumor Defense Against Immune Response
Quick Look S22
Apoptosis and Anti-Apoptosis in Multimodality Cancer
Treatment - Lucio Miele, MD, PhD S22
The Generation and Persistence of CD4 T Cell Memory -
Dick Dutton, PhD S23
Downregulation of Tumor Antigens by IFN - Caroline Le
Poole, PhD S23
Closing Comments - Allan Goldstein, PhD S24
Abstracts: Session I S26
Abstracts: Session II S27
Abstracts: Session III S28
Abstracts: Session IV S29
Abstracts: Session V S29
Abstracts: Session VI S31
Abstracts: Session VII S31
Glossary S33
The Albert B. Sabin Vaccine Institute Board and Councils
S37
Fourth Annual Walker’s Cay Colloquium Participants
S38
S2S3
About the Albert B. Sabin Vaccine Institute
The Albert B. Sabin Vaccine Institute is a nonprofit public organiza-
tion dedicated to continuing the work of Dr. Albert B. Sabin, who en-
visioned the tremendous potential of vaccines to prevent deadly dis-
eases. The Institute promotes rapid scientific advances in vaccine
development, delivery, and distribution worldwide. A vaccine think
tank, we are dedicated to finding innovative and effective solutions.
In particular, we attempt to build bridges of communication and dis-
covery between research, clinical medicine, academia, business, and
government.
Each year the Albert B. Sabin Vaccine Institute brings together
more than 30 leading cancer researchers at the annual Walker’s Cay
Colloquium on Cancer Vaccines and Immunotherapy. Vaccine scien-
tists from many disciplines participate in three days of open discus-
sion of ideas and data, some of them unpublished. The Institute con-
venes this annual colloquium in the hope of shortening the time
needed to translate scientific discoveries into meaningful treatments
for cancer. Participants share unpublished data and conclusions that
may generate new ideas.
The meeting site, Walker’s Cay, is a small island in the northern
Bahamas owned by Robert Abplanalp, Chairman and Chief Execu-
tive Officer of Precision Valve Corporation. Richard Nixon fre-
quently visited Walker’s Cay during his Presidency. He found the se-
cluded atmosphere conducive to decision making. President Nixon
was at Walker’s Cay when he decided to commit American resourc-
es to a War on Cancer.
H.R. "Shep" Shepherd, Chairman of the Sabin Vaccine Institute,
and Robert H. Abplanalp envisioned and launched the annual Walk-
er’s Cay Colloquium on Cancer Vaccines and Immunotherapy. The
War on Cancer accelerated basic biomedical research and opened the
door for molecular biology. In this tradition, scientists who partici-
pate in the colloquium are experimenting with vaccines that either
prevent cancer or elicit the body’s immune system to vanquish can-
cer cells.
The Colloquium is co-chaired by two distinguished cancer vaccino-
logists. The inaugural Colloquium in March 1999 was co-chaired by
James P. Allison, PhD, Director of Cancer Research Laboratories and
Professor of Immunology at the Howard Hughes Medical Institute at
the University of California, Berkeley, and Drew Pardoll, MD, PhD,
Professor of Oncology at the Johns Hopkins University School of
Medicine.
The second and third meetings, in March 2000 and March 2001,
were co-chaired by Ralph A. Reisfeld, PhD, Professor, Department
of Immunology, the Scripps Research Institute, and Jeffrey Schlom,
PhD, Head of the Tumor Biology Laboratory at the National Cancer
Institute.
The fourth colloquium, in March 2002, was co-chaired by W.
Martin Kast, PhD, Professor at Loyola University and the Cardinal
Bernardin Cancer Center in Chicago, and Malcolm S. Mitchell,
MD, of the Karmanos Cancer Institute at Wayne State University in
Detroit.Introduction
H.R. Shepherd, DSc, Chairman
The Albert B. Sabin Vaccine Institute
New Canaan, CT
In 2002, the Albert B. Sabin Vaccine Institute convened its
fourth annual think-tank colloquium attended by 30 of the
foremost cancer researchers from academia, government, and
industry. These scientists exchanged information in a free and
open atmosphere about progress toward developing second-
generation cancer vaccines, improving the immunotherapy of
cancer, and forming partnerships and consortia to advance the
field more rapidly.
S4
The following text is edited from the proceedings of
the Fourth Annual Walker’s Cay Colloquium on Cancer
Vaccines and Immunotherapy, which ran to well over 500
pages. In attempting to make the language and concepts
accessible to readers across many disciplines, we trust we
have remained faithful to the sense of the original.MARCH 7, 2002
SESSION I: Translational Research
QUICK LOOK
Reisfeld et al. showed that a DNA vaccine encoding carci-
noembryonic self antigen (CEA) given orally to CEA trans-
genic mice attenuated Salmonella typhimurium. This led to
cell-mediated protection against MC38 colon carcinoma,
without the induction of antibodies to lipopolysaccharide.
The construct consisted of a CMV promoter, ubiquitin, and
"survivin" in an IRES vector. An immune response was pro-
duced in the Peyer’s patches by this route, which protected
against the development of metastases in seven of eight mice.
Schlom et al. incorporated a gene for CEA into a vector,
which proved to be superior to either a peptide or a protein.
Poxviruses such as vaccinia and avipox were most useful,
particularly when costimulatory molecules were included in
the construct and the vector was varied during immunization
to avoid development of poxvirus antibodies. This vaccina-
tion scheme was used in a small clinical trial with nine pa-
tients per group. TRICOM vectors incorporating CD80,
ICAM-1 and LFA-2 were best with a DNA vaccine in prophy-
laxis. Treatment of metastases required GM-CSF injection in-
corporated within the avipox vector together with low-dose
IL-2 besides the vector+CEA. Mitchell reports on "ubiqui-
tous" tumor associated antigens that might be used for immu-
nization of large groups of patients, focusing particularly on
"MG50" originally isolated from melanomas. MG50 was
found in all breast cancer specimens examined, as well as the
majority of pancreatic carcinomas and glioblastomas. Sever-
al HLA-A2-restricted epitopes were found that elicited CD8+
cytotoxic T lymphocyte (CTL) reactive against peptide load-
ed targets, but which have not yet been tested against tumors
that display MG50. Of these, LLEAVPAV is perhaps the most
promising nonamer, for which epitope mimics will be produced.
DNA Vaccines and Immunocytokines
for Cancer Therapy
Ralph Reisfeld, PhD
The Scripps Research Institute, La Jolla, CA
Established metastases of CT-26 murine colon cancer were
eradicated by targeting EpCAM/KSA antigen, transduced in-
to CT26 cells with a KS1/4-IL2 fusion protein. Treatment
with this immunocytokine induced an MHC class I-restricted
CD8+
T cell response which effectively eradicated established
metastases in 50% of mice that also remained completely pro-
tected against tumor cell challenge for up to 6 months post-
treatment. Importantly, intravenous injections of two small
(5 µg) non-curative doses of the immunocytokine, 4 days af-
ter tumor cell challenge, increased this long-lived protective
immunity to optimal levels in 100% of experimental animals.
Cellular mechanisms involved in these events included a 14-
fold increase in CTL precursor frequency and the subsequent
induction of genes encoding cytokines produced by type 1
helper T cells, some of which differentiated into long-term
memory T cells. These increases correlated with increases in
tumor-protective immunity, indicating effective priming of ef-
fector T cells. Conclusions from these findings are (1) in-
creased frequency of precursor CTLs correlating with in-
creased T-cell memory and (2) an altered and more pro-
nounced responsiveness of memory T cells after a second en-
counter with the same or related antigen. Additional findings
indicated that long-lived CD8+
T cell memory does not require
long-term exposure to residual deposits of antigen, because
memory was maintained in the absence of tumor antigen.
In a parallel development, we induced tumor-protective im-
munity by using an orally administered DNA vaccine based
on oncofetal carcinoembryonic self-antigen (CEA) to break
peripheral T cell tolerance to CEA in CEA-transgenic mice.
This oral vaccine was delivered to Peyer’s patches in the gut
by attenuated Salmonella typhimurium and induced tumor-
protective immunity against murine colon carcinoma by
MHC class I-restricted CD8+
T cells. This treatment proved
most effective in a prophylactic setting, resulting in complete
tumor rejection, when boosted by 5×5-µg i.v. injection of
KS1/4-IL2 fusion protein 24 h after tumor cell challenge.
Immunological mechanisms involved included improved li-
gation of CD28 on activated T cells with B7.1/7.2 costimula-
tory molecules on dendritic cells and upregulation of proin-
flammatory cytokines IL-12, IFN-? and GM-CSF released
from activated T cells. T cell activation after immunocytokine
boost was also indicated by upregulation of T cell activation
markers CD25, CD28, CD69 and LFA-1.
We also developed a DNA-based vaccine against an inhibi-
tor of apoptosis, survivin, overexpressed in tumor cells. When
combined with ubiquitin and a secretory secondary lymphoid
chemokine (SLC) produced in the same expression vector,
this vaccine effectively induced a CD8+
T cell response and a
three- to fourfold increase in tumor cell apoptosis resulting in
the prevention of pulmonary metastases of Lewis lung carci-
noma in syngenic C57BL/6 mice. Taken together, these nov-
el immunotherapeutic approaches may lead to the rational de-
sign of more effective cancer treatment modalities.
Recombinant DNA Vaccines
Jeffrey Schlom, PhD
National Cancer Institute, Bethesda, MD
Examination of different strategies to enhance the immunoge-
nicity of a given antigen has shown that placing the gene of
the tumor antigen into a vector is much more efficient than us-
ing a protein or peptide.
Our studies have focused on two types of the poxvirus as
vectors: the replication-competent vaccinia virus and fowlpox,
which is a replication-incompetent avipox virus. Diversified
prime-and-boost - a vaccination strategy of priming with vac-
cinia and boosting with avipox- is more effective than giving
either alone. Placement of costimulatory molecules into these
vectors which can accept insertion of up to eight transgenes,
each on its own poxvirus promoter, produces a still stronger
antitumor effect.
Studies with a CEA-transgenic mouse model show that two
vaccinations with CEA protein and adjuvant has no effect,
S5whereas one or two vaccinations with rV-CEA induce a
strong CEA-specific CD4 or CD8 response. In this model the-
re is significant CEA expression in fetal tissue, in GI tissue at
greater levels than there is in humans, and CEA is secreted in
the blood at 5-100 µg/ml, which is the level observed in most
patients with colorectal cancer. In addition, CEA is expressed
in pancreatic cancer, gastric cancer, in 70% of non-small-cell
lung cancers, 50% of breast cancers, and the majority of head
and neck cancers.
A clinical, randomized trial examined the effect of the pri-
me-and-boost strategy - using vaccinia CEA and avipox CEA
- in patients with advanced CEA-positive carcinomas who
had failed to respond after two to six rounds of chemotherapy
with a variety of agents. Each patient was given vaccinia-
CEA followed by four vaccinations of avipox-CEA, or the re-
ciprocal. In most patients, a CEA-specific T-cell response was
induced.
Patients receiving four monthly rV-CEA vaccinations alone
had no extension of survival. Patients receiving the rV-CEA
vaccine with boosting with avipox-CEA achieved much better
survival and a statistically significant increase in CEA-specif-
ic T cells. Immune responses increased constantly with each
administration of avipox. Many patients continue to receive
monthly vaccination, with some having had over 20 vaccina-
tions. Survival duration, interestingly, was unrelated to pre-
vaccination CEA-specific T-cell levels. However, generation
of CEA-specific CTL responses was associated with increased
survival, after accounting for disease status. There seems to be
a relationship in this small cohort between survival and the
ability to mount a T-cell response. Now under investigation is
whether the patients who responded had a different previous
treatment from those whose disease remained in stasis.
New data indicate that when the boost is stopped, T-cell re-
sponse declines; the precursors go down but increase again
when vaccination is restarted. The optimal interval between
doses is yet to be determined, but the data show that the pri-
me-and-boost strategy is better than giving either avipox or
vaccinia alone. Continuing mouse studies also bear out the
benefit of combining rV-CEA vaccine and avipox-CEA
boost, and a still stronger antitumor response with the addi-
tion of costimulatory molecules, called TRICOM for TRIad
of COstimulatory Molecules, typically B7-1, ICAM-1, and
LFA-3. Each has a different ligand on the T cells and each ap-
pears to signal differently, suggesting a possible synergy.
Clinical studies bear out that use of three molecules in the
same vector produces antigen-specific T-cell responses.
Immunization Against Ubiquitous
Tumor-Associated Antigens
Malcolm S. Mitchell, MD
Karmanos Cancer Institute, Wayne State University, Detroit, MI
New understanding of possible cancer antigens has drawn at-
tention to two molecules: MUC1 and MG50. MUC1 is a mu-
cin that is ubiquitous in adenocarcinomas, including cancers
of the breast, ovary, colon, lung and pancreas. MG50 is a re-
cently discovered antigen originally identified on melanomas
(MG50 stands for melanoma gene 50), and is also found in
adenocarcinomas of the breast, pancreas, and colon, and in
glioblastomas, but not in hematopoietic tumors. MG50 is
therefore another ubiquitous molecule, perhaps even more
widely present than MUC1. Our work has attempted to deter-
mine the mechanisms of action of putative cancer antigens,
and their potential efficacy in immunization, as measured by
cytotoxicity.
MUC1 has been thought to be a non-MHC-restricted T-cell
stimulatory antigen, but this is not entirely correct. MUC1 has
at least three cytotoxic epitopes that are HLA-A2-restricted.
We have called the epitope with perhaps the greatest promise
M1.1. The strongest stimulatory response, as indicated by cy-
totoxicity generated in vitro with human T cells, was the com-
bination of M1.1 with PADRE, a pan-DR epitope that stimu-
lates helper CD4 T cells. M1.1 attached to a consensus signal
sequence was also immunogenic for CTL. The combination
elicited a CD8, HLA class-I-restricted response, but this was
aided by CD4 T cells. When we depleted CD4 cells in vitro,
we found poor reactivity with PADRE and peptide or M1.1 at-
tached to a signal sequence (fusion peptide). The best respons-
es occurred following several weekly stimulations. There was
a point of diminished return, however. If restimulation was
continued beyond two or three times, there was less cytotoxic-
ity and the number of cells decreased. On repeated restimula-
tion, the annexin V level rose, suggesting activation-induced
cell death (apoptosis). This may be why MUC1 has appeared
to be poorly immunogenic. We could limit cell death by de-
creasing the peptide concentration in the culture after the sec-
ond restimulation and by adjusting the IL-2 in the medium.
The second molecule of interest, MG50, was isolated by
subtractive hybridization of cDNA from a melanoma with
mRNA from a squamous cell lung carcinoma. We found 12
novel cDNA sequences, two of which were selected for fur-
ther study because they were found in melanomas but not in
normal tissues.
A survey of MG50 showed that it was more than just a mel-
anoma antigen. It was present in 18 of 18 breast cancer spec-
imens, 12 of 12 pancreatic carcinomas, 6 of 10 ovarian carci-
nomas and 3 of 10 colon carcinomas. It was not present in
normal breast or pancreatic tissue. It therefore seemed a good
antigen to mark those cancers.
The MG50 molecule contained HLA-A2.1-restricted CTL
epitopes. There were six such epitopes that encoded a 1496
amino acid protein. T cells immunized with these peptides,
especially peptide 624-632, led to CTL that lysed HLA-A2
melanoma cell lines. The CTL did not kill mutated fibroblast
or the non-A2 melanoma cell lines. Thus the epitopes were
naturally displayed on the tumor and were recognized in their
natural context.
Western immunoblotting of MG50 with serum from an im-
munized rabbit revealed two distinct size variants: 170 kDa,
which is close to the predicted size; and 80 kDa. The latter
may be a splice variant of the former. We also made mouse
monoclonal antibodies, which also recognized these two mo-
lecular weight variants. Most tumors to date have expressed
both weight variants. MG50 may be a good target for immu-
notherapy of adenocarcinomas and for further preclinical ex-
periments.
S6MARCH 7, 2002
SESSION II: Tumor Antigens
QUICK LOOK
Wilson and colleagues presented data on alternative peptide
ligands (epitope mimics). With a library of nonamers, they fo-
und extensive recognition by CD8 T cells of peptides with as
many as seven substitutions. The combination of specificity
and degeneracy has introduced an interesting complexity to
binding of peptides to the T-cell receptor, increasing the num-
ber of potential stimuli for CTL. Kan-Mitchell amplified this
point with HLA-A*0201-restricted Gag p17 peptide77-85 of
HIV1 (SL9). Of 75 analogs of the epitope synthesized and
tested for their ability to be recognized and to lead to lysis of
T2 cells, 20 were recognized by the donor cell line, PW1, and
12 of these were more potent than the original SL9 epitope.
Two mimics were immunogenic in vivo, stimulating CTL
cross-reactive to SL9 from various healthy donors. In a sec-
ond group of CTL derived from a different seronegative do-
nor, the mimics recognized were not the same as those identi-
fied by CTLs from donor PW1. This suggests a range of mim-
ics that must be studied in order to design a vaccine useful for
a significant number of patients. Salgaller described phase I
clinical trial with prostate cancer vaccines, with prostate-spe-
cific membrane antigens, prostate specific antigen, and pros-
tatic acid phosphatase as the antigens. Stability of disease for
at least 6 months was achieved in 54% of patients. Wei dis-
cussed c-erbB constructs for vaccines. Some degree of suc-
cess has been achieved in preventing the emergence of tumors
in mice with several of these constructs, particularly those
that retain most of the intracellular portion of the gene.
Berzofsky emphasized the importance of using high-affinity
peptide to increase the production of cytokines that skew the
TH response towards TH1. Optimizing CD4 help optimized
CTL generation, but the helper epitope had to be from the
same antigen and linked to the CTL epitope to produce an an-
amnestic response.
Identification and Optimization
of TCR-Specific Ligands for Vaccine Design
from Combinatorial Peptide Libraries
Darcy Wilson, PhD
Torrey Pines Institute for Molecular Studies, San Diego, CA
Large combinatorial peptide libraries are powerful tools for
identifying and optimizing peptide ligand antigens specific
for T cells having a clinically relevant specificity. This abili-
ty to identify and optimize such ligands is a vital tool for find-
ing optimal antigens for vaccine design. Optimized mimics
are not only good immunogens in their own right, but they in-
duce strong T cell immune responses to the native ligand.
We follow a three-step strategy for identifying peptide anti-
gens specific for T cells:
1.Scan peptide combinatorial libraries for sequences that
stimulate proliferation, cytokine release or lysis of pulsed
target cells. These scans optimize peptides for MHC and
TCR binding. This achieves a significant enhancement of
immunogenicity for poorly immunogenic antigens, such as
you see in autoimmune disease and for many tumor anti-
gens.
2.Synthesize candidate sequences and assess their capacity to
stimulate the original clone or the line in terms of its EC50,
which is the concentration that causes a half-maximal re-
sponse.
3.Perform preclinical studies of immunogenicity in vivo or of
the ability to prime transgenic mice.
The antigen under study was gp100 melanoma. It was chosen
because of extensive work by others that identified an antigen
and its fragment epitope, gp100 p209, responsible for stimu-
lating tumor infiltrating lymphocytes (TIL). The Rosenberg
group at the National Cancer Institute (NCI) then went on to
modify the 209 peptide and enhance its immunogenicity by a
critical substitution of methionine in place of threonine at po-
sition 2 (p209-2M).
We used two CD8+
T cell clones generated by the Rosen-
berg group at NCI to scan a nonamer peptide library. After
several scans, the various predicted peptides were synthe-
sized and tested at various dilutions for their activity on the
two index clones. This resulted in identification of a large
number of effective peptides, many having six amino acid
substitutions, that are active with EC50 values significantly
better than either the 209 or the 209-2M peptides. Work to da-
te shows that optimized mimics are highly immunogenic and
provoke good immune responses against the native ligands.
These combinatorial peptide libraries can be used to identi-
fy MHC and TCR binding residues for T-cell clones and epi-
tope selected T-cell lines.
Finally, the repertoire of CD4 T cells is promiscuous; a sin-
gle T cell clone can be stimulated by over one million differ-
ent decamer peptide sequences. This finding leads to the
question as to how degeneracy and T-cell specificity can co-
exist in the same repertoire.
Probing Degeneracy of Recognition
by Epitope-Specific Cytotoxic T cells (CTLs)
for Superagonists for Vaccine Design
June Kan-Mitchell, PhD
Karmanos Cancer Institute, Wayne State University, Detroit, MI
Antigen-specific cytotoxic T-lymphocyte (CTL) responses are
critical in the control of infection and are believed to be impor-
tant in cancer surveillance. Induction of CTL will play an im-
portant part in therapeutic and prophylactic vaccines. It is be-
coming clear that the quality as well as magnitude of the CTL
response is important. CTL responses to the immunodominant
HLA A2-restricted Gag epitope SLYNTVATL (SL9, residues
77-85) is uniquely prominent among chronically HIV-infect-
ed patients (75% of patients). For this reason, we have decid-
ed to use this as a prototype epitope to validate our premise
S7that mimic peptides of native epitopes can be found that will
elicit a more relevant and effective CTL response.
To identify "mimic peptides" (mimic peptide sequences) of
SL9 that are better immunogens, we used in vitro-primed
SL9-CTL lines to scan a large combinatorial nonapeptide li-
brary consisting of three trillion distinct peptides. CTLs spe-
cific to SL9 were primed and expanded from CD8+
naive T
cells from a healthy, seronegative donor (PW1, index line). Of
75 potential candidate peptides deduced, synthesized and
tested for their ability to be identified and lysed by the index
CTLs, 20 were recognized by PW1, and of these 12 were
more potent than SL9, five of which differed by five or six
residues. More importantly, CTLs from B.H. recognized six
common superagonist mimics, four of which differed in five
positions with two non-conservative substitutions. These
mimics apparently bind more stably to HLA-A*0201 than the
native epitope. Alternative peptide ligands with multiple
changes of sequence that include non-conservative substitu-
tions have not been readily predicted by any other method.
Two mimics were found to be immunogenic ex vivo, immu-
nizing CTLs crossreactive to SL9 from P.W., B.H. and heal-
thy donors R.G. and J.D. Of interest, J.L. SL9-specific CTLs
did not recognize the six common mimics predicted by PW1.
These results provide the proof-of-principle that probing
TCR degeneracy of de novo-primed CTLs with peptide li-
braries will identify superagonists potentially useful as vac-
cines for infections or cancers. These peptides are prototypes
of a new generation of APLs identified by the unbiased scan-
ning of synthetic peptide libraries. Specifically, they are
unique with respect to the position and the number of the sub-
stitution(s) on the nonameric peptide sequence. For the pur-
pose of vaccine design, thorough analysis of the overlap in
degeneracy may be necessary to select optimal crossreactive
peptide analogs.
Prostate Cancer Antigens and Vaccines
Michael L. Salgaller, PhD
Northwest Biotherapeutics, Inc., Seattle, WA
The discovery of prostate cancer antigens and treatment strat-
egies share a number of the same problems with other cancer
types, and presents its own unique challenges. As with many
cancer types, there is a lack of information on good candidate
targets and tumor rejection antigens. A lack of information on
the presence or absence of tumor-associated antigens on tu-
mors themselves is particularly challenging with prostate can-
cer. This situation is related to insufficient tumor material be-
ing available for investigation.
Our patients have had prostatectomies, and when tumors
can be excised, they are often small and difficult to isolate
from surrounding normal tissue. Also, the most common area
of metastasis is bone, and bone is a poor site from which to
generate cell lines, or to get T cells and TIL lines.
The patient group is sometimes not as amenable to research
participation as others. Many older patients with prostate can-
cer do not want to go through anything that is not directly re-
lated to treatment. So, we have had many patients, for exam-
ple, who have been through surgery and hormones and radia-
tion and chemotherapy, but have a problem with an extra
10-ml draw for immune monitoring. In addition, the mean
patient age is 60 to 70 years, and these patients have a poorer
immune response than younger patients.
As with other cancer types, there are possible tumor rejec-
tion antigens, which cover tumor suppressors, oncogenes, and
other expressed proteins. Prostate antigens that have under-
gone the most thorough clinical analysis are cell-based pro-
teins and glycoproteins: prostatic acid phosphatase (PAP),
prostate-specific antigen (PSA), and prostate-specific mem-
brane antigen (PSMA).
PSMA is membrane-bound, type II glycoprotein. It appears
to be an easier target to hit than PSA, since it does not move
around systemically throughout the body. Moreover, its ex-
pression increases with disease progression, especially when
patients no longer respond to hormone therapy.
An analogous technology uses dendritic cells with PAP as a
target. On a Western blot, its expression decreases with dis-
ease progression, and patients with end-stage disease - the
starting point for most FDA-approved trials - have the small-
est PAP expression.
Expression of PSA varies with disease state, and there is a
lot of controversy about PSA. The FDA allows you to use
PSA to include or exclude a patient, or to put a patient in a cer-
tain treatment group, but they will not use PSA levels to ap-
prove a product because something may lower PSA but not
improve survival or quality of life.
We investigated the capacity of a vaccine comprising autol-
ogous dendritic cells (DC) pulsed ex vivo with recombinant
PSMA (rPSMA) to generate clinically meaningful antitumor
immune responses in patients with hormone-refractory pros-
tate cancer (HRPC).
In 2000 and 2001, 32 patients with metastatic or non-meta-
static HRPC were enrolled in a phase I/II clinical trial. Their
peripheral blood mononuclear cells were isolated by leuka-
pheresis, matured to DC by in vitro culture with maturation
factors (GM-CSF, IL-4, and inactivated BCG) for up to
7 days, followed by rPSMA loading and harvesting of the
vaccine. Patients received four intradermal treatments of 5,
10, or 20 million rPSMA-loaded mature DC at monthly inter-
vals, followed by 6 months of observation. Measurement of
serum anti-PSMA antibodies, PSMA-stimulated lymphocyte
proliferation, and delayed-type hypersensitivity (DTH) skin
testing were carried out before, during, and after vaccination.
Clinical responses were assessed by CT/bone scans and labo-
ratory tests, including PSA levels.
More than 120 vaccine injections were well tolerated, with
skin irritation at the injection site being the most common ad-
verse reaction. Headache and fatigue were also reported. Al-
though shrinkage of tumor masses has not been observed,
positive results have been achieved. Overall, 54% of patients
achieved stability of their disease after 6 months, as assessed
by radiographic criteria, 83% had a PSMA-specific immune
response, 92% with stable disease had a PSMA-specific im-
mune response, and 46% had a decrease in PSA velocity.
Compared to baseline, 93% of 27 evaluable patients convert-
ed to DTH-positive against the BCG component of the vac-
cine. Of patients with disease progression but not metastatic
S8disease, none showed progression after 27 weeks. Of patients
with metastatic disease, 11% showed progression after
13 weeks, compared to 50% after 12 weeks in trials with oth-
er agents. Due to these promising initial findings, we have ini-
tiated a double-blind, placebo-controlled phase III clinical tri-
al with 495 patients. The primary objective of this 3-year tri-
al is disease progression as measured by radiographic criteria.
We will measure percent progression and overall survival at
52 weeks, and continue to monitor safety, PSA levels, and im-
mune responses.
Human Her-2/neu
Wei-Zen Wei, PhD
Karmanos Cancer Institute, Wayne State University, Detroit, MI
Human ErbB-2, or Her-2/neu, is a well-recognized member
of the epidermal growth factor receptor family and functions
as an oncogene when over-expressed. It is a preferred dimer-
ization partner with the other members of the epidermal gro-
wth factor receptor family, ErbB-1, 3 and 4. When the ligand
binds, an ErbB dimer is formed on the cell surface to trigger
tyrosine kinase activity which leads to cell proliferation. In
constructing ErbB-2 DNA vaccines, the kinase activity was
eliminated by point mutation or sequence deletion. To induce
selected immune reactivity, such as a CD8 T-cell response,
with or without antibody production, ErbB-2 DNA was mod-
ified so that the protein product was directed to different parts
of the cell for processing and presentation.
Five categories of DNA vaccines have been constructed:
1. Full-length ErbB-2 with a single substitution of the lysine
residue 753 to alanine, which eliminates the ATP binding
capacity and kinase activity. This molecule is not an onco-
gene, but inhibits the function of ErbB-2 oncogene.
2. A transmembrane form without the cytoplasmic, i.e. tyro-
sine kinase, region; thus a safer vaccine.
3. A secreted form that contains about 505 amino acids of the
extracellular domain.
4. A cytoplasmic form that contains the entire coding se-
quence, but does not have the ER signal. The protein prod-
uct is dropped into the cytoplasm and never makes it to the
surface.
5. A lysosomal form that is a fusion of the transmembrane
form with the cytoplasmic domain of lysosomal associated
membrane protein-1 (LAMP). The product is directed to
the lysosome by LAMP.
BALB/c mice immunized with the full-length form contain-
ing a point mutation, the tyrosine kinase-free transmembrane
form, or the secreted form of ErbB-2, showed antibody pro-
duction, primarily IgG2a. The secreted form induced both
IgG1 and IgG2a. Mice immunized two to three times rejected
tumor challenge. Co-vaccination with GM-CSF DNA always
induced a stronger protection. The cytoplasmic and lysoso-
mal form of vaccine induced significant protection only when
coinjected with GM-CSF DNA. When mice were immunized
with the cytoplasmic or lysosomal ErbB-2, which produce
proteins in the cytoplasm or lysosome, but not cell surface, T
cells, but not antibodies were activated. These vaccines, when
given together with GM-CSF DNA resulted in 75% to 80%
protection on a subsequent challenge. The protective effect
from the native form of ErbB-2 was CD-4 T-cell mediated.
The cytoplasmic version of the vaccine, however, is almost
entirely CD-8 T-cell mediated. These results show that it is
possible to construct a DNA vaccine that induces primarily a
CD4 or a CD8 T-cell response.
Immunization of ErbB-2 transgenic mice which express hu-
man ErbB-2 and are tolerant to ErbB-2, with the DNA vac-
cines, induced limited protection. When these mice were im-
munized with ErbB-2-positive allogenic tumor cells the result
was a striking immune response, and they were able to reject
the tumor.
One can proceed with clinical trials using allogenic tumor
cells. The preferred approach is to introduce foreign epitopes
into the DNA to mimic the allogeneic tumor cells, but retain
the well-defined quality of DNA vaccines. We have chosen to
work with tetanus toxin fragment C. In transgenic mice,
ErbB-2 fused to tetanus toxin fragment was much more effec-
tive than the native ErbB-2. The difference was less promi-
nent in normal mice which are not tolerant to ErbB-2. Inter-
estingly, normal mice immunized with the fusion vaccine and
rejected ErbB-2-positive tumors were protected much better
from a second challenge with ErbB-2-negative tumors than
mice immunized with native ErbB-2, indicating strong epi-
tope spreading, probably enabled by CD4 cells. Epitope
spreading protects mice from ErbB-2-negative tumors and is
important in long-term disease-free survival. Fusion vaccines
are being further developed to enhance the antitumor effect.
Helper Epitopes
Jay Berzofsky, MD, PhD
National Cancer Institute, Bethesda, MD
Work in our laboratory over the past 5 years on helper epi-
topes has focused on modification of amino acid side chains
that interact with the MHC molecule to improve the affinity
of the peptide for the MHC molecule without altering the sur-
face of the peptide-MHC complex seen by the T-cell receptor.
The hope is that this modification will result in a more potent
immunogen that would still induce T cells that would recog-
nize the natural sequence.
This kind of modification was first accomplished about
10 years ago when we were looking at a helper epitope from
the HIV envelope protein, called T1. We found that replacing
a glutamic acid E with an alanine A, removing the negative
charge, improved the binding to a class II MHC molecule in
the mouse that we studied.
This raised the question: If we alter the helper peptide so
that it has higher affinity, would it be more effective in induc-
ing help for a CTL response to a CTL epitope that is made of
one long peptide without altering the CTL epitope? In mice,
we found that the enhanced peptide resulted in about a 33-fold
increase in lytic units of CTL against targets expressing the
epitope.
S9To determine whether this approach would increase protec-
tion, we challenged mice with a recombinant vaccinia virus
that expresses the HIV envelope protein. At lower challenge
doses, the enhanced vaccine or the moderate-affinity wild-
type vaccine both reduced the virus to less than 102
pfu, but at
higher challenge doses, we saw virus reduction to 102
pfu on-
ly with the improved vaccine.
Examination of cytokines produced in response to this en-
hanced helper peptide or the wild-type showed whether the
help was not only quantitatively better, but also qualitatively
different. The enhanced peptide skewed the response to a Th1
phenotype, producing much more IFN-?, whereas the wild-
type peptide produced a mixed response.
The mechanism by which the enhanced helper cells work
appears to be related to increased CD40L expression stimu-
lating substantially more IL-12 production by the dendritic
cells than when the helper cells are stimulated by the wild-ty-
pe peptide. This might explain not only some of the increase
in CTL activity, but also the polarization toward Th1.
Comparison of the moderate-affinity wild-type peptide with
the high-affinity enhanced helper peptide shows a reciprocal
interaction between the helper cell and the dendritic cell. The
higher affinity peptide first induced much more CD40 ligand
on the helper cell. That, in turn, activated dendritic cells not
only to express more costimulatory molecules, but also to
greatly increase IL-12 production. The IL-12 production by
the dendritic cell then acts on the helper cell to make it a mo-
re polarized Th1-type helper cell.
The dendritic cell then more readily activates a CTL re-
sponse. Improving the affinity of the helper peptide results in
a more effective vaccine, not only by quantitatively increas-
ing the amount of CTL through this mechanism through the
dendritic cell, but also by qualitatively improving and skew-
ing the helper response toward Th1, which may be more pro-
tective in both tumor and virus models.
MARCH 7, 2002
SESSION III: Involvement
of HLA-Class II Molecules
in Tumor Rejection
QUICK LOOK
Ostrand-Rosenberg emphasized HLA class II presentation
by tumor cells as vaccines, while Celis presented binding al-
gorithms for DR1, DR4 and DR7 for prediction of the impor-
tant stimulatory epitopes. Storkus described work with
MAGE-6, which is found in melanomas, renal cell carcino-
mas and atypical nevi, and appears to be an early tumor-asso-
ciated antigen.
Activating Tumor-Specific CD4+ T Cells:
Tumor Cells as Antigen-Presenting Cells
and Vaccines
Suzanne Ostrand-Rosenberg, PhD
University of Maryland, Baltimore, MD
When we began our work on developing tumor cells as anti-
gen-presenting cells, our focus was on the potential develop-
ment of vaccines. We now believe that our approach will also
be beneficial to further understanding of how tumor cells
present antigens. We anticipate that this work will contribute
to understanding the role of tumor cells in tolerance induction
and activation/inactivation of CD4+
T cells in vivo.
Using basic molecular techniques, we inserted into tumor
cells the genes encoding MHC class II molecules and the ge-
nes encoding costimulatory signal molecules to directly acti-
vate CD4 cells. In mouse models, these vaccines consisted of
tumor cells transfected with CD80, and in some cases also
transfected with superantigens. The vaccines were effective
prophylactic agents in that 100% of mice that were later chal-
lenged with a tumor were protected. In mice with primary tu-
mors under 1 cm in diameter at the time of vaccine treatment,
we cured about 50%. When tumor size is greater than 1 cm,
the cure rate drops off due to immunosuppression. In animals
in which a mammary tumor had metastasized and was re-
moved, a postsurgical vaccine was less successful, but surviv-
al was significantly extended. We observed, however, a large
reduction in metastatic cells in the lung, brain, liver, and
lymph nodes.
Since efficacy of the MHC class II vaccines is due to their
ability to present endogenously encoded tumor antigens, we
have also investigated the role of MHC class II accessory
molecules, such as the class II-associated invariant chain pro-
tein and DM, which are known to impact antigen presentation
by MHC class II molecules. (DM is a class II-associated pro-
tein that regulates presentation of antigens by class II mole-
cules). We made tumor cell transfectants that express MHC
class II either with or without invariant chain and DM. These
transfectants were tested for presentation of an endogenously
synthesized antigen.
S10We found that MHC class II-positive cells that express class
II and do not coexpress invariant chain or DM are very immu-
nogenic and are not tumorigenic. They present endogenous
antigen in class II, and do not present exogenous antigen. If
transfectants are made with the class II transactivator, which
up-regulates MHC class II, DM and invariant chain, the tu-
mor cells retain their malignancy and are not immunogenic in
vivo. In vitro, they do not present endogenously synthesized
tumor antigens, but they are very good presenters of exoge-
nous antigen. So if you want the tumor cell vaccines to pres-
ent endogenously synthesized tumor antigen to stimulate an-
titumor immunity, then the tumor cells must express MHC
class II and not coexpress invariant chain and DM.
These studies were done with tumor antigens localized to
the endoplasmic reticulum using T-cell hybridomas as the
read-out for T-cell activation. We also used transgenic naive
T cells and found that activation of naive CD4+
T cells re-
quires coexpression of CD80 by the vaccines. Further work
with tumor antigens targeted to other subcellular compart-
ments shows that the vaccines also present tumor antigens
originating in the nucleus, cytoplasm or mitochondria.
In vivo experiments demonstrate that contrary to class I-tar-
geted tumor vaccines, the class II-modified vaccine cells are
the relevant antigen-presenting cells at the initial stage of
CD4 T-cell priming.
We are currently translating this immunotherapy strategy
for clinical use by transducing human ocular melanoma and
breast adenocarcinoma cells with appropriate HLA-DR and
CD80 genes. These vaccines will initially be tested in vitro
for their ability to activate tumor-specific CD4+
T cells.
The Role of Epitopes for Tumor-Reactive
T cells
Esteban Celis, MD, PhD
The Mayo Clinic, Rochester, MN
Helper T cells are going to play a key role in cancer vaccines
because of their ability to enhance CTL priming during the in-
duction of the immune response. They are also of interest al-
so because they might increase the generation of memory CT-
Ls, and augment the effector function of CTLs at the tumor si-
te and allow these cells to expand as they are killing tumor
cells. In some cases, helper T cells might even exhibit antitu-
mor activity directly against MHC class II-positive tumors or
indirectly to the production of cytokines during cross-presen-
tation at the tumor site. Over the past few years, we have iden-
tified a promiscuous epitope from HER-2/neu that binds to
several MHC class II alleles that comprise close to 70% of the
human population.
We were also able to identify a peptide from the MAGE3 tu-
mor antigen that is presented by these several MHC class al-
leles. In addition, we also identified a peptide from the mela-
noma-associated antigen gp100 that is presented by several
MHC class II molecules which are present in approximately
50% of the population. Within this peptide, there is an HLA-
A2 CTL epitope. By vaccinating with this peptide, we might
be able to elicit both cytotoxic T cells and T helper cells. A
clinical trial is underway to determine whether this peptide
enhances induction of both helper and CTL responses.
Recent work with the EBNA2 antigen involves targeting
post-transplant proliferative disease and B-cell lymphoma,
mainly in young adults and children who are getting solid or-
gan transplants and who are susceptible to these diseases be-
cause of severe immune suppression. We have identified a
peptide from EBNA2, which is one of the first genes to be ex-
pressed during infection. This epitope is restricted by a large
number of MHC class II molecules. This therapy may poten-
tially be extended to massive carcinomas.
We stimulated cells from many individuals and in many
cases produced strong proliferative responses. In two patients
because there was high background, we do not know wheth-
er those responses were real. The majority of individuals who
responded to this peptide could recognize their autologous
Epstein-Barr virus (EBV)-transformed B lymphoblastoid cells.
In some cases, these T cells induced a 97-99% inhibition of
infection of B cells and the subsequent proliferation that is in-
duced by EBV. The most successful inhibition observed took
place with the EBNA2-specific line, and in the DR-16-restrict-
ed cell line. The addition of cyclosporin up to the near-toxic
doses of >1 µg/ml showed that the vaccine still produces a sig-
nificant amount of inhibition of proliferation of T cells.
Some of these MHC class II-restricted T cells have cytolyt-
ic activity. The DR52 cell line has very high cytolytic activi-
ty against autologous EBV cells that are either pulsed or un-
pulsed with the EBNA2 peptide, so they do not require exog-
enous peptide. They can therefore kill quite well, whereas
negative controls, irrelevant EBV lines that do not express
DR52 are not killed.
Some cells actually only recognize the autologous EBV
cells if you pulse them with peptide, suggesting that there is
not enough peptide or that the affinity of this particular cell
line is not high enough to recognize the peptide. But interest-
ingly, this same cell line has the capacity to prevent the infec-
tion of the live virus, so it would still be therapeutic.
CD4+ T cell Responses in Melanoma/RCC
and DC-Based Repolarization
Walter Storkus, PhD
University of Pittsburgh School of Medicine, Pittsburgh, PA
Over the past decade, murine models have provided insights
into the impact of resident tumors on the functional status of
the immune system. Within the CD4+
T cell, there is an evo-
lution of the nature or quality of the type of CD4 response
over time as the tumor establishes itself and metastasizes.
This phenomenon is seen in the B16 model of melanoma in
B6 mice, where a Th0/Th1-type mixed response to the specif-
ic tumor or tumor lysate is seen early. It is followed 7 to
14 days after tumor inoculation with a predominance of the
Th2 functional phenotype, with a lot more IL-4 and IL-5 pro-
duced in response to tumor antigens. By about day 20, high
frequencies of CD25+
and CD4+
cells are observed and these
are capable of producing TGF-ß and IL-10, but not much
IFN-? or IL-4, in the spleen or tumor.
S11It has also been shown that some of these effects can be
blocked. Early injection of neutralizing anti-IL-4 or anti-IL-
10 antibodies systemically into these mice can obviate some
of the transition from the Th0/Th1 towards the Th2/Th3 func-
tional phenotype. Late injection of anti-TGF-ß, can also min-
imize some of the Th3/Tr effects that are observed after
day 14 of tumor growth.
Ablation of the CD4+
T-cell compartment allows reactiva-
tion of the cellular Th1-type arm and treated, the lesions in tu-
mor-bearing animals frequently regress. Treated animals
bearing B16 melanomas can also develop "therapeutic" viti-
ligo, as noted by pigmentation changes in their skin/fur.
We are now corroborating whether similar immune devia-
tion occurs in human patients with cancer. This has required
us to identify MHC class II-presented epitopes that are de-
rived from tumor-associated antigens. These can ultimately
serve as indicators to gauge the functional attributes of tumor-
reactive CD4+
T cells in patients with resident tumors.
We picked two model antigens for analysis, namely MAGE-
6 and EphA2. These antigens are both expressed broadly
across many lineages of cancers, and MAGE-6 is expressed
by the majority of melanoma and renal cell carcinomas as
well as in premalignant lesions. MAGE-6 is a cytoplasmic
protein of unknown function that is expressed in atypical ne-
vi, even though these are clinically not considered malignant.
This antigen is also upregulated early in other malignancies.
MAGE-6 therefore represents an early model antigen that
should be expressed at least in certain cancers from stage I
through stage IV of the disease.
EphA2 is a member of a large family of receptor tyrosine
kinases. It accumulates in cells that have lost or diminished
expression of E-cadherin. As a correlate, EphA2-overex-
pressing cancer cells become increasingly invasive into the
basal membranes in situ, displaying an enhanced metastatic
capability. EphA2 is therefore considered to be a "late anti-
gen" in disease progression.
To date, our work is furthest progressed for the MAGE-6
antigen. Three MAGE-6 epitopes have been identified, which
are all presented by HLA-DR4 to CD4+
T cells.
Our epitope selection involved the use of peripheral blood
T cells isolated from HLA-DR4+
patients that have rejected
their disease and who have remained disease-free for pro-
longed periods of time. We interrogated CD4+
T cells from
these patients as to which MAGE-6 peptides are immunolog-
ically relevant, and we have identified what we believe are the
three most dominant ones.
We evaluated responses to these epitopes in ten normal
HLA-DR4+
individuals. We generated dendritic cells in the
standard fashion, and found that those cells produce very little
IL-12 if their CD40 molecules are crosslinked or if they are
stimulated with LPS or SAC. In essence, they do not seem to
polarize or repolarize the T cell response coming directly out
of the patients in either a Th1 or Th2 direction, and in fact ap-
pear neutral in this respect. Most normal individuals are very
low responders or nonresponders to MAGE-6 peptides. In a
few instances, some donors did react to MAGE-6 epitopes, but
these responses displayed a mixed Th1/Th2 phenotype.
We then looked at 33 HLA-DR4+
patients with cancer,
15 with renal carcinoma and 18 with melanoma. Approxi-
mately half of the patients with renal cancer had stage I and
half had stage IV disease. Individuals with active disease
were all unequivocally Th2-biased in their responses and
produced only IL-5 (but no IFN-?) when stimulated with
the three MAGE-6 epitopes. Patients who had achieved
disease-free status, if they did respond, responded in a
completely Th1-biased manner. They produced IFN-?, but
not IL-5.
The vast majority of patients with melanoma had stage IV
disease, which did not allow any disease-stage correlation
with Th polarized responses. While the data provided for
MAGE-6 epitopes strongly suggest a disease-dependent rath-
er than a disease stage-dependent bias in CD4+
T-cell reactiv-
ity, individual data points are each deduced from independent
donors. These studies will be best performed longitudinally
within the same patient.
Based on a limited data set, we have had some individuals
with renal cancer who we can track pre- and post-therapy. We
looked at responses in stage I disease in one patient treated
only surgically and remaining disease-free for 2 months. ELI-
SPOT frequencies suggested a Th2 to Th1 shift in CD4+
func-
tional responses to the MAGE-6 epitopes. In another individ-
ual, we observed increased CD4+
T cell staining with fluores-
cent tetramers of HLA-DR4 loaded with the MAGE-6 Th
peptides. While this provides an index of specific T-cell fre-
quency, it is not revealing as to whether these T cells are func-
tionally Th1- or Th2-like. To address this, we are now com-
bining tetramer and cytokine staining approaches to be able to
deduce both frequency and function.
The issue boils down to a simple question: If tumor antigen-
specific Th2 cells are dominant in patients with active dis-
ease, and vaccines are not being applied to a level immuno-
logic playing field where both Th1- and Th2-type responses
are equally possible, what can we do to accentuate our ability
to develop Th1-biased responses in patients already predis-
posed not to react in this manner?
Based on the mouse therapeutic studies, we could try to de-
lete the non-Th1-type CD4+
T-cell responders completely and
subsequently apply a specific vaccine capable of promoting
Th1-biased immunity. Another possibility is based on the
finding that effector T helper cell subsets may not be termi-
nally committed to a Th1- or Th2-functional state, but can be
re-educated or re-tooled based on environmental pressures to
become functionally beneficial.
Other investigators have previously evaluated "committed"
allergen-specific Th2-type CD4+
T cell clones stimulated
with antigen-loaded autologous dendritic cells in the presence
of nothing, IL-4, or IL-12. If IL-4 or neutral conditions are ap-
plied, these cells remain functionally Th2-biased, but if rIL-
12 is placed into the antigenic stimulation, a majority of
these cells can be converted to a Th1 pattern of IFN-? secre-
tion, with some cells exhibiting a mixed Th1/Th2 pattern of
cytokine secretion, and a minority of responder cells prefer-
entially secreting the Th2-cytokine IL-4. This Th2-to-Th1
functional conversion mechanism appears to involve IL-12
upregulation of its own ß-2 subunit on these Th2 clones, as
well as the loss of the GATA-3 Th2-transactivator protein and
the gain of the T-bet Th1-transactivator protein in these par-
ticular treated cells.
S12We have recently evaluated the issue of whether CD4+
T
cells from patients with cancer can be reconditioned or repo-
larized in vitro. Peripheral blood CD4+
T cells were stimulat-
ed with peptide-pulsed immature DCs, DCs in the presence of
recombinant IL-12 or DCs conditioned to secrete high levels
of IL-12. We found that when these T cells were subsequent-
ly analyzed for their pattern of cytokine release in response to
MAGE-6 peptides, only in cases where IL-12 was present
during the stimulations were the original Th2-type dominated
CD4+
T cell responses converted into Th1-type responses af-
filiated with IFN-? secretion. While we have been unable to
discern Th3 or Tr1 regulatory T cell responses to MAGE-6
peptides in cancer patients, further examination of CD4+
T
cells within the tumor lesion itself and the tumor-draining will
need to be performed as these cell types are likely more prev-
alent in these locations.
Lastly, based on our results, we believe that peptide-based
vaccines will benefit from the use of DC1-type dendritic cells
that are forced to secrete high levels of IL-12. These antigen-
presenting cells will not only be able to prime Th1-type T cell
immunity to tumor antigens, they will have the capacity to re-
polarize non-Th1-type T-cell responses towards Th1-type im-
munity.
MARCH 7, 2002
SESSION IV: Discussion Session:
Regulatory Perspectives, Including FDA
QUICK LOOK
In a special invited lecture, Miele reviewed issues of interest
to academic scientists hoping to bring discoveries or inven-
tions to the marketplace. The interests and agenda of academ-
ic scientists has traditionally been very different from those
that concern industrial scientists, but now the two worlds are
merging, as the practical value of academic discoveries is be-
coming more widely recognized.
Developing Cancer Immunotherapies
for the Clinic: Regulatory Perspectives
Lucio Miele, MD, PhD
University of Illinois at Chicago
Moving from the therapeutic discovery phase into product de-
velopment requires a different mindset and organization com-
pared to a basic research laboratory, and carries pitfalls that
can trip up even the most careful investigator. There are sim-
ilarities to the funding process for National Institutes of
Health (NIH) grants and the investigational new drug (IND)
process, but important differences as well. Approval under
the IND process does not include grant money, and yet with-
holding of approval means you are absolutely forbidden from
conducting the study, even if you want to do it with your own
money. Even if you are approved, you could still be disap-
pointed and legally liable if something goes wrong. There are
also positive differences. With the IND process, reviewers are
not anonymous. You can talk to them and in many cases that
is encouraged.
The Food and Drug Administration (FDA) is also a com-
plex organization to navigate. There are five main sections.
The Center for Drugs Evaluation and Research (CDER) han-
dles essentially chemicals, drugs made with traditional organ-
ic chemistry approaches. The Chemical Biologics Evaluation
and Research (CBER) is of most interest to those developing
vaccines. The Center for Devices and Radiological Health
(CDRH) has authority over diagnostics, including monitoring
devices and diagnostic trials. The Center for Food Safety and
Applied Nutrition (CFSAN) and the Center for Veterinary
Medicine (CVM) are of less interest to those working in the
human vaccine field.
There is also a significant difference between "traditional"
drugs and biologics. With traditional drugs, the organic chem-
istry is clear and the primary issues are clinical. Once the for-
mulation is established, you can move to preclinical trials and
to the clinic. For biologics the process is more complex. Bio-
logics are agents over which the CBER has authority: recom-
binant proteins, cytokines, monoclonal antibodies and all the
engineered derivatives thereof, cell-based products, viruses
and virus-like particles, gene therapy products, blood prod-
S13ucts, tissue products including stem cells, and peptide vac-
cines. Because the types of biologics are more complex, the
CBER is organized by product type: Office of Therapeutics
(for most cancer vaccines), Office of Vaccines, and Office of
Blood Products.
Most immunotherapy agents are biologics reviewed by the
Office of Therapeutics: monoclonal antibodies, cytokines and
all kinds of recombinant chimeric proteins; cell and gene ther-
apy products; and combination products. Some products that
comprise combinations, such as dendritic cell agents, will
dictate which division reviews the total product. For example,
a dendritic cell transfected with two cytokines which also has
a recombinant protein may have three product reviewers.
The reviewers include a primary reviewer, who is generally
an expert in the type of product under study. He or she chairs
the committee and writes the review. A pharmacologist exam-
ines the preclinical animal model data and decides whether
data are adequate to support clinical development. There is al-
so a clinical reviewer who is a physician, generally with ex-
tensive experience in trial design and analysis. There are also
radiologists or nuclear medicine specialists for imaging prod-
ucts. Finally, ad hoc consultants review components of the
product that are not necessarily within the area of expertise of
the primary reviewer.
The review cycle starts a 30-day clock when the IND is re-
ceived. Each reviewer reads either his or her part of the IND,
or the entire team, and writes a critique. The team then meets
either in person or by e-mail. The primary reviewer collects all
the critiques and writes a comprehensive critique, as well as a
letter to the sponsor which summarizes the contents of the cri-
tique. The IND and the decision are presented to the Division
for further critique. The Office of Therapeutics meets weekly
and the divisions present their INDs. If reviewers agree that
the IND should be approved, there is a brief discussion of
whatever minor changes may be necessary, and then the re-
view letter is signed off by the Division Director or the Center
Director. If the IND is on clinical hold, there is an extensive
discussion with explanations for the hold decision. A Clinical
Hold Oversight Committee does quality control to ensure that
INDs are not put on hold for frivolous reasons. The FDA will
also go to great lengths to work with you to avoid putting you
on clinical hold, including advice on changes to prevent desig-
nation of a hold status. The primary reason for going on hold
is insufficient information to establish safety.
If you think you have developed a drug or biologic with
clinical potential, there are four key questions to answer be-
fore filing an IND:
1. Do you have a product? Have the active ingredients been
determined? Is purity of proteins at 90-95%? Is the agent
stable long enough for reproducible results? Is it sterile
and, for humans, free of HIV, hepatitis, etc? What is the po-
tency?
2. How well have you characterized the product?
3. What kind of preclinical safety information do you have on
this product and are these related to the intended clinical
use?
4. What are the possible safety issues in your clinical study
and how are they addressed in trial design?
Issues to cover include pharmacokinetics and functional po-
tency assays to better quantify patient exposure. A trial is best
conducted with a single lot of product to ensure consistency
of results. Retention of lot samples can help uncover prob-
lems related to inconsistency in subsequent lots. Detailed
pharmacokinetics and toxicity in animals is more relevant for
drugs than it is for biologics. Often here is no animal model
that is relevant to establish whether a human biologic agent is
either safe or effective. Dosing can be based on prior studies,
or a series of dose escalations.
Overall, the study will benefit if the investigators:
• Follow good clinical protocol
• Follow inclusion criteria and seek FDA permission before
undertaking an exception
• Keep good records and be prepared to show them
• Strictly follow the FDA rules on adverse event reporting
• Report all protocol violations as they happen.
Above all, the success of a trial depends on frequent and open
communication with the FDA and the primary reviewer. The
FDA has access to abundant privileged information about
what works and does not work, and can steer an investigator
to what is known to work best.
S14MARCH 8, 2002
SESSION V:
Strategies for Immunization
QUICK LOOK
Samuel presented data showing enhanced vaccine efficacy
through use of biodegradable polymer microspheres as vehi-
cles for immunization. MUC1 peptides have been used for
immunization of TH and CTL in animals, and will be tested
in humans. Mescher used 5 µm beads to present antigens to
T cells. The larger beads were required to get deregulation of
CTLs. Dutton discussed the two types of CTL in mice, CTL1
(parallel to TH1 cells) and CTL2 (like TH2), but noted that
neither cell type was the principal effector cell in vivo. Re-
cruitment of host macrophages, NK cells and B cells was con-
sidered to be most important in vivo. Kast showed that that
alphavirus vectors such as Venezuelan equine encephalitis vi-
rus avoided the problem of viral replication and pre-existing
immunity to the vector in immunizing against human papillo-
mavirus tumors in transgenic mice. An HLA-A2-restricted
protective response was generated in the mice. Treatment of
established tumors was also accomplished. Brandwein de-
scribed a complex adjuvant which, when injected into the
perilymphatic areas, together with oral indomethacin, low-
dose cyclophosphamide and zinc, improved the survival of
patients with advanced head and neck cancers. The promising
regimen has not yet been used in a controlled trial. Lachman
described work with viral replicons in which the structural
protein-coding site was replaced with a site for inserting the
gene of interest. The resulting replicon was an infectious rep-
lication-deficient virus that effectively protected against the
development of tumors, similar to Kast’s findings. Mammary
tumors were prevented up to 14 weeks after treatment with
neu-containing constructs. Lyerly attempted to monitor the
immune response in clinical trials. He found that ELISPOT is
far more sensitive than the tetramer assay or intracellular cy-
tokine assay, being able to detect one cell in 10,000 peripher-
al blood cells against MART-1 or CMV.
PLGA Nanosphere Delivery of Peptides
and Lipopeptides to Dendritic Cells
John Samuel, PhD
University of Alberta, Edmonton, AB
Tiny spheres of a biodegradable and biocompatible polymer can
be used as a delivery system for targeting antigens to dendritic
cells and to elicit the desired type of immune responses against
a wide range of incorporated materials. The material under in-
vestigation is poly(D,L-lactic-co-glycolic acid) (PLGA), a poly-
mer comprising lactic acid and glycolic acid. It is the same poly-
ester used to make nontoxic resoluble surgical sutures.
Nanospheres of PLGA can encapsulate a broad range of mol-
ecules, from water-soluble material to highly lipophilic materi-
al. They can also incorporate peptides, proteins, and plasmid
DNA, as well as viruses. This flexibility makes it a highly effi-
cient delivery system for targeting antigens to dendritic cells.
PLGA nanospheres were taken up by about 80-90% of human
and mouse dendritic cells in culture in vitro. They were also ef-
ficiently taken up by mouse dendritic cells in vivo.
PLGA nanospheres provide an attractive platform for can-
cer vaccines. We can induce a strong Th1 response, provided
we coencapsulate monophosphoryl lipid A or CpG oligonu-
cleotides along with the antigen. We have recently shown that
PLGA nanosphere delivery can be used to achieve a Th1 ty-
pe of responses against a peptide that usually induces a Th2
response. Immunization of mice with a Th2 peptide in tradi-
tional adjuvants such as alum or CFA can be used to achieve
a Th2 imprint. A subsequent immunization with PLGA nano-
spheres containing the same peptide can bias the immune re-
sponses towards a Th1 type, despite the previous Th2 imprint.
Thus this delivery system may be used to alter an already es-
tablished Th2 responses to achieve a Th1 response.
In mice with lung cancer expressing MUC1 mucin, immu-
nization with nanospheres containing a MUC1 lipopeptide
(BLP25) and monophosphoryl lipid A induced significant an-
ticancer effects. Virtually complete protection was achieved
when the vaccine was given prophylactically, i.e., before es-
tablishment of the tumor. Therapeutic immunization of mice
with established tumors induced lower levels of protection,
but significantly greater than those observed for the negative
control groups, with response varying with the extent of tu-
mor burden.
PLGA nanospheres offer an efficient and flexible vaccine de-
livery system for inducing effective anticancer immune re-
sponses.
Large Multivalent Immunogen (LMI)
Immunotherapy
Matt Mescher, PhD
University of Minnesota School of Medicine, Minneapolis, MN
An innovative mode of delivering immunotherapy was sug-
gested by experiments in which class I antigen protein was
absorbed onto different sized beads of silica, polysulfated la-
tex or polyanhydride polymer, from 5 to 1 µm in diameter,
and their ability to trigger degranulation by CTL.
Bead size was found to have a significant affect on the abil-
ity of the CTL to sense and respond to the antigen. Antigen
placed on 4-µm or 5-µm particles is effective, but effective-
ness falls off dramatically as particle size diminishes. At
1-µm bead size, the CTLs show no response to the antigen.
Class I alloantigen placed on beads called large multivalent
immunogen (LMI), placed in vivo, stimulated no detectable
CTL response. The response to allogeneic or syngeneic tumor
could be augmented, however, with beads having the same
membrane antigen on the surface as was present on the cell
that the animal was challenged with. For example, if an exper-
iment challenged an H-2b mouse with an H-2d allogeneic tu-
mor, H-2d had to be on the LMI to be effective. This augmen-
tation of tumor-specific CTL responses resulted in reduction
of growth in a variety of syngeneic tumor models in mice.
S15The physical form of the antigen is also critical for augmen-
tation. No significant augmentation was observed when using
the same membrane antigen as free membranes or mixed with
adjuvants such as CFA, or with irradiated tumor cells. LMI
administration did not have to be at the site of the tumor. It
was effective when the beads were delivered intraperitoneal-
ly, intravenously, or subcutaneously.
One murine trial examined efficacy of the therapy on estab-
lished murine tumors, in which tumors were visible and pal-
pable and metastasis had occurred. Treatment with just micro-
spheres produced little effect, but microspheres combined
with chemotherapy produced synergistic effects. Tumors
grown for 8 days and treated with just Cytoxan shrank over
the next few days. But after another day or two, the tumors be-
gin to grow aggressively and there was very little extension of
survival due to Cytoxan alone. In contrast, Cytoxan followed
2 days later with a single injection of beads with the tumor an-
tigen on their surface resulted in no tumor progression follow-
ing Cytoxan treatment. In the majority of mice, tumor became
undetectable within the next 5 to 10 days. The mice had a
long-lived immune memory: if they were rechallenged with
live tumor, they rapidly rejected the tumor. Similar results
were obtained in several different established tumor models.
Ongoing studies are employing adoptive transfer of tumor-
specific TCR-transgenic CD8 T cells to develop a better un-
derstanding of the mechanism responsible for the effects of
LMI on CTL activation and tumor growth.
A small phase I clinical trial at the University of California
San Diego (conducted by Dr. Malcolm Mitchell) used plasma
membranes isolated from two melanoma lines used to prepare
antigen and to coat the cell-sized beads. Patients received the
antigen treatment only. There were no toxicity problems. Of
16 patients, 10 showed increased melanoma-specific precur-
sor frequency pre- and post-immunization. The disease was
stabilized in 4 of 17 patients and one patient went into partial
remission.
A second trial is underway at the University of Minnesota
Cancer Center for patients with melanoma and renal carcino-
ma. The patients’ tumors are resected, and used to isolate the
membrane antigen to coat onto the LMI. Patients are treated
with one or more combinations of cyclophosphamide, LMI
(107 beads per injection, with a minimum of two injections),
Cytoxan and a subsequent short course of low-dose IL-2 on
an outpatient basis. Although the results are still coming in,
observations so far show no adverse reaction to the beads. Of
the 12 melanoma patients who have been treated at this point,
about half are progressing. Five remain in remission. Two
have vitiligo, which in other studies correlated with remis-
sion. In the patients with renal carcinoma, only four have be-
en treated. Two have progressed, one appears to be stable, and
one remains in remission after 8 months.
The Role of the Effector
in Antitumor Response
Dick Dutton, PhD
Trudeau Institute, Saranac Lake, NY
A novel approach in vaccine research attempts to determine
which form of the immune response is the most effective at
controlling tumor growth and find ways to initiate that re-
sponse.
CD8 T cells from T cell receptor transgenic mice specific for
a peptide derived from ovalbumin are adoptively transferred
into animals that have tumors transfected to express ovalbu-
min, the B16ova melanoma and the EG7 (EL4ova) thymoma.
The melanoma, injected intravenously, grows as lung metas-
tases. The thymoma, injected intradermally, grows as a readi-
ly measurable compact mass. The tumors are injected on day -
7. The CD8 effectors are generated from naive CD8 T cells by
4-day in vitro culture with antigen-presenting cells (APCs)
and ovalbumin and injected on day zero. We first tested wheth-
er Tc1s or Tc2s worked better in controlling tumor growth.
We put in grade numbers of either Tc1s or Tc2s. At low
numbers of transferred cells nothing happens. At one million
cells, the transfer starts to be effective and the mice survive
an extra 30 days. Higher numbers have little additional effect
in prolonging survival. Interferon-secreting Tc1 effectors be-
come effective at about 20 times fewer cells than Tc2s. Ef-
fector cells generated in vitro efficiently kill the tumor target
and that killing is 95% perforin-dependent and about 5% Fas
ligand-dependent. So we expected that they would also kill
the tumor by perforin-mediated cytolysis in vivo. But, to our
surprise, effectors generated from OT-1 perforin-knockout
mice were as effective as those from OT-I wild-type both in
the Tc1 and Tc2 model. They could kill the tumor by per-
forin-mediated lysis but that is not what happened in vivo.
Tc1 control was dependent on IFN-?. We showed that B16
melanoma treated with interferon in vitro upregulates TNF
receptors. If TNF is added, the cells are killed in vitro. But
CD8 effector cells from OT-1 mice crossed to TNF-a-defi-
cient mice are equally effective in Tc1 as those from wild-
type and in further studies we found Fas ligand to be only
marginally important.
We are thus somewhat at a loss to explain how the CD8 T
cells control tumor growth. We speculate that they are sum-
moning up NK cells, neutrophils, macrophages, and the actu-
al killing of the tumor is being carried out by these cells. The
CD8 T cell merely finds the tumor, makes cytokines and che-
mokines and orchestrates a host response. We suspect the kill-
ing is mediated by something other than the T cells based al-
so on observations in the EG7 model. Only when Tc1s and
Tc2s are adoptively transferred do you get control of the tu-
mor. The donor cells go into the draining lymph node, not the
contralateral, and also to the spleen. This produces an accel-
erated accumulation of host cells, particularly neutrophils and
NK cells, and also B cells.
In the absence of adoptive transfer there is a substantial host
response but it is ineffective. Following adoptive transfer,
something happens that causes the host response to become
effective. Presumably, the adoptive transferred cells play a
S16key role, but it is also essential that the host do something to
ensure that tumor growth is controlled.
There are several possible explanations:
• The host makes a response, but it is not big enough.
• The host response is good but the effector cells do not mi-
grate to the tumor.
• Regulatory cells or the tumor control or kill the effector
cells when they arrive at the tumor site.
We speculate also that effector cells generated in the absence
of the adoptive transfer may somehow not be effective be-
cause the tumor may send out signals that it is a "self" cell,
and the responding CD8 T cells leave the tumor unharmed.
There are several clinical implications of these possible sce-
narios. If the response is simply insufficient, perhaps repeat-
ed adoptive transferring of effector cells will keep the attack
on the tumor going. If we need to provide inflammatory sig-
nals at the tumor site to attract the effector cells, these could
be provided. If we need to remove regulatory cells, these
could be removed. Prior studies have showed that removal of
CD4 cells in a number of murine tumor models resulted in tu-
mor rejection. It is our hope that further studies will reveal
which options to pursue.
Venezuelan Equine Encephalitis Virus
for Treatment of Cervical Cancer
W. Martin Kast, PhD
Loyola University, Chicago, IL
Alphaviruses are being investigated for potential use in treat-
ment of cervical cancer. They present several attractive fea-
tures for this application:
• A very high protein expression level.
• An RNA-based vaccine is inherently safer than DNA-based
vaccines.
• The alphavirus under examination, Venezuelan equine en-
cephalitis alphavirus is a lytic virus, which means it is self-
eliminating, and that also adds to its safety.
• It is a universal carrier. Indeed, like any vector, you can put
other things of your choice into it.
• Humans have no preexisting immunity to the virus. This is
extremely important because you can at least start vaccinat-
ing with this vector without being confronted with neutral-
izing immunity up front.
This vector delivery system was used in the classical human
papillomavirus (HPV)-16-induced mouse model, and induced
strong CTL responses toward targeted cells. In addition there
is strong long-lasting protection. This model gives you tumors
in almost all cases. If you immunize with viral replicon parti-
cles containing GFP, you have no protection, and you have
100% protection if you use E7-VRPs. It is CD8-mediated.
The C57BL/6 mice are totally protected. A CD8-knockout
mouse is not protected. If you use a class II-knockout mouse,
which is a mouse that has no CD4 cells, you still have full pro-
tection, but that does not mean that CD4 cells are not impor-
tant, especially for memory response induction.
We have gone up to three vaccinations with GFP-VRPs be-
fore we came in with E7-VRPs and still there is complete pro-
tection against tumor cells. Whereas if you do it with MVA
and you first come with GFP-MVA and then inject E7-MVA,
you will lose your protection. Using E7-VRPs in mice with a
subcutaneous tumor, six of eight mice were made tumor-free
in 7 days.
Overall, E7/E6 VRPs induce very potent CTL against HPV
and protect mice against HPV-16-induced tumors. It can ef-
fectively treat and eradicate established tumors. Vaccination
with VRPs does not abrogate the effect of subsequent vacci-
nations.
We have recently also generated a new HPV-16-induced tu-
mor cell line in HLA A2 transgenic mice. We think that this
cell line will be in high demand because there is a lack of A2-
mediated tumor protection models.
So far we have shown proof of the concept in a pure B6
background mouse model and have moved into the clinic with
the A2 epitopes. We are not curing patients yet; we need a
model for the transition from mouse to humans.
IRX-2: A Natural Cytokine Stimulant
for Cancer Vaccines
Harvey Brandwein, PhD
Immuno-Rx, Inc., New York, NY
Over the last several years there has been much promising
work on the identification and application of tumor antigens
to stimulate an immune response. The next breakthrough in
the development of effective cancer vaccines, however, re-
quires further boosting or stimulating the immune system,
particularly T cells and the cellular immune system to re-
spond appropriately and robustly to these antigens in order to
achieve durable immunizations and tumor regressions.
Our efforts are focused on stimulating a coordinated cellu-
lar immune response, which includes in vivo stimulation of
both dendritic cells and T cells as well as providing a Th1-
specific microenvironment as an adjuvant for cancer vac-
cines. This approach has been used with IRX-2, our natural
cytokine product in a 50-patient clinical trial for late-stage
head and neck cancer. IRX-2 is a natural cytokine formulation
consisting of several cytokines and chemokines produced un-
der Good Manufacturing Practices (GMP) by human mono-
nuclear cells in tissue culture. Active ingredients include IL-
2, Il-1ß, interferon-? and TNF-a.
This clinical trial was conducted at the National Cancer In-
stitute of Mexico (INCAN) in Mexico City under the direc-
tion of Dr. Jaime De la Garza, Institute Director, where there
was an opportunity to accrue a significant number of late-
stage head and neck cancer patients who had not yet been
treated with chemotherapy, an experimental practice widely
used in the US yet increasingly being seen as without added
survival benefit.
The IRX-2 treatment regimen consisted of daily 1-ml (peri-
lymphatic) injections of IRX-2 for 10 days preceded by a sin-
S17gle low (nonchemotherapeutic) dose of cyclophosphamide
and daily indomethacin. Both of these agents were given as
contrasuppression to mediate the highly immunosuppressive
effects of such tumors. Zinc was also given daily to counter
certain nutritional deficits common in such patients with head
and neck cancer which is important for effective immunity.
Following the 10-day course of IRX-2 treatment, the pa-
tients were taken to surgery on day 21 and tumors and lymph
nodes were examined. The clinical results were excellent and
we observed a 75% disease-free survival of IRX-2-treated pa-
tients, compared with only a 40% survival in control (i.e. sur-
gery-only) patients at 24 months.
Histology studies on the tumors showed that IRX-2 treat-
ment changes the composition of the tumors in that the amo-
unt of solid tumor mass decreases dramatically as the tumor
becomes fragmented from the massive infiltration of cytotox-
ic T cells and other leukocytes. Lymph nodes that received the
IRX-2 treatment showed an increased density of T and/or B
cells, which paralleled the increases in these cells seen in the
tumors. In a number of lymphocytopenic patients, the IRX-2
treatment also led to an increase in the number of new naive
T cells in circulation. These results indicate that IRX-2 is ab-
le to stimulate a robust and durable immunization against en-
dogenous tumor antigens present in the lymph node of pa-
tients with head and neck cancer, which then leads to tumor
regression and improved survival.
Our future studies are now being aimed at expanding this
work with US-based clinical trials for head and neck cancer,
and further work exploring the potential use of IRX-2 as a "T-
cell adjuvant" for other tumor (peptide) antigen and DNA
vaccines being developed for several types of cancer includ-
ing melanoma, and prostate and cervical cancer.
Gene Vaccination Against HER-2/neu
Lawrence B. Lachman, PhD
University of Texas M.D. Anderson Cancer Center, Houston, TX
HER-2/neu is a therapeutic target and an excellent target for
DNA vaccines because it is overexpressed in 20-30% of
breast cancer patients and in many patients with other cancers
including ovarian, stomach, lung, and even head and neck.
One area of gene vaccine research relating to HER-2/neu fo-
cuses on giving a DNA vaccine, then boosting it with some-
thing else, for example a protein antigen, a live, attenuated, or
inactivated microorganism, a virus-like particle, or a replicon.
A category of virus of special interest is alphaviruses, which
are positive-strand RNA, nonpathogenic and carried by mos-
quitoes. There is no preexisting immunity.
Work at Chiron Corporation has focused on RNA-DNA ma-
nipulation to create a bacterial expression plasmid with a unit
from the Sindbis alphavirus. The envelope proteins have
been replaced with the gene for rat neu. This plasmid then
transfects an antigen-presenting cell or a dendritic cell. The
RNA is transported to the nucleus. The replicase is translated
and starts producing positive-strand RNA. The RNA is again
replicated for the gene of interest, then it is transcribed and
translated into protein inside the cell that was transfected.
This system is intentionally maximized to be tropic for den-
dritic cells.
In normal BALB/c mice vaccinated once with 1 µg of the
circular plasmid ELVIS-neu, there is an antibody response af-
ter 2 weeks. The response is more powerful if the vaccine
dose is increased to 100 µg and administered three times at
2-week intervals. Vaccination with ELVIS-neu results in sup-
pression of tumor growth and metastasis of neu-expression
mammary tumor cells. Experiments in neu-transgenic mice
vaccinated subcutaneously with ELVIS-neu showed that
when 90% of the vaccinated mice were tumor free, 50% of the
controls were already tumor-bearing. When 80% of the con-
trol mice were dead, 70% of the vaccinated mice were still
surviving.
Studies of tetramer-specific CD8+
T cells show a definite in-
duction of antigen-specific cells following ELVIS-neu vacci-
nation. Therapeutic vaccination does not increase survival of
tumor-bearing mice. A more powerful vaccination is re-
quired, such as a prime-and-boost.
There has been a lot of discussion about the difference be-
tween a viral particle replicon (VPR) and a replication-com-
petent virus. A replicon is an infectious virus incapable of
replication, because the VPR has no envelope genes.
Magnitude and Duration
of Antigen-Specific
Immune Response May Affect Clinical Response
H. Kim Lyerly, MD
Duke University Medical Center, Durham, NC
Monitoring of human immune responses has looked into the
function of cellular immunity following some form of immu-
notherapy, via vaccine or cytokines. The latest phase of work
has identified for the first time specific responses against self-
antigens. We are seeing immune responses against specific
peptide antigens that we had not really been able to describe
clearly before. The key question is whether the magnitude,
specificity, quality or durability of a given response is suffi-
cient to lead to a clinical benefit. Much progress has been ma-
de in stimulating an immune response, whether CD8, CD4,
Th1 or Th2. Today, real progress is being made with tools that
allow us to measure these responses quantitatively.
There are a number of camps within immunotherapy. One
camp says: "The kind of immune response is irrelevant. If we
do not see clinical responses, it does not really matter." An-
other school looks at response rate as the gold standard, and
that finding a high-level response rate should translate into a
clinically effective therapeutic.
Statistics from small scale studies with a dozen or two doz-
en patients or animals can be highly misleading, because high
response rates in small studies can shrink to almost a zero re-
sponse rate when the sample size increases. The problem with
doing 60- or 80-patient trials, however, is that they typically
take 2 to 3 years and may cost $2-3 million. Given this prohib-
itive timing and cost, vaccine schemas can be prioritized based
on what can be measured as an indicator of specific immunity.
S18Tools do exist to measure immune responses. ELISPOT
analysis, for example, shows very clear signals in the case of
Epstein-Barr virus (EBV). We use six-well replicates for all
of our ELISPOTs because of noise levels with phytohemag-
glutinin. Intracellular cytokine analysis of blood samples
shows a population of cytokine-secreting cells for EBV pep-
tide. Tetramer analysis show a nice population of EBV-specif-
ic T cells. For patients who are EBV-seropositive and have an
EBV-specific T-cell response, we tend to have concordance
between ELISPOT, intracellular cytokine, cytokine, and
tetramer assays.
A newer technique that is receiving attention is in vitro
stimulation and detection of T-cell responses, which helps de-
termine the degree of specificity. A lot of data have shown
that after 3 weeks of in vitro stimulation and multiple cycles
of dendritic cells, you get 0.5% of the T cells specific for that
antigen. This is often claimed to be "an enormous immune re-
sponse." It is not really clear whether it is "enormous", but it
is specific. The more important issue is being able to detect in
vivo responses, and quantifying them may help us choose the
types and strategies of DC vaccination.
To investigate pre- and post-vaccine responses, we have
looked at CMV seropositive and seronegative patients. This is
a useful system because about half the people in the US are
seropositive and half seronegative. We screened patients for
HLA-A201 and did genotyping. We analyzed the immune re-
sponses of 18 patients by tetramer, ELISPOT, and intracellu-
lar cytokine assays, and everything worked exactly the way
we thought it should. Except for one person, there was always
a detectable response by tetramer, ELISPOT or intracellular
cytokine assays. The CMV response was fairly stable for at
last 10 months.
The key issue is: What is the target immune response that
you’re seeking to quantify and reliably detect? If that re-
sponse is 1 in 10,000, I do not think there are any assays that
can reliably detect that. But if we assume that a clinically rel-
evant or a physiologically reasonable immune response is
akin to something that protects us against viral infections,
then I think it becomes an issue of signal-to-noise ratio. The
ability to reliably detect 1 in 200 or 1 in 500 cells with these
assays is clearly different from the ability to reliably detect 1
in 10,000 or subthreshold 1 in 5000 cells. It is a challenge to
quantify such differences. That is why the answers may be
different depending on what we seek to achieve.
Measuring immune responses and trying to generate im-
mune responses is going to require coordinating a number of
groups. It may be that we need to include a vaccination pha-
se, something to stimulate T-cell expansion with additional
cytokines, and perhaps something to eliminate the immuno-
regulatory elements. I think we are poised to begin to do the-
se studies, but we need to do them in the context of careful
measurements of the immune responses.
MARCH 8, 2002
SESSION VI: Costimulatory Molecules
QUICK LOOK
Chen discussed the 4-1BB (CD137) costimulatory molecule
on T cells. Naive T cells developed this molecule when they
became primed effectors, and later memory T cells. An ago-
nist antibody to CD137 can be used for therapy as well. This
anti-CD137, in combination with HPV-16 E7, can cause re-
gression of established tumors, provided that CD137 is also
upregulated, and specific peptide is also administered to stim-
ulate the T-cell receptor. Kipps discussed the TNF family of
molecules, which includes CD40 ligand (CD154). Adenovi-
rus-CD154-infected CLL B cells have been used in early hu-
man trials to treat B cell lymphoma, with encouraging prelim-
inary results. Schoenberger discussed work on T-cell help for
cytotoxic T lymphocyte (CTL). Alphavirus replicons are Th
independent, while cross-priming, adenoviruses and VSV are
Th dependent. Naive CD8 T cells that require T-cell help but
do not receive it, upon encountering APC with antigen, ex-
pand and acquire cytotoxic function in vivo. However, they
fail to expand on secondary stimulation.
CD137 (4-1BB), More than a Costimulator
Lieping Chen, MD, PhD
The Mayo Clinic, Rochester, MN
Costimulation phenomena are generally interpreted by a two-
signal model, which says that T-cell receptors receive signal
1 from the MHC-tumor antigen complex, while signal 2 is
from a group of costimulatory molecules that specifically
bind to receptors on the T cells.
This two-signal model has been modified now. It is now
known that naive T cells, after receiving signal 1 and signal 2,
move to the next stage, becoming pre-effector cells, also cal-
led primed T cells. Evidence indicates that in this stage, T
cells consist of two functional populations: one may not de-
pend on costimulation to move to effector cell status and be-
come fully active, and the other requires further costimulation
to mature. We also know that most effector cells will die and
very few will become memory T cells. Every step of T-cell
maturation is probably regulated, either positively or nega-
tively, by many molecules. Likewise, there is no "dominant"
costimulator. We used to think the B7/CD28 pathway was
dominant, but now we believe this molecule works only at a
certain stage of T cell differentiation or maturation.
The molecule of interest is CD137, a member of the TNF re-
ceptor superfamily. CD137 was originally found on activated
T cells. Later it was found also on NK cells as well as macro-
phages and dendritic cells. CD137 on T cells can be engaged
by its natural ligand (CD137L). TRAF 1 and TRAF 2 medi-
ate intracellular signals and activating NF-K B.
We reported a couple of years ago that agonistic monoclo-
nal antibodies, administered as two 100-µg injections, were
able to cause regression of mouse mastocytoma and sarcoma.
S19Continuing work in mouse models indicates that some tumors
respond to this therapy but others are refractory.
Responsiveness to the anti-CD137 mAb did not correlate
with the expression of MHC class I. Common to all refracto-
ry tumors is the absence of any primed T cells at the outset,
and therefore no signal 1. Evidence supporting this comes
from further studies in which 11 of 16 refractory tumors re-
gressed when treated with a combined therapy, in which pep-
tide primed some T cells and anti-CD137 further boosted
those T cells. The treatment involved subcutaneous peptide
administered simultaneously with 100 µg of antibody, intra-
venously or intraperitoneally. A second antibody dose was
administered 3 days later. Among mice with C3 tumors
(which are epithelial cell in origin), 40% were cured and 60%
showed slowed tumor growth. In addition, anti-CD137 also
inhibited autoimmunity in several mouse models. It consis-
tently stimulates CD8 in all the models, but its effect on CD4+
T cells varies in different models. In some models, we ob-
serve stimulation of T cell proliferation while in others, CD4+
T cells undergo apoptosis. We have further demonstrated that
CD137 is also expressed on dendritic cells and NK cells and
direct ligation of CD137 promotes activation of NK cells and
dendritic cells. A challenge in moving to clinical trial will be
to develop humanized CD137 monoclonal antibody with ago-
nistic activity.
Immune Gene Therapy Using Recombinant
Proteins of the Tumor Necrosis Factor Family
Thomas J. Kipps, MD, PhD
UCSD Cancer Center, La Jolla, CA
Proteins of the tumor necrosis factor (TNF) family play criti-
cal roles in the regulation of the immune system. Production
of the prototypic molecule, TNF-a, at the site of infection can
cause vascular permeability, diapediesis of immune and in-
flammatory cells, cellular activation, and/or cell clearance,
enhancing the immune response, for example, against an in-
vading microorganism. Other members of the TNF family,
such as Fas ligand or TNF-related apoptosis-inducing ligand
(TRAIL) can function to induce clearance of autoreactive or
defective immunocytes. Some molecules, such as BAFF
(Blyss, TANK) are important for inducing differentiation
and/or survival of lymphocytes, such as B cells. While others,
such as CD154 (CD40 ligand) play critical roles in inducing
activation of antigen-presenting cells, allowing these cells to
induce T cell activation to presented antigens.
Many members have the capacity to function as a cytokine or
a cell-surface ligand. TNF, for example, is released from the
plasma membrane by a matrix metalloproteinases such as
TACE (TNF-alpha-converting enzyme) to become a soluble fac-
tor that, when complexed with two other TNF molecules, forms
a homotrimeric cytokine that potentially can effect signaling in
cells bearing TNF receptors, CD120a or CD120b. However,
TNF also can exist as an active cell-surface ligand that is capa-
ble of interacting with these same receptors on neighboring cells.
In fact, the specific biologic activity of such cell-surface proteins
appears greater than that of the soluble cytokine.
TNF, and other members of the TNF family, can be engi-
neered to enhance its stability and biologic activity at the plas-
ma membrane, thereby avoiding the potential for systemic
toxicity that can be observed upon systemic release of the sol-
uble factor. Gene transfer of such recombinant forms into
cells can effect high local concentration of these membrane-
bound factors. Transduction of genes encoding recombinant
factors that can trigger immune activation, such as CD154,
can generate cellular vaccines of tumor cells and/or effect ac-
tivation of antigen-presenting cells (APC), thereby enhancing
the capacity to generate an immune response against weak tu-
mor-associated antigens.
For example, infection of leukemia cells from patients with
chronic lymphocytic leukemia (CLL), a B cell leukemia, with
replication-defective adenovirus encoding recombinant
CD154 (Ad-CD154) produces changes in transduced and by-
stander CLL cells that render these cells highly proficient at
antigen presentation to autologous T cells. Moreover, the
transduced cells can induce generation of autologous cytotox-
ic T cells capable of killing either transfected or non-trans-
fected CLL cells in vitro.
Because of this, we performed a phase I study in which
groups of patients received successively larger single bolus
infusions of autologous Ad-CD154-infected CLL cells. We
observed very encouraging biologic and clinical effects.
Treated patients generated high levels of Th1-type cytokines,
such as IL-12 and IFN-?. The amount of cytokines produced
appeared to be related to the number of CD4 T cells in the
blood at the time of infusion and not to the dose of adminis-
tered cells. Bystander leukemia cells that did not express the
transgene also were induced to express immune accessory
molecules, such as CD80 or CD95 (Fas) within a day after the
infusion of autologous Ad-CD154-infected leukemia cells.
On average, the patients experienced about a 400% increase
in the absolute numbers of T cells comprising both CD4 and
CD8 T cells. ELISPOT and analyses of the T-cell repertoire
of these T cells indicated that treatment increased significant-
ly the numbers of leukemia-reactive T cells. This was associ-
ated with reductions in lymph node size and leukemia cell
counts. Dramatic reductions in leukemia cell counts also oc-
curred within days after the infusion that preceded the in-
crease in absolute T-cell counts. Recent studies indicate that
such cells might be cleared through innate immune effector
mechanisms and latent sensitivity of bystander leukemia cells
to apoptosis dependent on CD95 (Fas).
No dose-limiting toxicity was observed. Side effects con-
sisted mostly of ‘flu-like symptoms that appeared to correlate
with the levels of endogenous cytokine generated in response
to therapy. Clinical and laboratory toxicities were grade 2 or
less and lasted less than 10 days. Because of this, we initiated
a phase II study in which patients receive repeated biweekly
injections of autologous Ad-CD154-infected CLL cells. This
trial currently is underway. To date we have observed signif-
icant reductions in leukemia cell counts and lymphadenopa-
thy in treated patients. In addition to observing cellular im-
mune activation against autologous leukemia, we also ob-
served direct effects on transduced and bystander CLL cells
that might account for some of the noted clearance of tumor
cells in vivo. These studies suggest that gene transfer of mod-
S20ified members of the TNF family could find useful applica-
tion in the immune gene therapy of neoplastic disease.
T Help for CTL
Stephen P. Schoenberger, PhD
La Jolla Institute for Allergy and Immunology, San Diego, CA
Cytotoxic T lymphocytes (CTLs) are an extraordinarily good
"gun" for cancer because they can recognize the class I pep-
tide. T help has been observed to assist CTL, but the question
remains: What is T help for? We do not know if T help is re-
quired for the expansion of CTL, for their differentiation and
acquisition of function, or for their survival.
We showed a number of years ago that an agonistic anti-
body against anti-CD40 given at or near the time of immuni-
zation would restore the ability to get effector CTL 2 weeks
later. This suggests that the help message is transmitted from
the CD4 cell to the antigen-presenting cell (APC) via
CD40/CD40 ligand interaction.
What happens when the naive CTL (that is, the naive CD8
cell) sees the APC that had the antigen but there was no help
around is unknown. It is possible that if CD40 is required to
mature the APC, prior to the CD40 signal the APC was not
mature.
There are three scenarios:
1. The CTL could see the APC and not respond at all.
2. The non-activated APC might express signal 1 without sig-
nal 2 and somehow directly kill that CTL.
3. The APC may transmit a signal to the naive CTL that re-
sults in a biological response - in this case expansion -
but because of the way that we look for effector CTL, we
do not see it.
We decided to look earlier than the conventional 2-week pe-
riod after immunization and directly at the primary CTL with-
out an in vitro restimulation. We took CD4-depleted or class
II knockout mice, immunized them, and on days 7, 14, 21, 28,
etc., did intracellular cytokine staining or ELISPOT for IFN-
? and TNF, and a primary or secondary cytotoxicity assay. Af-
ter 14 days, we saw a nice population of IFN-?-producing
CD8 cells when the immune, but not naive animal, was stim-
ulated with the right peptide.
The somewhat surprising result is that in the absence of
help, helper-dependent CTL do actually arise. They are pre-
sent at 30% to 60% of the wild-type levels on days 7, 14, and
21. They seem to make a little less IFN-? compared to the
ones primed with help. ELISPOT analysis confirms that
IFN-?-producing cells are present in the absence of help.
To determine whether these CD8+
cells are cytotoxic as pri-
mary effectors, we used an in vivo cytotoxicity assay to look
at primary cytotoxic effector functions within the animal. We
found that the CTL primed without help are cytotoxic in vivo
on days 7, 14, or 28. By day 28, however, there is less killing
in the wild-type, and the primary effector seems to be gone in
the helper-deficient animal.
With respect to secondary restimulation, we found that on
day 7 these cells killed in the secondary one in vitro, but by
day 14 the cells did not kill anymore. This was a head-scratch-
er because the primary in vitro CTL assay showed that there
was killing on days 7 and 14, but the in vitro restimulation
said they could only kill on day 7. To clarify this seeming in-
congruity, we applied intracellular cytokine staining before
and after restimulation, direct ex vivo, 6 days after restimula-
tion on a class I-positive/class II-negative. We saw no costim-
ulatory molecule, and we can find no added cytokines. The
CTL that were primed in the presence of help expand beauti-
fully - 15-fold to 22-fold. Those primed in the absence of help
actually decrease; they do not expand at all.
We found that restimulation on day 7 results in CTL in the
presence of B7-1 or B7-2, but there is absolutely nothing in
the absence of B7-1 and B7-2. In the presence of help, these
basically all seem equivalent. If you restimulate - keeping in
mind that the cells primed without help are not going to ex-
pand upon secondary encounter - you still have enough cells
around to give you a somewhat positive result in the absence
of B7-1 and B7-2.
Our work to date shows that:
• Helper-dependent CTL will grow and proliferate and ac-
quire effector function, but they cannot expand upon sec-
ondary encounter with antigen.
• Both the helped and the help-less phenotype depend on B7-
1/B7-2, so the cause cannot be simply the presence or ab-
sence of B7-1 or B7-2. This is something that would not be
predicted by the most stringent interpretation of the two-
signal model.
• Agonistic antibodies against CD28 or 4-1BB given during
priming can restore the capacity for secondary expansion.
• There are alternative ligands for B7-1 and B7-2 expressed
on T cells, on CTL at least, that are distinct from CD28 and
CTLA-4. These transmit a positive costimulatory signal
that our data suggest can confer survival during the initial
round of division. This behaves much more like what
Kevin Lafferty would say is signal 2. We have more data
showing that that effect of the alternative ligand is seen on-
ly for a short period, and then CD28 signals take over. The-
re is more than one signal 2, but that is becoming increas-
ingly obvious.
S21MARCH 9, 2002
SESSION VII: Tumor Defense Against
Immune Response
QUICK LOOK
Miele discussed the Notch family of genes, found from Dro-
sophila upwards through the phyla. In normal cells, Notch ge-
nes control the timing of differentiation and provide tempo-
rary survival signal. In cancer cells, degenerate Notch genes
provide a permanent survival signal. Other genes such as ras
collaborate, by cleaving Notch and retaining it in the cell.
Notch-1 downregulation has an antineoplastic effect in vivo.
Notch inhibitors include antisense, RNA interference, and re-
combinant decoy protein. Dutton discussed CD4 memory
cells, which can persist without further restimulation. Effec-
tor cells become memory cells spontaneously in class II
knockout mice. CD25 (expression) decreases to normal. On
the other hand, CCR7 is lost as the cells divide and become
effectors. Proliferation is not required to generate memory
from effector cells. These memory cells persist in the lym-
phoid tissue, with very few in the periphery. Le Poole de-
scribed downregulation of tumor antigens by IFN-? in mela-
noma and murine colon carcinoma cells. IFN-? decreases the
expression of MART-1, gp-100 and TRP-1. T-cell recognition
is decreased after IFN-? treatment.
Apoptosis and Antiapoptosis in Multimodality
Cancer Treatment
Lucio Miele, MD, PhD
University of Illinois at Chicago
The highly changeable nature of cancer is a leading challenge
in treatment of patients. There is genetic heterogeneity among
histologically similar tumors, and even within individual tu-
mors within individual patients. Some single-agent treat-
ments do work. Gleevac is the most recent of them - but you
also get resistance, and recurrence.
We have chosen to work on apoptosis of tumor cells be-
cause it is a common mechanism of many treatment modali-
ties. Several families of chemotherapy agents - DNA-damag-
ing agents, microtubule-targeting agents, topoisomerase in-
hibitors, radiation therapy, and also some growth factor inhi-
bition - will produce apoptosis. CTLs do kill using this and
also perforin- and granzyme-independent and Fas-indepen-
dent mechanisms.
Apoptosis can be affected through trimeric ligands of the
TNF TRAIL and Fas family, which form a death-inducing
cell complex at the surface of the cell. This complex results in
the activation of upstream caspases, which execute cell kill-
ing. The pathway is modulated by decoy receptors, which of-
ten operate within the transforming cells.
Apoptosis can also be triggered by mitochondria through
the release of cytochrome-C, which forms an apoptotic zone
together with APAF-2 and Pro-caspase 9, resulting in the ac-
tivation of caspase 9. Mitochondria can also trigger apoptosis
through otherwise less-understood apoptosis-inducing fac-
tors. This is modulated by antiapoptotic BH family proteins,
such as Bcl-x and Bcl-2.
The DNA damage-control pathway is linked both to the mi-
tochondrial and to the membrane apoptosis pathways through
p53 and p73. This explains why a number of tumor cells are
more sensitive to apoptosis than normal cells, even though
they may overexpress some antiapoptotic proteins.
An area of investigation in the control and characterization
of apoptosis is Notch molecules, originally found in Drosoph-
ila. Notch molecules are transmembrane, ligand-activated
signaling modifiers. Notch signaling participates in many
cell-fate decisions during development and postnatal life.
Notch signaling is a potentially valid target for apoptosis
modulation in cancer for several reasons.
In mammals, there are four Notches, and the constitutively
active forms of each are transforming. This constitutively ac-
tive Notch is present in a small number of human tumors, and
in 10% of T-cell ALL, in which it causes disease. It has been
shown that if you transduce this into bone marrow precursor
cells and put them back into irradiated mice, you do get a T-
ALL. More interestingly, there are wild-type Notch receptors
and ligands that are overexpressed in a number of solid tu-
mors, including tumors that are of interest to tumor immunol-
ogists, such as cervical cancer and melanoma and some hema-
topoietic malignancies.
The ligands are expressed on the surface of neighboring
cells, so these receptors work mostly in the context of cell-cell
contact. When the ligand interacts with Notch, it results in
cleavage of Notch, releasing from the plasma membrane a
Notch intracellular domain. After ligand engagement, the
Notch is cleaved and releases a domain I called NIC (Notch
intracellular), which moves into the nucleus, where it inter-
acts with a transcriptional repressor, CBF-1.
In many normal cells, Notch signaling assists in control of
the timing of differentiation. It provides a temporary survival
signal within the context of differentiation. This seems to be
the case in the thymus, and in human skin. Evidence indicates
that Notch in T cells seems to assist CD8 development. In can-
cer cells, however, excessive activation of Notch prevents dif-
ferentiation and aids tumor survival.
In murine studies in which we used a Notch-1 antisense
construct, mice that had been infected with the vector alone
all developed tumors. The tumor in two of them regressed and
six were dead by the end of the experiment, a matter of weeks.
Among mice that received cells transduced with antisense
Notch, only two developed palpable tumors. By the end of the
experiment all mice were tumor-free.
Conclusions to date:
• Notch signaling is deregulated in many malignancies, in-
cluding some that are good targets for tumor vaccines.
• Inhibition of Notch signaling causes apoptosis and does
have antineoplastic activity in vitro and in vivo.
• This strategy indeed enhances the effectiveness of immuno-
therapy, and its opposite may enhance immunotherapy in
different ways.
S22The Generation and Persistence
of CD4 T Cell Memory
Dick Dutton, PhD
Trudeau Institute, Saranac Lake, NY
Studies over the last few years have indicated that once naive
CD4 T cells differentiate to active effector cells, the popula-
tion can give rise to memory cells. These memory cells share
the cytokine polarization of the effectors from which they we-
re generated. This concept of a direct pathway from effectors
to memory has been further strengthened by our studies
showing that after the initial stages of naive CD4 T cell acti-
vation, neither exposure to antigen nor to a major histocom-
patibility complex is needed for the transition of effector cells
to memory cells or for their persistence in vivo.
Effectors polarized in vitro, and put into recipient mice will
become polarized memory cells months later. So if you want
Th1 memory cells you need to make Th1 effectors, and for
Th2 you need to make Th2 effectors.
We determined the mechanism that enabled the recipient to
maintain this memory population. We made in vitro effectors,
and adoptively transferred them to hosts that have no possibil-
ity of antigen presentation. Using a Thy1 marker, we looked at
the recovery of these cells at various times in the two different
recipients. They survived well in the absence of any antigen or
class II stimulation, which means that a population of memo-
ry cells can persist without any restimulation by antigen.
Our studies also shed some light on how a cell gets from be-
ing an effector cell to being a memory cell. It turns out that to
get a memory cell, you leave an effector cell alone. We found
that effector cells generated by in vitro culture divide about
every 6 to 8 h and express CD25 and CD44. When injected in-
to animals, the effectors divide furiously, but they soon stop
dividing and then divide hardly at all in the next up to 70 days.
When we take away stimulation by reculturing cells in medi-
um alone for 2-3 days, the "rested" effectors divide a little,
but considerably less than the non-rested effectors.
We conclude that you go from effector to memory cell just
by becoming a resting cell and downregulating the many ac-
tivities of effector cells. This occurs when you take away all
possible signals. Thus, it is a default pathway. But the ques-
tion remains as to whether there are other routes to memory
cells that do not go all the way through the effector phase. A
number of people have tried to devise ways to show that
memory cells express genes previously expressed by effec-
tors. However, these are population studies which do not re-
flect individual cell potential, and it is still possible that there
are several earlier time points in which you can go back to a
resting state and persist as a memory cell.
Animal studies indicate that by day 4 following viral infec-
tion, there are already large numbers of responding cells in the
draining lymph nodes, and these cells expand rapidly up to
day 6-8. We know from other studies that the cells become
competent to leave the lymph nodes and migrate to the lung,
where they act as effectors and help clear virus.
After flu infection, although the dendritic cells presenting an-
tigens migrate into the draining lymph nodes, they go beyond
since a response takes place in both the spleen and the non-
draining lymph nodes. There is a substantial response systemi-
cally in sites way away from the source of the antigen. Only
those cells that have differentiated most extensively have gone
into the site of the infection. When the response is over and vi-
rus is cleared, a proportion of the whole spectrum of effector
cells become memory and can be found 50 days after the flu
infection. At the sites of the original infection, there are still
quite a number of cells but most are now in secondary lym-
phoid sites especially the spleen. In the lymphoid organs, there
are a surprising number of cells that still have gone through rel-
atively few divisions and persist for a great period of time.
It therefore appears that on the pathway toward effectors,
before they have gone to the full highly differentiated state,
cells can go back to resting cells that you can find in the ani-
mal 50 days after their exposure to antigen.
Downregulation of Tumor Antigens by IFN-?
Caroline Le Poole, PhD
Loyola University, Chicago, IL
An immune escape mechanism has been identified that appears
to be mediated by IFN-?, as revealed by studies of M14 human
melanoma cells. These cells can have highly variable expres-
sion of different target antigens. That makes them an ideal cell
line in which to study factors that could affect expression or the
level of expression of target antigens by tumor cells.
Because melanoma tumors are frequently heavily infiltrat-
ed by T cells, our study focused on the effects of IFN-?. This
cytokine appears to stimulate down-modulation of both TRP-
1 and more so of MART-1 as shown by immunostaining. Af-
ter prolonged exposure to IFN-?, we see less and less of the
MART-1-encoding transcript. Even for gp100, transcript lev-
els eventually go down in the presence of IFN-?. Downregu-
lation of MART-1 was observed at a range of IFN-? concen-
trations from 1000 IU/ml down to 62.5 IU/ml. MART-1 ex-
pression bounced back up at IFN-? concentrations of about
30 IU/ml.
If we removed IFN-? from the culture medium and let the
cells recover for about 3 days, the expression came back up.
Thus IFN-? can down-modulate melanosomal target antigens,
but continuous presence of the cytokine is required to do so.
So this is not something that permanently shuts off the gene.
IFN-? can also upregulate expression of MHC molecules at
the cell surface, both HLA class I and class II. So that leads us
to the next question: Is this going to affect the recognition by
T cells in a positive way or in a negative way? We reacted a
series of patient-derived melanoma cell cultures with the
HLA-matched A42 MART-1-restricted T cell clone. We not-
ed a markedly reduced recognition of the melanoma cells
once they had been pretreated with IFN-?. If the peptide
AAGIGILTV is added to the culture it restores the recognition
of the melanoma cells. This shows that we are not looking at
other mechanisms also induced by IFN-?, but that reduced
T cell reactivity results from the loss of expression of the tar-
get antigen.
To show that there is IFN-? expressed within the tumor en-
vironment, we used quantitative RT-PCR. For primary mela-
S23nomas there is not much change compared to normal skin, ex-
cept for one that had about a tenfold increase in the number of
transcripts for IFN-? in it. However, the metastasis had con-
sistently about a 200-fold increase in the number of tran-
scripts for IFN-?. This indicates an abundance of IFN-? with-
in that tumor environment.
Conclusions so far indicate:
• Antigen loss is an immune escape mechanism that plays a
role particularly in tumors where the antigens are not essen-
tial for viability of the tumor cells.
• The microenvironment that these tumor cells find them-
selves in actually contains factors that will alter the physi-
ology of the cells and downregulate the levels of target an-
tigen expression.
• IFN-? is an important candidate compound to suppress ex-
pression of melanosomal target antigens.
• Downregulation occurs at the level of transcription.
The relevance of this mechanism is supported by the elevat-
ed expression of IFN-? within the tumor environment as well
as by the reduced levels of MART-1 expression observed in
tumor cells that are in close proximity to the infiltrating T
cells.
MARCH 9, 2002
Closing Comments
Allan Goldstein, PhD
Albert B. Sabin Vaccine Institute
On behalf of the Sabin Vaccine Institute, we are all grateful
that you have been able to join us and to freely exchange in-
formation. It has been a great meeting.
Over the last several years that we have been gathering here,
more than 100 thought leaders in cancer vaccines have con-
tributed to these meetings. What has occurred over the last
several years in this area is extraordinary. Investigators have
been using the breakthroughs in genetics and proteomics to
enhance the pace of discovery in this area.
The Sabin Vaccine Institute is at the point of asking the key
question: Is it possible to really focus efforts to develop a can-
cer vaccine initiative? I believe so. I believe it is time to
move your work up to a higher priority and initiate an inter-
national vaccine initiative, with the specific goal of shorten-
ing the time needed to translate scientific discoveries into
meaningful treatments for the prevention and cure of cancer.
In order to do that, we have been establishing a number of
specific goals and targets. We need your help to make this
happen. Working with Ed Neiss, H.R. Shepherd and many
others, we have identified five objectives to present to the
Sabin board of trustees:
• Establish a President’s Council that would include interna-
tional membership by chief executive officers of pharma-
ceutical and biotech companies currently developing can-
cer vaccines, adjuvants, and delivery systems.
• Organize a cancer vaccine scientific advisory board, com-
prising leading scientists and clinicians in academe, gov-
ernment, and industry to advise the Sabin Vaccine Institute
of the scientific, medical, and regulatory issues needed to
accelerate and shorten the period of time from the laborato-
ry bench to the bedside to approval.
• Develop a speakers’ bureau to help inform the public of the
progress being made in the cancer vaccine area.
• Develop a series of highly focused workshops and think
tanks, such as the one here today, that will define the scien-
tific issues and problems yet to be resolved, ranging from
assay validation to merit combinations of different cancer
vaccines and adjuvants.
• Establish a research fund to encourage and support scientif-
ic studies designed specifically to support innovative, high-
risk projects that would have difficulty being supported by
traditional government funding agencies. This fund would
also support projects that would use combinations of differ-
ent cancer vaccines - as an example, an adjuvant - that are
scientifically sound but would also have difficulty being
funded.
To fund these initiatives, we have ambitious financial goals:
we want to raise $5 million in the first year, $10 million on the
second year, and $15 million in the third.
To do this, we need your support to guide us to the best con-
tacts in your organizations. Obviously, we will seek support
S24from the pharmaceutical and biotech industries. But there
are other, less-obvious contributors. The insurance industry,
for example, has a vested interest in the success of what you
all are doing. Any contacts you have, as well as your
thoughts on the priority issues, would be useful for us to
have.
I would like to thank Martin Kast and Malcolm Mitchell
for their extraordinary work in preparing this meeting. They
spent the better part of the past year working on this. I would
also like to acknowledge someone who is not here, that is
H.R. Shepherd. Shep is normally sitting right here, and for
him not to be here has been very difficult for him. He has had
a number of health issues this year. But he is the heart and soul
of not only the Sabin Vaccine Institute but of this particular
meeting and this initiative. We hope that next year he is up
here doing his thing.
S25Abstracts
Abstracts: Session I
DNA vaccines and immunocytokines for cancer therapy
R.A. Reisfeld1
, R. Xiang1
, U. Pertl
1
, A.G. Niethammer1
,
J.M. Ruehlmann1
, Y. Ba1
, and S.D. Gillies2
1
The Scripps Research Institute;
2
Lexigen Pharmaceuticals Corporation
We induced tumor protective immunity against colon carcinoma
with an orally administered DNA vaccine based on oncofetal carci-
noembryonic self-antigen (CEA) that broke peripheral T-cell toler-
ance to CEA in CEA-transgenic C57BL/6 J mice. This oral vaccine,
delivered intralymphatically to Peyer’s patches of the gut by an at-
tenuated strain of Salmonella typhimurium, induced tumor-protec-
tive immunity mediated by class I MHC antigen-restricted cytotox-
ic CD8+
T cells. Activation of these CTLs was indicated by increased
secretion of proinflammatory cytokines IFN-? and IL-12 and by spe-
cific and complete tumor rejection in 50% of vaccinated CEA-trans-
genic mice after a lethal challenge with murine MC38 colon carci-
noma cells. These tumor cells had been doubly transfected with CEA
and the human pan-epithelial cell adhesion molecules Ep-
CAM/KSA and consequently served as a docking site for an im-
munocytokine consisting of IL-2 fused with a recombinant antibody
that specifically recognizing KSA. Importantly, the efficacy of the
tumor-protective immune response was markedly increased by
boosts with this immunocytokine, resulting in complete tumor rejec-
tion in 75% of experimental animals and increased expression of co-
stimulatory molecules B7.1 and B7.2 and intracellular adhesion
molecule I on antigen-presenting dendritic cells. Consequently, the
release of the inflammatory cytokines IFN-?, IL-12 and granulocyte-
monocyte colony-stimulating factor from activated T cells of suc-
cessfully vaccinated CEA-transgenic mice was intensified. In-
creased T-cell activation mediated by boosts with the immunocyto-
kine after tumor cell challenge was further indicated by expanded
expression of T-cell activation markers CD25, CD28, CD69 and
LFA-1.
These data suggest that immunologic mechanisms are responsible
for the effective immunization with the DNA vaccine. Thus, the
marked increase in expression of CD28 on T cells and of B7.1 and
B7.2 on dendritic cells is important since the activation of naive T
cells requires two independent signals: (1) binding of the peptide-
MHC complex by the T-cell receptor, producing a signal to T cells
indicates antigen recognition; and (2) ligation of CD28 with B7.1 or
B7.2, producing a second signal and thereby initiating T-cell re-
sponses and production of armed effector T cells. Taken together,
our findings suggest that the application of such CEA-based DNA
vaccines may be useful in combination therapies directed against hu-
man carcinomas expressing CEA self-antigens.
Recombinant anti-carcinoma vaccines
Jeffrey Schlom
National Cancer Institute/NIH
We have developed several approaches to enhance the immunoge-
nicity of tumor-associated antigens (TAAs), and have developed an-
imal models to evaluate these strategies. These strategies include (a)
delivery of the TAA via recombinant poxviral vectors, (b) diversi-
fied prime-and-boost vaccination regimens, (c) the use of multiple T
cell costimulatory molecules, (d) design of agonist TAA epitopes,
and (e) the use of cytokines as biological adjuvants. Studies have
demonstrated that two types of recombinant poxvirus vectors are
very effective in enhancing antigen-specific T-cell responses: the
replication-competent vaccinia (rV) and the replication-defective
avipox viruses. A small randomized phase II trial was conducted in
which patients with advanced CEA-expressing carcinomas, who had
failed conventional therapies, received either rV-CEA (V) prime
vaccination followed by three monthly avipox-CEA (A) booster
vaccinations (i.e., the VAAA regimen), or the reciprocal AAAV reg-
imen. Patients vaccinated with the VAAA regimen demonstrated the
generation of significantly higher levels of CEA-specific T cells and
longer survival than patients randomized to the AAAV regimen. We
have demonstrated that the insertion of the transgenes for a triad of
costimulatory molecules (B7-1, ICAM-1 and LFA-3, designated
TRICOM) into these vectors, along with the TAA transgene, could
markedly enhance T-cell responses to the TAA, and the elimination
of well-established tumors in animal models. Collaborative clinical
trials in patients with advanced carcinoma have now been initiated
with rV-CEA/TRICOM and rF-CEA/TRICOM vectors. These vec-
tors also contain a CEA agonist epitope described previously.
Immunization against cancers with ubiquitous antigens:
MUC1 and MG50
Malcolm S. Mitchell, Daniel Compagno, and Alexey Glazyrin
Karmanos Cancer Institute, Wayne State University School of Medicine
Tumor-associated antigens are generally altered-self antigens, usual-
ly differentiation antigens, rather than unique tumor-specific mole-
cules. While in the past a tumor was thought to have antigens that
differed from those of another tumor, it is now clear that several an-
tigens are shared among tumors from different individuals, some
among tumors of different histotypes.
MUC1 is a ubiquitous mucin antigen of adenocarcinomas, which
is a potentially useful immunogen for those cancers but is of weak
immunogenicity. Although the response to MUC1 may perhaps in-
volve an atypical, non-MHC-restricted CTL response, MHC-re-
stricted epitopes have also been identified. Three of these are SAP-
DTRPAP, APDTRPAPG and STAPPAHG, all HLA-A2 restricted,
with the last restricted by HLA-A11 too. We have previously report-
ed that linkage of a signal sequence from adenovirus or IFN-? to the
nonamer can improve the generation of CTL against a MART-1
(melanoma) epitope. The fusion peptide is conducted into the endo-
plasmic reticulum of dendritic cells where it associates with nascent
HLA class I molecules. We inserted fusion peptides comprising a
consensus signal sequence and each of the three HLA-A2-restricted
MUC1 nonamers into autologous dendritic cells. These were used to
generate CTL from naive CD8+
T cells, within a population of pe-
ripheral blood mononuclear cells. CD4+
T cells and IL-2 were essen-
tial for this immunization, and the appearance of activated
CD4+
/CD25+
cells was associated with the development of CTL. Fu-
sion peptides obviated the need for adding a CD4+
T cell-stimulating
peptide such as PADRE. However, PADRE was helpful in increas-
ing immunity when used with nonamer alone. Specific CTL against
MUC1 decreased sharply after two or three restimulations, due to ac-
tivation-induced apoptosis, which could be avoided by decreasing
the concentration of peptide to 5 µg/ml.
MG50 is a novel antigen found in a variety of cancers, which we
isolated from melanoma and sequenced. MG50 is an 8.1 kb gene en-
coding a 1496 amino acid protein. MG50 has been found in most
melanomas, but in all breast and pancreatic cancers, as well as in 4
of 4 glioblastomas, 4 of 6 ovarian carcinomas, and 4 of 12 colon car-
cinomas. There has been little or no staining of normal tissue coun-
terparts. MG50 appears to be an IL-1 receptor antagonist, of which
several isoforms have been demonstrated previously. We have iden-
tified six HLA-A2.1-restricted epitopes that can immunize CTL to
recognize them in vitro. CTL directed against these epitopes also re-
act with HLA-A2.1+
melanoma cell lines, showing that the epitopes
are naturally expressed. Polyclonal and monoclonal antibodies to
MG50 react with fixed and frozen specimens of human tissues, su-
pernatant fluids from growing melanoma, breast and pancreatic can-
cer cell lines and mouse breast cancers. With these antibodies, by
Western immunoblotting at least two isoforms of MG50 in tumors,
170 kDa and 80 kDa, are identified. Since MG50, in contrast to most
other tumor-associated antigens, is not altered self, but rather repre-
sents an apparently "aberrant" molecule, it is very possible that the
barriers to successful immunization with this molecule may not be
as significant as with MUC1, telomerase or similar antigens with a
wide distribution. Targeting MG50 may also alter the function of the
tumor cell that it resides in, because its function may be to protect the
tumor cell from immunological destruction.
S26Abstracts: Session II
Identification and optimization of TCR-specific ligands
for vaccine design from combinatorial peptide libraries
D.B. Wilson1
, K Schroder1
, K Osawa1
, C. Boggiano1
,
D.H. Wilson1
, and M. Mitchell
1
1
Torrey Pines Institute for Molecular Studies;
2
Karmanos Cancer Institute,
Wayne State University School of Medicine
T-cell clones and lines of clinically relevant specificity can be used
to scan combinatorial peptide libraries to identify large panels of
peptide ligands that best stimulate immune function by these clones
and lines. These ligand sequences are optimized with respect to mo-
tifs most appropriate for MHC binding and interaction with TCR.
They are strong immunogens, but most importantly, they provoke
strong T-cell-mediated immune responses against the native ligand
(when known) used to generate the T-cell clone or line. This presen-
tation will discuss how these libraries were used to define a panel of
peptide ligands specific for two human T-cell clones reactive to pep-
tide 209, a fragment of the melanoma-associated antigen, gp100, and
how these ligands are effective in ex vivo priming protocols in pro-
voking CTL reactivity against gp100+
melanoma cell lines. Finally,
we compare specificity degeneracy in CD4 and CD8 T cells, an in-
teresting problem of how specificity and degeneracy can coexist in
the CD4 TCR repertoire.
Probing degeneracy of recognition by epitope-specific cytotoxic
T cells (CTLs) for superagonists for vaccine design
J. Kan-Mitchell
1
, R. Hegde1
, K. Schaubert
1
, B. Bisikirska1
,
S.E. Blondelle2
, and D.B. Wilson2
1
Karmanos Cancer Institute, Wayne State University School of Medicine;
2
Torrey Pines Institute for Molecular Studies
We have probed the degeneracy of a de novo primed epitope-specif-
ic CTL line with a large synthetic combinatorial nonapeptide library
consisting of about 3 trillion distinct peptides for the purpose of
identifying mimetopes (mimics) that may be useful for the design of
an effective vaccine. The HLA A*0201-restricted CTLs specific to
the HIV Gag p1777-85 epitope were primed and expanded from CD8+
naive T cells from a healthy, seronegative donor (PW1, index line).
Of 75 potential candidate peptides deduced, synthesized and tested
for their ability to be identified and lysed by the index CTLs as well
as CTLs from two other seronegative A*0201+
donors (B.H. and
J.L.), 20 were recognized by PW1, and 12 of these were more potent
than SL9, 5 of which differed by five or six residues. More impor-
tantly, CTLs from B.H. recognized six common superagonist mim-
ics, four of which differed in five positions with two non-conserva-
tive substitutions. These mimics apparently bind more stably to
HLA-A*0201 than the native epitope. Alternative peptide ligands
with multiple changes of sequence that include non-conservative
substitutions have not been readily predicted by any other method.
Two mimics (no. 11 and no. 41) that have been studied to date
were found to be immunogenic ex vivo, immunizing CTLs crossre-
active to SL9 from P.W., B.H. and healthy donors R.G. and J.D. Of
interest, J.L. SL9-specific CTLs did not recognize the six common
mimics predicted by PW1. Moreover, neither no. 11 nor no. 41 in-
duced CTLs from J.L. PBMC.
These results provide the proof-of-principle that probing TCR de-
generacy of de novo primed CTLs with peptide libraries will identi-
fy superagonists potentially useful as vaccines for infections or can-
cers. They also demonstrated at least two patterns of overlap by SL9-
specific CTLs among the subjects under study. Therefore, for the
purpose of vaccine design, thorough analysis of the overlap in de-
generacy may be necessary to select optimal crossreactive peptide
analogs.
Prostate cancer antigens and vaccines
Michael L. Salgaller1
, Abdel-Aziz Elgamal
1
, Marnix Bosch1
,
Anne Lodge1
, Gopi Shankar1
, Alton Boynton1
,
Arie Belldegrun2
, Christopher Logothetis3
,
and Christos Papandreou3
1
Northwest Biotherapeutics, Inc.;
2
UCLA Medical Center;
3
University of
Texas M.D. Anderson Cancer Center
The clinical development of prostate cancer vaccines presents sever-
al challenges. Reagents are more limited and difficult to obtain as
compared with other tumor types. The advanced age of the patient
population presents the researcher with subjects having diminished
immune systems and who are often less willing to undergo proce-
dures for research purposes. Consequently, the majority of research
has involved those cancers for which tumor and immune cells are
readily available. Despite these hurdles, new and novel approaches
are improving the poor overall survival rates through the develop-
ment of antigen-based treatment options. These efforts are particu-
larly important in the realm of hormone-refractory prostate cancer
(HRPC), since no therapy exists with significant clinical impact.
This is a major issue for the 36,000 men who will die from the
disease annually, despite transient responses to secondary treatment
such as hormone ablation therapy.
During the past few years, candidate target antigens for experi-
mental vaccines have been identified in several laboratories. These
include oncogenes, overexpressed proteins, and carbohydrates.
Three of the furthest in clinical development are well-established
clinical markers of prostate cancer: prostate-specific membrane an-
tigen (PSMA), prostate-specific antigen (PSA), and prostatic acid
phosphatase (PAP). Following conclusive preclinical evidence indi-
cating that the human body responds immunologically to prostate
antigens, clinical trials have been underway for many years with
PSMA, PSA, and PAP as targets.
We investigated the capacity of a vaccine composed of autologous
dendritic cells (DC), pulsed ex vivo with recombinant PSMA (rPS-
MA), to safely generate clinically meaningful antitumor immune re-
sponses in HRPC patients. In 2000 and 2001, 32 patients with meta-
static or non-metastatic HRPC were enrolled in a phase I/II clinical
trial. Their peripheral blood mononuclear cells were isolated by leu-
kapheresis, matured to DC by in vitro culture with maturation fac-
tors (GM-CSF, IL-4, and inactivated BCG) for up to 7 days, fol-
lowed by rPSMA loading and harvesting of the vaccine. Patients re-
ceived four intradermal treatments of 5, 10, or 20-million rPSMA-
loaded mature DC at monthly intervals, followed by up to a total of
6 months of observation. Measurement of serum anti-PSMA anti-
bodies, PSMA-stimulated lymphocyte proliferation, and delayed-
type hypersensitivity (DTH) skin testing were carried out before,
during, and after vaccination. Clinical responses were assessed by
CT/bone scans and hematochemical laboratory tests, including PSA
levels.
More than 140 total vaccine injections were well tolerated; no clin-
ical signs of autoimmunity or serious adverse events were observed.
Overall, 54% of patients achieved stability of their disease at
=6 months follow-up, as assessed by radiographic criteria, and 83%
of patients had a PSMA-specific immune response, 92% of patients
with stable disease had a PSMA-specific immune response, and 46%
of patients had a decrease in PSA velocity. Compared to baseline,
93% of 27 evaluable patients converted to DTH-positive against the
BCG component of the vaccine. Due to these promising initial find-
ings we have initiated a double-blind, placebo-controlled phase III
clinical trial
(© 2002 Northwest Biotherapeutics, Inc. All rights reserved).
Human ErbB-2 (Her-2/neu) vaccines
Wei-Zen Wei
Karmanos Cancer Institute, Wayne State University School of Medicine
A panel of DNA vaccines has been generated to encode recombinant
human ErbB-2 (E2) full-length protein or fragments that are free of
S27tyrosine kinase activity and are directed to specific subcellular com-
partments to activate selected immune responses. In ErbB-2 K?A
(E2A), the ATP-binding lysine (K) residue 753 was substituted with
alanine (A) to eliminate kinase activity. SecE2 is a secreted protein
containing the N-terminus 505 amino acids. E2TM contains the ex-
tracellular and transmembrane domain of ErbB-2. E2-LAMP is
E2TM fused to the cytoplasmic tail of lysosomal associated mem-
brane protein (LAMP-1). In cyt ErbB-2 (cytE2), the ER signal se-
quence was deleted so that the full length ErbB-2 is directed to the
cytoplasm. These vaccines demonstrated striking antitumor activity
in BALB/c or C57BL/6 mice. CD4 T cells were the primary antitu-
mor effectors induced by E2/E2A DNA and CD8 T cells were the ef-
fectors induced by cytE2 DNA. Vaccine-induced antibody is dis-
pensable in tumor rejection. Epitope spreading was induced via CD4
T cells during tumor rejection and a second challenge with ErbB-2-
tumor was rejected. The same DNA vaccines were weak immuno-
gens in human ErbB-2 transgenic (Her-2 Tg) mice which express
ErbB-2 regulated by whey acidic protein promoter. Immunization of
Her-2 Tg mice with allogeneic cells expressing human ErbB-2 in-
duced a significant anti-ErbB-2 response, indicating that reactivity
to foreign antigens engages immune response to self or weak anti-
gens. Therefore, ErbB-2 DNA induced antitumor immunity in nor-
mal mice and exogenous antigens copresented with ErbB-2 are re-
quired for effective vaccination in ErbB-2 transgenic mice.
A mechanism by which helper peptide analogues can increase
CTL responses and skew help toward Th1 cytokines
Jay A. Berzofsky
National Cancer Institute/NIH
Using a helper T-cell epitope from the HIV-1 envelope protein as a
model antigen, we sought to determine the effect of amino acid se-
quence modification to improve binding affinity for the class II
MHC molecule ("epitope enhancement") on the ability of this pep-
tide to provide help for a covalently linked CTL epitope. The higher
affinity analogue peptide induced both a higher CTL response and
greater protection against a recombinant vaccinia virus expressing
the antigen HIV-1 gp160. Moreover, it skewed the T-helper response
toward Th1 cytokine production, with increased IFN-?, and de-
creased IL-4 and IL-10. Knowing that one mechanism of CD4+
T
cell help for a CD8+
CTL response is via activation or conditioning
of antigen-presenting cells such as dendritic cells (DC), we exam-
ined the mechanism of this effect of epitope enhancement using a
two-step culture system. Specific helper T cells were incubated with
purified DCs in the presence of the wild-type or enhanced helper
peptide, and then separated. The helper T cells from this first culture
were studied for level of CD40L expression. The DCs isolated from
the first culture not only were studied for levels of costimulatory
molecules and for IL-12 production, but also were placed in a sec-
ond culture with primed purified C8+
CTL precursors, to stimulate a
CTL response in the presence of the minimal CTL epitope, without
any further source of help. We found that the higher affinity en-
hanced helper peptide induced more CD40L on the helper T cells,
and in turn induced more costimulatory molecule expression, and es-
pecially more IL-12 production, by the DCs. Further, the DCs isolat-
ed from the first culture with the higher affinity peptide were more
effective at stimulating the induction of CTL. This correlation repre-
sented cause and effect, because the increased induction of CTL in
the second culture could be inhibited by blockade of CD40L in the
first culture. We conclude that the higher affinity peptide results in a
reciprocal interaction between the helper T cell and the DC, induc-
ing more CD40L on the DC, which in turn stimulates more IL-12
production by the DC, making it a more polarizing DC, which in turn
skews the T helper cells toward Th1 phenotype. The greater induc-
tion of B7-1 and B7-2 and IL-12 production by the DCs also makes
them more effective at stimulating CTL activity in the absence of ad-
ditional help. Because both CD4+
Th1 cells and CD8+
CTL may be
important for clearance of both viruses and tumors, this type of epi-
tope enhancement of CD4+
help may be valuable in the design of
second-generation vaccines for many such diseases.
Abstracts: Session III
Activating tumor-specific CD4+
T cells:
tumor cells as antigen-presenting cells and vaccines
S. Ostrand-Rosenberg
University of Maryland
CD4+
T helper lymphocytes are an essential component for effective
responses against many tumors. To facilitate generation of tumor-
specific CD4+
T cells we have designed "vaccines" that target the ac-
tivation of these cells. The vaccines consist of tumor cells that ex-
press syngeneic MHC class II molecules, CD80, plus other accesso-
ry molecules that enhance antigen presentation. Our hypothesis is
that such genetically modified tumor cells serve as antigen-present-
ing cells (APC) for endogenously synthesized tumor-encoded pep-
tides. In vivo antigen presentation experiments confirm this hypoth-
esis. In vivo tumor rejection experiments in a mouse sarcoma mod-
el demonstrate that the vaccines are efficacious prophylactic and im-
munotherapy agents for the prevention of solid tumors and for the re-
jection of established primary solid tumors up to 1 cm in diameter.
The vaccines are also effective immunotherapy agents in a post-sur-
gery setting for mice with established, disseminated mammary car-
cinoma metastasis. In vitro antigen presentation experiments clarify
the mechanism and intracellular trafficking pathways of MHC II-re-
stricted peptide presentation, and demonstrate that tumor-encoded
peptides from all tested subcellular compartments (nuclei, mito-
chondria, endoplasmic reticulum, cytoplasm) are presented. Howev-
er, coexpression of invariant chain (Ii) by the vaccines can inhibit
presentation of peptides derived from certain subcellular locations.
Because of the therapeutic efficacy of the vaccines seen in mouse
models, we are currently generating reagents to extend this approach
to the clinic. We are using ocular melanoma as a test system. Within
5 years of diagnosis, 50% of ocular melanoma patients develop
metastatic disease. Our strategy is to immunize patients with prima-
ry tumors, but without overt metastasis with panels of non-autolo-
gous but HLA-DR-matched, CD80+
HLA-DR+
Ii
-
ocular melanoma
cells. Until recently we have had technical problems generating the
vaccines and obtaining sufficient DR expression. Recent production
of retroviral constructs containing DR alpha and beta genes flanking
an internal ribosomal entry site (IRES) have overcome this problem.
We are currently testing the vaccines in vitro for their ability to acti-
vate tumor-specific CD4+
T cells.
Defining MHC class II T-cell epitopes for tumor antigens
H. Kobayashi
1
, R. Omiya1
, B. Sodey1
, E. Appella2
, and E. Celis1
1
Mayo Clinic;
2
National Cancer Institute/NIH
There is little doubt that CD4+
helper T lymphocytes (HTL) play an
important role in the establishment of effective antitumor immuni-
ty. During the induction phase of T cell-mediated immune respons-
es, HTL participate in the induction of antigen-specific CD8+
cyto-
toxic T lymphocytes (CTL), which are the main effector immune
cells against malignant cells. In addition, HTL may also be impor-
tant in the maintenance of long-lived CTL responses, which is crit-
ical for the prevention of relapses and in the maintenance of im-
mune memory. In addition, CD4+
HTL are capable of exhibiting an-
titumor effector function by directly recognizing and killing MHC
class II
+
tumor cells that present HTL epitopes on their surface. For
all the above reasons, the inclusion of both CTL and HTL epitopes
in antitumor vaccines should be seriously considered in order to
maximize the possibility of attaining optimal therapeutic effective-
ness.
Numerous MHC class I epitopes from tumor-associated antigens
(TAA) such as HER2, CEA, MAGE and several laboratories have
identified gp100 over the past 10 years. On the other hand, few MHC
class II epitopes for these TAA have been defined. In our laboratory
the focus has been on the identification of MHC class II-restricted
epitopes using two approaches: (1) reverse immunology (predictive
approach) and (2) a combined genetic/proteomic approach using tu-
mor-reactive HTL. We will present a summary of our most recent re-
S28sults analyzing the strengths and weaknesses of these methods and
discuss how we plan to take this knowledge into the clinic.
CD4+
T-cell responses in melanoma/RCC
and DC-based repolarization
Walter J. Storkus
Departments of Surgery and Molecular Genetics and Biochemistry,
University of Pittsburgh Medical Center and the University of Pittsburgh
Cancer Institute
Th1-type CD4+
antitumor T cell "help" appears critical to the induc-
tion and maintenance of antitumor CTL responses in vivo. In con-
trast, Th2- or Th3/Tr-type CD4+
T-cell responses may subvert Th1-
type cell-mediated immunity, providing a microenvironment condu-
cive to disease progression. We have recently identified "helper" T
cell epitopes derived from the MAGE-6 gene product, a tumor-asso-
ciated antigen expressed by most melanomas and renal cell carcino-
mas. In the current study, we assessed whether peripheral blood
CD4+
T cells from HLA-DRß1*0401+
patients are Th1- or Th2-bi-
ased to MAGE-6 epitopes using IFN-? and IL-5 ELISPOT assays,
respectively. Strikingly, the vast majority of patients with active dis-
ease were highly skewed towards Th2-type responses against
MAGE-6-derived epitopes, regardless of their stage (stage I vs stage
IV) of disease. In marked contrast, normal donors and cancer pa-
tients with no current evidence of disease, tended to exhibit either
mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6
peptides, respectively. CD4+
T-cell secretion of IL-10 and TGF-ß1
against MAGE-6 peptides was not observed, suggesting that specif-
ic Th3/Tr-type CD4+
subsets were not common events in these pa-
tients. Our data suggest that immunotherapeutic approaches will
likely have to overcome systemic Th2-dominated, tumor-reactive
CD4+
T-cell responses to provide optimal clinical benefit.
Recent reports of studies in allergen-specific T cell models suggest
that "committed" human Th2-type CD4+
T cells can be repolarized
towards Th1-type, IFN-?-dominated responders, if appropriately re-
stimulated in vitro with autologous type-1 dendritic cells (DC1s).
Our preliminary studies suggest that DCs conditioned or transfected
to produce high levels of IL-12 or DCs in the presence of exogenous
rhIL-12 can drive a Th2?Th1 conversion in MAGE-6-reactive
CD4+
T cells isolated from RCC patients with active disease. These
results suggest that combinational immunotherapies implementing
DC1 and specific tumor antigens may yield optimally efficacious
Th1-type therapeutic immunity.
Abstracts: Session IV
Developing cancer immunotherapies for the clinic:
regulatory perspectives
Lucio Miele
University of Illinois at Chicago
Academic investigators and start-up biotechnology companies are
generating cutting edge research in cancer immunotherapy. Howev-
er, therapeutics development requires a mindset and organization
that are different from "discovery-driven" science, and for good rea-
son. Bringing new agents to the clinic under Investigational New
Drug (IND) application presents challenges that investigators are not
always prepared to meet. These challenges involve more than just
bureaucracy and red tape. In most cases, a good idea and a poorly
characterized agent are not enough. What is needed is a clearly de-
fined product, and proof that it can be manufactured within reason-
able specifications of purity, potency, stability and freedom from
infectious agents. From the clinical standpoint, trial design must in-
corporate specific safeguards against unexpected adverse events. In
recent years, the Food and Drug Administration (FDA) has been
very sensitive to the need to bring new agents to market as quickly
as possible. However, the FDA is bound by specific statutes and by
the Code of Federal Regulations, and it is accountable not just to
investigators, but to Congress and the public at large. Thus, it needs
to strike a balance between speedy review and patient safety.
pEspecially in the area of biologics, often the best strategy is to dis-
cuss development plans and the scientific justifications behind them
with FDA officials in pre-IND meetings, rather than filing and IND
only to discover that it is inadequate. This presentation focuses on
early development of immunotherapeutic agents by academic inves-
tigators or small companies. Pitfalls to avoid in IND submissions for
specific agents and strategies to prevent and overcome problems in
product characterization and trial design will be discussed.
Abstracts: Session V
PLGA nanosphere delivery of peptides and lipopeptides
to dendritic cells
John Samuel
University of Alberta
Poly(D,L-lactic-co-glycolic acid) (PLGA) is a biodegradable and
biocompatible polymer approved for human administration. PLGA
nanospheres are suitable for encapsulation of a wide variety of anti-
gens such as recombinant proteins, peptides, lipopeptides, and plas-
mid DNA. We investigated PLGA nanospheres for delivery of
MUC1 antigens to dendritic cells in vitro and in vivo. PLGA nano-
sphere formulations of a lipopeptide, BLP25, were shown to over-
come self-tolerance and induce Th1 immune responses in MUC1-
transgenic mice. PLGA nanospheres were phagocytosed efficiently
by human and mouse dendritic cells in vitro. PLGA nanospheres ad-
ministered to mice by the intradermal route, could be identified with-
in dendritic cells of the draining lymph nodes. Human dendritic cells
pulsed with PLGA nanosphere formulations of BLP25 can induce
primary T cell proliferative responses. PLGA nanosphere delivery
system offers an efficient method for targeting antigens to dendritic
cells in vitro and in vivo.
Large multivalent immunogen for immunotherapy
Matthew Mescher1
, Julie Curtsinger1
, Jeffrey Miller1,2
,
Malcolm Mitchell
3
, and Ian Okazaki
2
1
Center for Immunology and
2
Department of Medicine,
University of Minnesota Medical School;
3
Karmanos Cancer Institute,
Wayne State University School of Medicine
Studies of murine CD8 T cells have demonstrated that class I/pep-
tide antigen complexes must be displayed on a cell-size surface for
effective recognition and activation to occur [1]. CD8 T cells are op-
timally stimulated by antigen on 5-µm diameter microspheres, and
do not respond when the same antigen is displayed on 1-µm diame-
ter beads. This finding prompted experiments to examine whether in
vivo delivery of antigen on cell size beads, termed ‘large multivalent
immunogen’, might activate or enhance CTL responses [2]. Admin-
istration of class I alloantigen on beads did not initiate responses, but
dramatically augmented CTL responses generated upon challenge
with allogeneic tumor cells. A similar augmentation of CTL re-
sponse, and concomitant reduction of tumor growth, was observed
when LMI were prepared by coating beads with plasma membranes
isolated from tumor cells and administered to mice at the same time
that they were challenged with live syngeneic tumor. When mice
bearing established, progressing subcutaneous P815 mastocytoma
(7 to 12 days) were treated with LMI, there was little effect on tumor
growth or survival. However, combined treatment with Cytoxan and
LMI had highly synergistic effects, resulting in prolonged reduction
of tumor growth and significant extension of survival [3]. In some
experiments tumor became undetectable in the majority of treated
mice; these mice survived indefinitely and rapidly rejected rechal-
lenge with tumor. Similar results were obtained in experiments ex-
amining survival of mice bearing established fibrosarcoma in a lung
metastasis model. Antigen delivery on LMI was uniquely effective
in these experiments. Antigen in the form of irradiated tumor cells or
plasma membrane in adjuvant was ineffective, and free plasma
S29membrane antigen (not on microspheres) had only a marginal effect.
Based on the results obtained in murine models, a small phase I tri-
al for melanoma was done. Two in vitro grown melanoma cell lines
were used as the source of plasma membrane antigen to prepare the
LMI. Three dose levels were tested and no significant toxicity was
found. Of 16 patients, 10 had an increased frequency of pCTL reac-
tive to the melanoma cell line following therapy. One partial re-
sponse was obtained, and disease was stable for more than
12 months in three patients. A second trial has recently been initiat-
ed for melanoma and renal carcinoma plasma membrane isolated
from autologous tumor is used for preparation of the LMI. Patients
are being randomized to one of three groups: LMI only, Cytoxan and
LMI, or Cytoxan, LMI and a short course of low-dose IL-2.
Ongoing murine studies are employing adoptive transfer of TCR
transgenic CD8 T cells to allow visualization of CTL responses to
tumors. Experiments using this approach have demonstrated that IL-
2 can be effectively used to sustain tumor-specific responses follow-
ing their initiation, but that the dose and duration of administration
must be limited. Additional results are suggesting that class I/peptide
tetramers can be used to prepare LMI that are effective in augment-
ing tumor growth reduction, suggesting that LMI immunotherapy
using well-defined tumor antigens may be feasible.
References:
1. Mescher MF (1992) Surface contact requirements for activation
of cytotoxic T lymphocytes. J Immunol 149:2402
2. Rogers J, Mescher MF (1992) Augmentation of in vivo cytotoxic
T lymphocyte activity and reduction of tumor growth by large
multivalent immunogen. J Immunol 149:269
3. Mescher MF, Rogers JD (1996) Immunotherapy of established
murine tumors with large multivalent immunogen and cyclophos-
phamide. J Immunother Emphasis Tumor Immunol 19:102
The role of CD8 T cells in the control of tumor growth:
the three surprises
Mark Dobrzanski, Brian Helmich, and Richard Dutton
Trudeau Institute, Inc.
We have focused on the effector arm of the antitumor response. The
response to antigen can take many forms and we wanted to deter-
mine which form vaccines should elicit for effective control of tumor
growth. We chose to look at tumor-specific Tc1 and Tc2 CD8 effec-
tor T cells in an adoptive transfer model using cells from T-cell re-
ceptor transgenic mice specific for a tumor neoantigen. We found
that perforin-mediated cytolysis was not required for the control of
tumor growth and there was only a modest dependence on Fas ligand
expression. CD8 Tc1 effectors from TNF-a-deficient mice were as
effective as wild-type, but IFN-?-deficient were much less effective.
Tc2 effectors required both IL-4 and IL-5 but elicited and were de-
pendent on an IFN-?-dependent host response. Untreated mice made
a sizeable response to tumor. This response was ineffective before
adoptive therapy but became effective and essential following ther-
apy.
Eradication of established tumors by vaccination
with Venezuelan equine encephalitis virus replicon particles
delivering human papillomavirus 16 E7 RNA
W. Martin Kast
Cardinal Bernardin Cancer Center, Loyola University Chicago
The etiological role of human papillomaviruses (HPV) in cervical
and other cancers suggests that therapeutic vaccines directed against
requisite viral antigens may eradicate tumors or their precursors. A
Venezuelan equine encephalitis (VEE) alphavirus vector delivering
the HPV-16 E7 RNA was evaluated for antitumor efficacy using a
murine HPV-16 E7+
tumor model. Vaccination with VEE replicon
particles expressing E7 (E7-VRP) induced class I-restricted CD8+
T-
cell responses as determined by IFN-? enzyme-linked immunospot
(ELISPOT), tetramer, and cytotoxicity assays. E7-VRP vaccination
prevented tumor development in all of the mice and effectively elim-
inated 7-day established tumors in 67% of tumor-bearing mice. Al-
so HLA- A*0201-mediated protection was observed in HLA-
A*0201 transgenic mice against challenge with a newly developed
HLA-A*0201+
HPV-16-transformed tumor (HLF 16) in which the
immunodominant H-2Db-restricted HPV-16 E7 epitope is knocked
out. The induction of protective T-cell responses was dependent on
CD8+
, but not CD4+
T cells. Long-lasting T-cell memory responses
developed in E7-VRP-vaccinated mice as determined by complete
protection from tumor challenge 3 months after the final vaccina-
tion. Remarkably, no neutralization was observed when mice were
prevaccinated with empty alphavirus vectors before being vaccinat-
ed with recombinant HPV-16 E7 RNA-expressing vectors. These
promising results highlight the potent CD8+
T-cell-mediated antitu-
mor effects elicited by VEE replicon-based vectors and support their
further development toward clinical testing against cervical intraepi-
thelial neoplasia or carcinoma.
Novel complex adjuvant
H. Brandwein
Immuno-Rx, Inc
IRX-2 is a uniform, well-defined set of natural Th1 cytokines and
monokines produced by human mononuclear cells in culture. Precli-
nical and clinical studies demonstrated IRX-2 to be a potent stimu-
lant of cellular immunity capable of initiating a robust and durable
immunization against tumors. To induce immune regression of squa-
mous cell cancer of the head and neck, 50 human patients received
ten daily perilymphatic injections of IRX-2 along with a contrasup-
pressive regimen of a single, a low (non-chemotherapeutic) dose of
cyclophosphamide and daily oral indomethacin and zinc. Following
this treatment protocol, on day 21 patients were taken to surgery for
resection of their tumors. The IRX-2 immunotherapy induced lym-
phocyte mobilization and infiltration into the tumors, associated
with a 40% clinical response rate and over 90% histological response
rate. Evidence suggests that IRX-2 is acting as an adjuvant for en-
dogenous antigens of head and neck cancer acting on both dendritic
cells and T cells of the immunized patients. Additional clinical trials
of IRX-2 in head and neck cancer are planned, in addition to the use
of IRX-2 as a T-cell adjuvant for cancer vaccines using defined (ex-
ogenous) tumor antigens.
DNA vaccination against HER2/neu using alphavirus-based
vectors
Lawrence B. Lachman
Department of Bioimmunotherapy, University of Texas M.D. Anderson
Cancer Center
When biopsy reveals that breast cancer cells express the transmem-
brane protein HER2/neu the prognosis for the patient is poor. The
mechanism by which HER2/neu, a member of the tyrosine kinase re-
ceptor family, accelerates the growth and metastatic potential of
breast cancer cells is not yet understood and thus options for treat-
ment are reduced. A vaccine that targets HER2/neu-expressing cells
could have significant therapeutic and preventative application by
controlling the proliferation and metastasis of the highly aggressive
HER2/neu+
cells. Gene vaccines have demonstrated that antitumor
immunity can be developed by immunizing with bacterial expres-
sion plasmids that encode the DNA sequence for tumor antigens
such as HER2/neu. Gene vaccines have been shown to induce strong
lasting immunity that includes the generation of cytotoxic T lympho-
cytes (CTL) the main mechanism for immunological control of tu-
mor growth. We have shown that vaccination of mice with an alpha-
virus-based plasmid expressing the sequence for HER2/neu protect-
ed mice from challenge with a HER2/neu-expressing murine breast
tumor cell line. The mice were protected from challenge with tumor
cells injected directly into the mammary tissue and intravenous in-
jection, a model of tumor metastasis. The vaccination also prolongs
the life of neu+
-transgenic mice and induces antigen-specific T cells
S30as demonstrated by tetramer analysis and interferon-? induction. The
plasmid we are using, called ELVIS, was created by Chiron Technol-
ogies (San Diego, CA), and is the first step of a two-stage vaccina-
tion protocol that uses viral replicon particles (VRP) as the second
step. The non-pathogenic VRP are similar to attenuated viruses kno-
wn to induce long-lasting immunity when used for childhood diseas-
es. We have found VRP-neu to be more effective than ELVIS-neu in
the assays described above, and we anticipate that a strategy of pri-
mary vaccination with ELVIS-neu followed by boosting with VRP-
neu will be maximally effective in controlling tumor growth and me-
tastasis. We hope to perform a phase 1b clinical trial in breast and
ovarian cancer patients using either ELVIS-neu or VPR-neu in the
near future.
Abstracts: Session VI
CD137 (4-1BB), more than a costimulator
Lieping Chen
Mayo Clinic
CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) re-
ceptor superfamily. It was reported that CD137 expresses on the sur-
face of activated T cells and signaling through CD137 costimulates
T cell growth and induces regression of established tumors. We dem-
onstrated that CD137 is expressed at high levels on natural killer
(NK) cells and CD137 signaling by an agonistic monoclonal anti-
body (mAb) promotes the activation of and IFN-? production from
NK cells in the presence of IL-2. These results support a direct role
of CD137 signaling in NK cell activation. In addition, freshly isolat-
ed mouse splenic dendritic cells (DCs) and bone marrow-derived
DCs express CD137 on the cell surface and in soluble form. Trigger-
ing CD137 increased the secretion of IL-6 and IL-12 from DCs. Mo-
re importantly, infusion of an agonistic monoclonal antibody to
CD137 into naive mice enhanced the ability of DCs to stimulate T-
cell proliferation in response to both alloantigens and a nominal an-
tigen in vitro. This enhancement of DC function is not mediated
through activation of T cells since the effect was also observed in
RAG-1 knockout mice that lack T cells. Our findings implicate
CD137 as an important receptor involved in the modulation of NK
and DC function, in addition to costimulatory function for T cells.
Latent sensitivity to Fas-mediated apoptosis after CD40-
ligation may explain activity of CD154 gene therapy in chronic
lymphocytic leukemia
Thomas J. Kipps
UCSD Cancer Center
Patients with chronic lymphocytic leukemia (CLL) treated with Ad-
CD154 (CD40L) gene therapy experience reductions in leukemia
cell counts and lymph node size associated with induction of the de-
ath receptor Fas (CD95). CD4 T cell lines can induce apoptosis of
CD40-activated CLL cells via a mechanism dependent on the CD95
ligand (CD95-L). To examine whether CD95-L was sufficient to in-
duce cytolysis of CD40-activated CLL cells, we used CHO cells
transfected with CD95-L as cytotoxic effector cells. CD40-activated
CLL cells were initially resistant to CD95-mediated apoptosis de-
spite high levels of expression of CD95. However, after 72 h, CLL
cells from seven of seven patients became increasingly sensitive to
CD95-mediated apoptosis. This correlated with a progressive de-
cline in FLIP and TRAF1 protein levels, which were induced within
24 h after CD40-ligation. Downregulation of FLIP with an anti-sen-
se oligonucleotide or a pharmacological agent, however, was not
sufficient to render CLL cells sensitive to CD95-mediated apoptosis
in the 24-72 h after CD40 activation. Although the levels of pro-
caspase-8 appeared sufficient, inadequate levels of FADD and
DAP3 may preclude assembly of the death-inducing signaling com-
plex (DISC). By 72 h after CD40 ligation, sensitivity to CD95 and a
progressive increase in FADD and DAP3 were associated with the
acquired ability of FADD and FLIP to coimmunoprecipitate with the
DISC following CD95 ligation. Collectively, these studies reveal
that CD40 ligation on CLL B cells induces a programmed series of
events in which the cells initially are protected and then sensitized to
CD95-mediated apoptosis through shifts in the balance of the anti-
apoptotic and proapoptotic proteins FLIP and FADD.
T help for CTL
Stephen P. Schoenberger
La Jolla Institute for Allergy and Immunology
CD8+
T lymphocytes (CTL) constitute a powerful effector popula-
tion for the immunological control of tumors and their activation re-
mains a key target for therapeutic vaccines. Although the majority of
CTL responses in vivo depend on ‘help’ from CD4+
T cells (Th), it is
unknown whether help is required for the expansion, functional de-
velopment, or survival of CTL. We have therefore used a robust and
well-characterized model tumor vaccination system to investigate
the fate of Th-dependent CTL cross-primed in the absence of CD4+
T cells. We find that Th-dependent do arise in the absence of help, de-
velop primary effector function such as cytokine secretion and in vi-
vo cytotoxicity, but in contrast to CTL primed in the presence of T
help, fail to expand upon secondary encounter with antigen. Priming
of CTL in the presence or absence of T help required B7-1/B7-2 on
antigen-presenting cells (APC), and the requirement for CD4+
T
cells could be replaced by agonistic antibodies against either CD28
or 4-1BB. In contrast to the B7-1/B7-2 requirement, priming of Th-
dependent CTL in the absence of CD4+
T cells did not depend on
CD28 or CTLA-4, indicating that CTL express novel costimulatory
ligand(s) for B7-1/B7-2 that control survival during initial expan-
sion. These data indicate that, based on the presence or absence of T
help, CTL can initiate distinct developmental programs, each begin-
ning with proliferation and effector function, but which differ in their
secondary response to antigen as memory cells. Both programs re-
quire B7 as a second signal, but early survival is mediated by inter-
action of B7 with a new ligand on CTL.
Abstracts: Session VII
Apoptosis and anti-apoptosis in multimodality cancer treatment
Lucio Miele
University of Illinois at Chicago
The induction of cancer cell death is a common goal of cytotoxic
chemotherapy and immunotherapy agents, as well as radiotherapy.
Programmed cell death, or apoptosis, is induced by various signaling
cascades that are triggered by membrane receptors, DNA damage,
cellular stress and other stimuli. Transformed cells frequently over-
express antiapoptotic proteins such as Bcl-2 or its homologues or
transcription factor NF-kB. Yet cancer cells are often more sensitive
than their untransformed counterparts to certain apoptosis-inducing
stimuli due to defects in the cell cycle arrest/DNA repair machinery.
Thus, modulating apoptosis pathways is a very attractive strategy to
increase the effectiveness of multimodality cancer treatments. In the
context of immunotherapy, cancer cell apoptosis can be induced by
CTL via the perforin/granzyme pathway or via FAS ligand, by CD4
cells via TRAIL, and by monoclonal antibodies via antibody-depen-
dent cell-mediated cytotoxicity, antibody-triggered signaling or an-
tibody-conjugated radionuclides or cytotoxic agents. Multimodality
strategies that enhance tumor cell sensitivity to apoptosis in combi-
nation with apoptosis-inducing immunotherapeutics are promising
avenues to increase the clinical effectiveness of immunotherapy.
This presentation will briefly discuss some general aspects of apop-
tosis regulation in transformed cells and present data on apoptosis
modulation through a novel strategy targeting the Notch signaling
pathway.
S31The generation and persistence of CD4 T cell memory
Susan L. Swain
Trudeau Institute
Our published studies over the last few years have indicated that on-
ce naive CD4 T cells have differentiated to active effector cells, the
population can give rise to memory cells. The memory cells which
develop share the cytokine polarization of the effectors from which
they were generated. This concept of a direct pathway from effectors
to memory has been further strengthened by our studies which show
that after the initial stages of naive CD4 T cell activation, neither ex-
posure to antigen nor exposure to major histocompatibility complex
is needed for the transition of effectors to memory or for their persis-
tence in vivo. Recently we provided direct evidence that effectors
can become memory cells without further division and that this tran-
sition can occur with great efficiency. Together these studies support
the direct default of effector to memory CD4 T cells and suggest that
the factors that shape the potential of memory cells act mostly dur-
ing the generation of effectors. This is important from the perspec-
tive of vaccines because effectors found in blood can likely be char-
acterized and should be good predictors of memory. Recently we
have begun studies of the factors regulating the generation of distinct
types of effector and by inference memory cells. We find that, in an
acute anti-influenza response in vivo, a heterogeneous population of
effector is generated which represents a continuum of different ro-
unds of division, different expression of phenotypic markers and dif-
ferent cytokine-producing potential. Only the most differentiated ef-
fectors can migrate to an inflammatory site. Migration to other non-
lymphoid sites is also restricted to highly differentiated CD4 effec-
tors. We suggest memory heterogeneity and location reflects that of
the in vivo-generated effector population, and describe a pattern of
properties which characterize the effectors which can disperse and
act in the tissues.
Downregulation of tumor antigens by IFN-?
I. Caroline Le Poole
Loyola University Chicago
T cells infiltrating melanoma tumors are frequently reactive with dif-
ferentiation antigens. Consequently, immune escape can occur when
melanoma cells dedifferentiate and loose expression of commonly
recognized antigens such as MART-1, gp100 or tyrosinase. A change
in microenvironment, encountered when transformed melanocytic
cells emigrate from the basal layer of the epidermis, may provide the
incentive for reduced expression of these target antigens. In the pres-
ent study, the contribution of IFN-? exposure to reduced expression
of melanosomal antigens was investigated. Immunostaining and
FACS analysis of M14 melanoma cells treated with 100-1000 U/ml
IFN-? revealed a >65% suppression of gp100, TRP-1 and MART-1
protein levels. A similar reduction was observed by Northern blot-
ting, suggesting that IFN-? affects melanosomal gene expression at
the transcriptional level. As inflammatory cytokine IFN-? can simul-
taneously enhance cell surface expression of HLA molecules, the
consequences of IFN-? exposure for the efficacy of recognition by
MART-1-reactive T cells was explored. In spite of elevated MHC
class I expression, cytotoxicity towards HLA-matched melanoma
cells (but not melanocytes) was reduced by up to 78% by IFN-? pre-
treatment, and was restored by addition of MART-1 peptide AAGI-
GILTV. This indicates that exposure to IFN-? can suppress recogni-
tion by melanoma-reactive T cells. The actual abundance of IFN-? in
tumor tissue was compared to that in control skin by qRT-PCR, and
revealed a 200-fold increase in transcripts within metastatic tumor
tissue. Laser capture microdissection and immunohistology localized
the majority of IFN-?-generating T cells to the tumor stroma. Tumor-
infiltrating T cells were frequently surrounded by a halo of reduced
MART-1 expression, suggesting that IFN-? generated by activated T
cells may interfere with successful recognition of melanoma target
cells in vivo. Taken together, these results indicate that IFN-? may act
as a two-edged sword, enhancing inflammatory responses yet ham-
pering effective recognition of melanoma cells.
S32Glossary
A
Active immunity: The production of antibodies against a specific disease by
the immune system. Active immunity can be acquired in two ways, either
by contracting the disease or through vaccination. Active immunity is usu-
ally permanent, meaning immune individuals are protected from the dis-
ease for the duration of their lives.
Adaptive immunity: Components of the immune system that are acquired
after birth. The response of antigen-specific lymphocytes to antigen, in-
cluding the development of immunological memory. Also referred to as
learned or acquired immunity.
Adjuvant: A substance sometimes included in a vaccine formulation to en-
hance or modify the immune-stimulating properties of a vaccine.
Alleles: Any one of a series of two or more different genes that may occupy
the same locus on a specific chromosome. As autosomal chromosomes are
paired, each autosomal gene is represented twice in normal somatic cells.
If the same allele occupies both units of the locus, the individual or cell is
homozygous for this allele. If the alleles are different, the individual or cell
is heterozygous for both alleles.
Allo-: Other; differing from the normal or usual.
Alloantigen: An antigen that occurs in some but not other members of the
same species. Isoantigen is sometimes used in this sense.
Amino acid: One of the building blocks of proteins.
Antibody: An infection-fighting protein molecule in blood or secretory flu-
ids that tags, neutralizes, and helps destroy pathogenic microorganisms
(e.g., bacteria, viruses) or toxins. Antibodies, known generally as immuno-
globulins, are made and secreted by B-lymphocytes in response to stimula-
tion by antigens. Each specific antibody binds only to the specific antigen
that stimulated its production.
Antibody-mediated immunity: Immunity that results from the activity of
antibodies in blood and lymphoid tissue (also called humoral immunity).
Antibody isotypes: Also known as immunoglobulin isotypes (see immuno-
globulin). Different immunoglobulin isotypes are called IgM, IgD, IgA,
and IgE.
Antigen: Any substance which can generate the formation of a specific antibody
(a protein created by the immune system to protect the body). For vaccines, the
term antigen refers to a vaccine component that induces protection for one sin-
gle disease (e.g., the measles antigen induces protection against measles).
Antigen processing: Before the immune system can respond to an antigen,
it must be able to recognize the antigen. It is able to do so through antigen
processing. Macrophages are the major antigen-processing cells; others in-
clude B lymphocytes.
Antigen-presenting cell: Antigen-processing cells ingest antigen and chop
it into small fragments that are then packaged within the major histocom-
patibility complex molecules and shuttled to the surface of the cell mem-
brane for presentation. Professional antigen-presenting cells are dendritic
cells, macrophages, and B cells.
Anti-idiotype: Antibodies to the variable regions of the antibody-binding
site. Anti-idiotypes contain the internal image of the tumor antigen and are
capable of inducing antibody responses to the original antigen.
Antitoxin: Antibodies capable of destroying microorganisms including vi-
ruses and bacteria.
Apoptosis: Single deletion of scattered cells by fragmentation into mem-
brane-bound particles that are phagocytosed by other cells; believed to be
due to programmed cell death. [G. a falling or dropping off, fr. apo,
off+ptosis, a falling]
Association: The degree to which the occurrence of two variables or events
is linked. Association describes a situation where the likelihood of one
event occurring depends on the presence of another event or variable. How-
ever, an association between two variables does not necessarily imply a
cause and effect relationship. The terms association and relationship are
often used interchangeably.
Attenuated: Weakened or treated in such a way as to decrease the ability of
a microorganism (such as a parasite or a virus) to cause infection or disease.
Attenuated strain: A strain of microorganism that has been altered in viru-
lence to exhibit diminished virulence.
Attenuated vaccine: A vaccine in which live viruses are weakened through
chemical or physical processes in order to produce an immune response
without causing the severe effects of the disease. Attenuated vaccines cur-
rently licensed in the United States include measles, mumps, rubella, polio,
yellow fever and varicella. Also known as a live vaccine.
Autoimmunity: The condition in which one’s own tissues are subject to del-
eterious effects of the immune system, as in autoallergy and in autoimmune
disease. Specific humoral or cell-mediated immune response against the
body’s own tissues.
Autologous: Occurring naturally and normally in a certain type of tissue or
in a specific structure of the body. Sometimes used to denote a neoplasm
derived from cells that occur normally at that site, e.g., squamous cell car-
cinoma in the upper esophagus.
Avidity: The binding strength of an antibody for an antigen. [L. avidus,
eager, greedy, fr. aveo, to crave]
B
B cells: Small white blood cells that help the body defend itself against infec-
tion. These cells are produced in bone marrow and develop into plasma
cells that produce antibodies. Also know as B lymphocytes.
Bacteria: Bacteria are single-celled organisms that live in and around us.
Bacteria may be helpful, but in certain conditions may cause illnesses such
as strep throat, most inner ear infections, and bacterial pneumonia. Pl. a
unicellular prokaryotic organism that usually multiplies by cell division
and has a cell wall that provides a constancy of form. They may also be aer-
obic or anaerobic, motile or non-motile and free-living, saprophytic, para-
sitic or pathogenic.
Bacterial vaccine: Vaccine derived from bacteria.
Bacterium: The singular form of bacteria.
C
Carcinoembryonic antigen (CEA): A glycoprotein constituent of the gly-
cocalyx of the embryonic endodermal epithelium, generally absent from
adult cells with the exception of some carcinomas. It may also be detected
in the serum of patients with colon cancer.
CD: Abbreviation for clusters of differentiation. Groups of monoclonal anti-
bodies that identify the same surface molecule. The cell surface molecule
is designated CD followed by a number (e.g. CD4 and CD8)
CD4: The cell surface protein CD4 is important for recognition by the T cell
receptor of antigenic peptides bound to MHC class II molecules. It acts as
a coreceptor by binding to the lateral face of MHC class II molecules.
CD8: The cell surface protein CD8 is important for recognition by the T cell
receptor of antigenic peptides bound to MHC class I molecules. It acts as a
coreceptor by binding to the lateral face of MHC class I molecules.
Cell-mediated immunity: Describes any adaptive response in which anti-
gen-specific T cells have the main role. It is defined operationally as all
adaptive immunity that cannot be transferred to a naive recipient with se-
rum antibody.
Challenge: In vaccine experiments, the deliberate exposure of an immunized
animal or person to the infectious agent.
Chemokines: A group of specific chemotactic polypeptides all of which
have a similar structure (4-cystein structure), involved in the migration and
activation of cells, especially phagocytic cells and lymphocytes. They
have a central role in inflammatory responses.
Clinical trial: A scientifically controlled study carried out under specific
conditions, usually to test the effectiveness of a new treatment.
Clone: A clone is a population of cells all derived from a single progenitor
cell.
CTL: Abbreviation for cytotoxic T lymphocytes.
Cytoplasm: The living matter within a cell (excluding the nucleus) that is re-
sponsible for the function of the cell (for example, protein synthesis).
Cytokines: Hormone-like low molecular weight proteins secreted by many
different cell types, which regulate the intensity and duration of immune re-
sponses. Cytokines amplify some parts of the immune system and suppress
other parts. Many cytokines have been identified such as interferon-a, in-
terferon-ß, tumor necrosis factor (TNF) and granulocyte colony stimulat-
ing factor (G-CSF). Cytokines made by lymphocytes are often called
lymphokines or interleukins (abbreviated IL).
Cytotoxicity: Degree to which a substance is poisonous to cells.
Cytotoxic T cells: T cells that can kill other cells. Most cytotoxic cells are
MHC class I-restricted CD8 cells, but CD4 T cells can also kill in some
cases. Cytotoxic T cells are important in host defense against cytosolic
pathogens.
D
Dendritic cells: Also known as interdigitating reticular cells, are found in T
cell areas of lymphoid tissues. They have a branched or dendritic morphol-
ogy and are the most potent stimulators of T cell response. Non-lymphoid
S33tissues also contain dendritic cells but these do not seem to stimulate T cell
responses until they are activated and migrate to lymphoid tissues. The den-
dritic cell derives from bone marrow precursors. It is distinct from the fol-
licular dendritic cell that presents antigen to B cells.
E
Efficacy: In vaccine research, the ability of a vaccine to produce a desired
clinical effect, such as protection against a specific infection or disease, at
the optimal dosage and schedule in a given population. A vaccine may be
tested for efficacy in phase III trials if it appears to be safe and shows some
promise in smaller phase I and II trials.
Efficacy rate: A measure used to describe how good a vaccine is at prevent-
ing disease.
ELISA: Abbreviation for enzyme-linked immunosorbent assay. A serologi-
cal assay in which bound antigen or antibody is detected by a linked en-
zyme that converts a colorless substrate to a colored product.
ELISPOT assay: An adaptation of ELISA in which cells are placed over an-
tibodies or antigens attached to a plastic surface. The antigen or antibody
traps the cells’ secreted products, which can then be detected by using an
enzyme-coupled antibody that cleaves a colorless substrate to make a local-
ized colored spot.
Epitope: A site on an antigen recognized by antibody. Epitopes are also cal-
led antigenic determinants. A T cell epitope is a short peptide derived from
a protein antigen. It binds to an MHC molecule and is recognized by a par-
ticular cell.
Epitope spreading: Describes the fact that responses to autoantigens tend to
become more diverse as the response persists.
Expression system: In genetic engineering, the cells into which a gene has
been inserted to manufacture desired proteins.
F
Functional antibody: An antibody that binds to an antigen and has an effect
that can be demonstrated in laboratory tests.
G
Genome: The complete set of genes present in a cell, parasite, or virus.
GM-CSF: Granulocyte macrophage colony-stimulating factor is a cytokine
involved in the growth and differentiation of myeloid and monocytic lin-
eage cells, including dendritic cells, monocytes and tissue macrophages
and cells of the granulocyte lineage.
Good management practice: Routine practices that minimize risk from
harmful antimicrobial resistant bacteria or resistance genes through good
farm management and hygiene practices (e.g., optimal housing conditions
and feeding strategies) and other non-antimicrobial disease preventive
strategies, whilst maximizing the productivity of food animal production.
Growth factor: Proteins involved in cell differentiation and growth.
H
Helper T cells: Also known as CD4 T cells or helper CD4 T cells. These are
cells that can help B cells make antibody in response to challenge. The most
efficient helper cells are also known as Th2, cells that make the cytokines
IL-4 and IL-5. Some experts refer to all CD4 cells as helper cells, regard-
less of function. Function determination requires cellular assays that show
that some CD4 T cells kill the cells they interact with, and therefore not all
CD4 cells are helper cells.
Host: A plant or animal harboring another organism.
HPV: Human papillomavirus, the causative agent of genital warts (condylo-
mata acuminata) a sexually transmitted infection.
HPV-16: Serotypes 16 and 18 commonly associated with progression to can-
cer of the cervix in women.
Humoral immunity: The antibody-mediated immunity made in a humoral
immune response. Humoral immunity can be transferred to nonimmunized
recipients by using immune serum containing specific antibody.
I
ICAM: Abbreviation for intercellular adhesion molecule. ICAM-1, ICAM-
2, and ICAM-3 are cell-surface ligands for the leukocyte integrins and are
crucial in the binding of lymphocytes and other leukocytes to certain cells,
including antigen-presenting cells and endothelial cells. They are members
of the immunoglobulin superfamily.
IFN: Abbreviation for interferon. Interferons are cytokines that can induce
cells to resist viral replication. Interferon-a and -ß are produced by leuko-
cytes and fibroblasts, respectively, as well as by other cells.
Ig: Standard abbreviation for immunoglobulin.
IL: Interleukin, a type of cytokine produced by leukocytes that influences a
variety of cells.
Immune response: The reaction of the immune system to foreign sub-
stances.
Immune system: The complex system in the body responsible for fighting
disease. Its primary function is to identify foreign substances in the body
(bacteria, viruses, fungi or parasites) and develop a defense against them.
This defense is known as the immune response. It involves production of
protein molecules called antibodies to eliminate foreign organisms that in-
vade the body.
Immunity: Protection against a disease. There are two types of immunity,
passive and active. Immunity is indicated by the presence of antibodies in
the blood and can usually be determined with a laboratory test. See active
and passive immunity.
Immunization: The process or procedure by which a subject (person, ani-
mal, or plant) is rendered immune, or resistant, to a specific disease. This
term is often used interchangeably with vaccination or inoculation, al-
though the act of inoculation does not always result in immunity.
Immunogen: A substance capable of provoking an immune response. Also
called an antigen.
Immunogenicity: The ability of an antigen or vaccine to stimulate immune
responses.
Immunosuppression: When the immune system is unable to protect the bo-
dy from disease. This condition can be caused by disease (like HIV infec-
tion or cancer) or by certain drugs (like those used in chemotherapy). Indi-
viduals whose immune systems are compromised should not receive live,
attenuated vaccines.
Immunoglobulin: A synonym for antibody.
Immunotherapy: Cancer treatment that produces antitumor effects primari-
ly through the action of natural host defense mechanisms or by the admin-
istration of natural mammalian substances.
In vitro: In an artificial environment, referring to a process or reaction occur-
ring therein, as in a test-tube or culture medium. Cf. in vivo. [L. in glass].
Inactive vaccine: A vaccine made from viruses and bacteria that have been
killed through physical or chemical processes. These killed organisms can-
not cause disease.
Interferon (abbreviation IFN): A class of small glycoproteins that exert anti-
viral activity at least in homologous cells through cellular metabolic process-
es involving synthesis of double-stranded RNA, which is an intermediate in
replication of RNA viruses. IFNs are classified into three groups, alpha, be-
ta, and gamma, based on their reactivities with antibodies as well as their
physicochemical properties and their cells of origin and method of induction.
Intracellular vaccines: Vaccines within a cell or cells.
Intramuscular injection: An injection made into the body of a muscle.
Investigational vaccine: A vaccine that has been approved by the Food and
Drug Administration (FDA) for use in clinical trials on humans. However,
investigational vaccines are still in the testing and evaluation phase and are
not licensed for use in the general public.
IRB (Institutional Review Board): A committee of physicians, statisti-
cians, community advocates and others that reviews clinical trial protocols
before they can be initiated and is responsible for monitoring the safety of
clinical trials at that institution. IRBs ensure that the trial is ethical and that
the rights of participants are adequately protected.
IR genes: Immune response genes are genetic polymorphisms that control
the intensity of the immune response to a particular antigen. Virtually all IR
phenotypes are due to the differential binding of peptide fragments of anti-
gen to MHC molecules, especially MHC class II molecules. The term is lit-
tle used now. An immune response gene defect is usually, but not always,
due to failure to bind an immunogenic peptide, so that no T cell response is
observed.
K
Killer T cell: Another term used for cytotoxic T cells.
L
Leukocyte: A general term for a white blood cell. Leukocytes include lym-
phocytes, monocytes, and polymorphonuclear leukocytes.
LFA-3: Lymphocyte function-associated antigen-3 is a molecule found on
many cells that is the ligand for CD2 (also known as LFA-2). It is a mem-
ber of the immunoglobulin superfamily.
Listeria monocytogenes: A species of the genus Listeria that is aerobic, mi-
croaerophilic, motile peritrichous bacterium. The bacteria are found in the
S34feces of humans and other animals, in vegetation and in silage, and are par-
asitic on poikilothermic and warm-blooded animals including humans.
Listeriosis: A sporadic disease causing meningitis septicemia, endocarditis
etc. Sometimes involved in infections of immunocompromised individuals
and in neonatal infections.
Lymphocytes: Small white blood cells that help the body defend itself
against infection. These cells are produced in bone marrow and develop in-
to plasma cells, which produce antibodies. Also know as B cells.
M
Macrophages: Large cells that help the body defend itself against disease by
surrounding and destroying foreign organisms (viruses).
Melanoma: A malignant cancer derived from pigment-producing cells (mel-
anin-producing) of the skin or any part of the body characterized by the
ability to frequently metastasize widely.
Metastasis: The shifting of disease or its local manifestations from one part
of the body to another via lymphatics, blood vessels, or by direct extension.
MF59 adjuvant: An oil-in-water emulsion of various particulate immuno-
stimulators used in vaccines recently (e.g., Fluad vaccine licensed in Eu-
rope) that results in the recruitment of antigen-presenting cells to the site of
administration.
MHC class I: These molecules present peptides generated in the cytosol to
CD8 T cells.
MHC class II: Molecules of MHC II present peptides degraded in intracel-
lular vesicles to CD4 T cells.
MHC (major histocompatibility complex): The gene cluster that controls
certain aspects of the immune response. Among the products of these ge-
nes are the histocompatibility antigens, such as HLA class I antigens,
which are present on every cell with a nucleus and serve as markers to dis-
tinguish self from non-self.
MHV-68: A serotype of mouse herpes virus.
Monoclonal antibody: Custom-made, identical antibody that recognizes on-
ly one epitope of an antigen.
Monocyte: A large white blood cell in the blood that ingests microbes or oth-
er cells and foreign particles. When a monocyte passes out of the blood-
stream and enters the tissues, it develops into a macrophage.
Monovalent vaccine: A vaccine that contains only one antigen.
MUC1: Abnormally glycosylated mucin found in breast and pancreatic tu-
mors.
N
Naked DNA: Antigen encoding DNA plasmid molecules that result in de no-
vo production of correctly folded antigen at the site of delivery.
NK cells: Natural killer cells, a type of lymphocyte that is large, usually gran-
ular, non-T, non-B lymphocyte that can kill certain microbes and cancer
cells. NK cells are important in innate immunity to viruses and other intra-
cellular pathogens, as well as in antibody-dependent cell-mediated cytotox-
icity (ADCC).
O
-oma: A tumor.
Oncology: The field of medicine concerning the diagnosis, treatment and
study of cancer.
Organism: Any living thing. Organisms include humans, animals, plants,
bacteria, protozoa and fungi.
-osis: A condition or a process, especially one that is abnormal.
P
Passive immunity: Protection against disease through antibodies produced
by another human being or animal. Passive immunity is effective, but pro-
tection is generally limited and diminishes over time (usually a few weeks
or months). For example, maternal antibodies are passed to the infant prior
to birth. These antibodies temporarily protect the baby for the first
4-6 months of life.
Pathogens: Bacteria, viruses, parasites, or fungi that can cause disease.
Pathogenesis: The origin and development of disease. More specifically, it
is the way a microbe (bacterium, virus, etc.) causes disease in its host.
patho-, -pathy: Abnormality.
PCR: Polymerase chain reaction is a technique for amplifying a specific se-
quence in DNA by repeated cycles of synthesis driven by pairs of recipro-
cally oriented primers.
Phagolysosome: A cell organelle that is made by the fusion of a phagosome
and a lysosome. When bacteria are phagocytosed by cells such as macro-
phages, they are taken up into a vacuole called a phagosome, which is a rel-
atively hospitable environment. The phagosome, however, quickly fuses
with a lysosome that is capable of making antimicrobial compounds that
can kill the bacteria.
Phase I clinical trial: An experiment designed to evaluate adverse reactions,
optimal dose, and best route of administration.
Phase II clinical trial: A scientifically controlled study carried out usually
to test the effectiveness of a new treatment.
Phase III clinical trial: These are pilot efficacy studies aimed at generating
statistically relevant data.
Prime-boost: Administration of one type of vaccine, such as a live-vector
vaccine, followed by or together with a second type of vaccine, such as a
recombinant subunit vaccine. The intent of this combination regimen is to
induce different types of immune responses and enhance the overall im-
mune response, a result that may not occur if only one type of vaccine we-
re to be given for all doses.
Priming: Giving one vaccine dose(s) first to induce certain immune respons-
es, followed by or together with a second type of vaccine. The intent of
priming is to induce certain immune responses that will be enhanced by the
booster dose(s).
R
Randomized trial: A study in which participants are assigned by chance to
one of two or more intervention arms or regimens. Randomization mini-
mizes the differences among groups by equally distributing people with
particular characteristics among all the trial arms.
Receptor: A protein molecule on the surface of a cell that serves as a recog-
nition or binding site for antigens, antibodies or other cellular or immunol-
ogy components.
Recombinant DNA technology: The technique by which genetic material
from one organism is inserted into a foreign cell in order to mass-produce
the protein encoded by the inserted genes.
Registration (Licensing, Authorization, Approval): The process of ap-
proving a drug for marketing in a country/region. Includes assessment us-
ing particularly the criteria of safety, quality, and efficacy. As a conse-
quence of inadequate local capacity many developing countries rely on
"third party certification," i.e. granting market authorization to products
approved in certain developed countries.
Regulatory authority: A government agency responsible for codifying and
enforcing rules and regulations as mandated by law.
Resistance: The ability of an organism to develop strains that are impervious
to specific threats to their existence. For example, the malaria parasite has
developed strains that are resistant to drugs such as chloroquine. The
Anopheles mosquito has developed strains that are resistant to DDT and
other insecticides.
S
SAGE: Abbreviation for Serial Analysis of Gene Expression.
Sarcoma: A connective tissue neoplasm, usually highly malignant, formed
by proliferation of mesodermal cells.
SCAN: A process for the rapid isolation and functional characterization of
tumor-infiltrating and tumor-associated T cells.
SCID: Severe combined immunodeficiency is an immune deficiency disease
in which neither antibody nor T cell responses are made. It is usually the
result of T cell deficiencies. The SCID mutation causes severe combined
immune deficiency.
Serial analysis of gene expression: SAGE is a high throughput differential
gene expression methodology that permits the identification of candidate
tumor antigen within a subset of differentially expressed genes that are over
expressed in cancer cells.
Solid phase epitope recovery: SPHERE, a combinatorial, peptide library
screening technology that enables the identification of the minimal epitope
recognized by T cells.
Strain: A specific version of an organism. Many diseases, including
HIV/AIDS and hepatitis, have multiple strains.
Statistical significance: The probability that an event or difference occurred
as the result of the intervention (vaccine) rather than by chance alone. This
probability is determined by using statistical tests to evaluate collected da-
ta. Guidelines for defining significance are chosen before data collection
begins.
S35T
T cells: A subset of lymphocytes defined by their development in the thymus
and by heterodimeric receptors associated with the proteins of the CD3
complex. Most T cells have aß heterodimeric receptors but ?dT cells have
a ?d heterodimeric receptor.
TRP-2: Abbreviation for tyrosine related protein-2, a melanogenesis-
related protein shown to be immunogenic in an animal model and
humans.
Tumor: (a) A swelling; a morbid enlargement. (b) A neoplasm; i.e., a mass
of new tissue, physiologically useless, growing independently of its sur-
roundings.
V
Vaccination: The administration of a killed or weakened infectious organism
in order to prevent the disease.
Vaccine: A product that produces immunity to protect the body from a disease.
Vaccines are administered through needle injections, by mouth or by aerosol.
Vaccinia virus: Virus causing cowpox, used by Edward Jenner in the suc-
cessful vaccination against smallpox, which is caused by the related vario-
la virus.
Vector: A carrier.
Virulence factor: A specific factor possessed by an organism rendering the
organism pathogenic, or established in the host.
Virus: A tiny organism that multiplies within cells and causes disease such
as chickenpox, measles, mumps, rubella, pertussis and hepatitis. Antibiot-
ics, the drugs used to kill bacteria, do not affect viruses.
Virus-like particle: An assembly of capsid proteins into a shell-like struc-
ture without nucleic acid and thus non-infectious. These empty shells can
display conformational epitopes that are not present on individual purified
capsid proteins.
S36Board of Trustees
Chairman
H. R. Shepherd
Co-Chairman
William R. Berkley
Founding President
Philip K. Russell, MD
Secretary/Treasurer
Michael E. Whitham, Esq.
Mary Ann Chaffee
Allan L. Goldstein, PhD
Nancy Gardner Hargrave
Lewis A. Miller
Edward S. Neiss, MD, PhD
Michael T. Osterholm, PhD, MPH
Carol Ruth Shepherd
Leadership Council
Jason Berman
Zev Braun
Betty F. Bumpers
Francis Cano, PhD
Phyllis Freeman, JD.
Sister Colette Mahoney
Harvey S. Sadow, PhD
Ronald J. Saldarini, PhD
Andrea K. Scott, Esq.
Georges C. St. Laurent, Jr.
Patricia Thomas
Donna J. Twist, PhD
Stephen G. Valensi, Esq.
James D. Watson, PhD
Lawrence J. Wilker, PhD
Barbara Wilson
Scientific Advisory Council
Chairman
Peter J. Hotez, MD, PhD
Francis E. Andre, MD
John V. Bennett, MD
Barry R. Bloom, PhD
Walter E. Brandt, PhD
Fred Brown, PhD, FRS
Robert M. Chanock, MD
John R. David, MD
Ciro A. de Quadros, MD, MPH
Dr. Jonathan M. Fine MD
Bruce G. Gellin, MD, MPH
John L. Gerin, PhD
Lance K. Gordon, PhD
Neal A. Halsey, MD
Scott B. Halstead, MD
Maurice R. Hilleman, PhD, DSc
Stephen L. Hoffman, MD, PhD, DSc
Samuel L. Katz, MD
Col. Patrick W. Kelley, MD, DrPH
Gerald T. Keusch, MD
Adel A.F. Mahmoud, MD, PhD
Mark Miller, MD
Carol Anne Nacy, PhD
Erling Norrby, MD, PhD
Sir Gustav J. V. Nossal, MD, PhD
Stanley A. Plotkin, MD
Jerald C. Sadoff, MD
Allan Saul, PhD
Steven E. Schutzer, MD
Michael Sela, PhD
Donald S. Shepard, PhD
Alan Sher, PhD
John P. Woodall, PhD
S37
The Albert B. Sabin Vaccine Institute Board and CouncilsJean-Pierre Abastado, PhD
Vice President, Scientific Affairs
IDM (Immuno-Designed Molecules)
Neil L. Berinstein, MD
Associate Vice President, Clinical Oncology
Program Director, Cancer
Aventis Pasteur Ltd.
Jay A. Berzofsky, MD, PhD
Chief, Molecular Immunogenetics and Vaccine Research
National Cancer Institute, NIH
Harvey Brandwein, PhD
President
Immuno-Rx, Inc.
Claudine Bruck, PhD
Director, Cancer Vaccines Program
GlaxoSmithKline Biologicals
Esteban Celis, MD, PhD
Professor, Department of Immunology
Mayo Clinic
Lieping Chen, MD, PhD
Mayo Clinic, Department of Immunology
Richard W. Dutton, PhD
Trudeau Institute, Inc.
Allan L. Goldstein, PhD
Professor and Chairman
Department of Biochemistry and Molecular Biology
The George Washington University
June Kan-Mitchell, PhD
Associate Professor, Department of Immunology
and Microbiology and Pathology
Karmanos Cancer Institute
Wayne State University
W. Martin Kast, PhD
Professor, Microbiology and Immunology
and Pharmacology
Cardinal Bernardin Cancer Center
Loyola University Chicago
Thomas J. Kipps, MD, PhD
Professor and Head, Division of Hematology/Oncology
Deputy Director of Research
UCSD Cancer Center
Lawrence B. Lachman, PhD, MBA
Professor, Department of Bioimmunotherapy
University of Texas M.D. Anderson Cancer Center
Caroline Le Poole, PhD
Assistant Professor, Pathology
Loyola University Medical Center
H. Kim Lyerly, MD
Duke University Medical Center
Matthew F. Mescher, PhD
Professor and Kimmelman Chair in Immunology
Director, Center for Immunology
University of Minnesota Medical School
Lucio Miele, MD, PhD
Associate Professor, Department of Pharmaceutics
and Pharmacodynamics
University of Illinois at Chicago
Lewis A. Miller
Chairman
Intermedica, Inc.
Malcolm S. Mitchell, MD
Professor of Medicine, Immunology and Microbiology
Hudson-Webber Cancer Research Center
Karmanos Cancer Institute
Wayne State University
Edward S. Neiss, MD, PhD
Vice Chairman, President and CEO
Almedica International, Inc.
Suzanne Ostrand-Rosenberg, PhD
Professor, Department of Biological Sciences
University of Maryland
Ralph A. Reisfeld, PhD
Professor, Department of Immunology
The Scripps Research Institute
Michael L. Salgaller, PhD
Vice President, Clinical and Research Affairs
Northwest Biotherapeutics, Inc.
John Samuel, PhD
Professor, Faculty of Pharmacy and Pharmaceutical
Sciences
University of Alberta
Jeffrey Schlom, PhD
Chief, Laboratory of Tumor Immunology and Biology
National Cancer Institute, NIH
Stephen P. Schoenberger, PhD
Division of Immune Regulation
La Jolla Institute for Allergy and Immunology
H. R. Shepherd, DSc
Chairman
Albert B. Sabin Vaccine Institute
Walter J. Storkus, PhD
Associate Professor, Department of Surgery
University of Pittsburgh School of Medicine
Wei-Zen Wei, PhD
Professor, Breast Cancer Program
Karmanos Cancer Institute
Wayne State University
Darcy B. Wilson, PhD
Scientific Director
Torrey Pines Institute for Molecular Studies
S38
Fourth Annual Walker’s Cay Colloquium Participants
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