TRICOM vector based cancer vaccines.
ABSTRACT For the immune system to mount an effective antitumor T-cell response, an adequate number of T-cells specific for the antigens expressed by the malignancy must be activated . Since most antigens expressed by tumors are "self"-antigens, tumor antigens often lack endogenous immunogenicity and thus do not sufficiently activate T-cells to levels that can mediate tumor eradication. In addition, virtually all solid tumor cells lack the costimulatory molecules necessary to activate tumor-specific T-cells. Approaches that stimulate immune responses to these tumor antigens have the potential to alter this poor responsiveness. This theory has promoted the use of active immunotherapy to generate immune responses against tumor-associated antigens (TAAs) for the treatment of cancer. As one such vaccine strategy, we have utilized poxviruses as delivery vehicles for TAAs in combination with T-cell costimulatory molecules. Initial studies have demonstrated that the insertion of costimulatory molecule trangenes into viral vectors, along with a TAA transgene, greatly enhances the immune response to the antigen. Using this approach, a TRIad of COstimulatory Molecules (TRICOM; B7-1, ICAM-1 and LFA-3) has been shown to enhance T-cell responses to TAAs to levels far greater than any one or two of the costimulatory molecules in combination. In this article, preclinical findings and recent clinical applications of TRICOM-based vaccines as a cancer immunotherapy are reviewed.
- SourceAvailable from: Fabrizio RomanoPancreatic Cancer - Clinical Management, 03/2012; , ISBN: 978-953-51-0394-3
- Current Pharmaceutical Design 01/2006; 12(3):259-260. · 3.29 Impact Factor
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ABSTRACT: Cancer-testis (CT) antigens are tumor-associated antigens attracting immunologists for their possible application in the immunotherapy of cancer. Several clinical trials have assessed their therapeutic potentials in cancer patients. Breast cancers, especially triple-negative cancers are among those with significant expression of CT genes. Identification of CT genes with high expression in cancer patients is the prerequisite for any immunotherapeutic approach. CT genes have gained attention not only for immunotherapy of cancer patients, but also for immunoprevention in high-risk individuals. Many CT genes have proved to be immunogenic in breast cancer patients suggesting the basis for the development of polyvalent vaccines.Immunotherapy 02/2014; 6(2):165-79. · 2.39 Impact Factor
Current Pharmaceutical Design, 2006, 12, 351-361351
1381-6128/06 $50.00+.00© 2006 Bentham Science Publishers Ltd.
TRICOM Vector Based Cancer Vaccines
Charlie T. Garnett, John W. Greiner, Kwong-Yok Tsang, Chie Kudo-Saito, Douglas W.
Grosenbach, Mala Chakraborty, James L. Gulley, Philip M. Arlen, Jeffrey Schlom* and
James W. Hodge
Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, MD, USA
Abstract: For the immune system to mount an effective antitumor T-cell response, an adequate number of T-cells specific
for the antigens expressed by the malignancy must be activated . Since most antigens expressed by tumors are “self”-
antigens, tumor antigens often lack endogenous immunogenicity and thus do not sufficiently activate T-cells to levels that
can mediate tumor eradication. In addition, virtually all solid tumor cells lack the costimulatory molecules necessary to
activate tumor-specific T-cells. Approaches that stimulate immune responses to these tumor antigens have the potential to
alter this poor responsiveness. This theory has promoted the use of active immunotherapy to generate immune responses
against tumor-associated antigens (TAAs) for the treatment of cancer. As one such vaccine strategy, we have utilized
poxviruses as delivery vehicles for TAAs in combination with T-cell costimulatory molecules. Initial studies have demon-
strated that the insertion of costimulatory molecule trangenes into viral vectors, along with a TAA transgene, greatly en-
hances the immune response to the antigen. Using this approach, a TRIad of COstimulatory Molecules (TRICOM; B7-1,
ICAM-1 and LFA-3) has been shown to enhance T-cell responses to TAAs to levels far greater than any one or two of the
costimulatory molecules in combination. In this article, preclinical findings and recent clinical applications of TRICOM-
based vaccines as a cancer immunotherapy are reviewed.
Key Words: TRICOM, costimulation, vaccination, TAA, cancer immunotherapy.
Stimulation and activation of T-cells is essential for a
successful adaptive immune response to an antigen. Tumor-
associated antigens (TAAs) are often self-antigens that are
over-expressed in tumor tissues as compared to normal tis-
sues , and a sufficient immune responses to TAAs may
result in direct attack of tumor. Unfortunately, even though T
lymphocytes specific for TAA are often present in tumor
bearing hosts, these immune responses are often ineffective.
As such, a primary goal of cancer immunotherapy is to re-
verse this state and elicit an immune response capable of
tumor destruction. Such strategies are aimed at shifting
TAAs from a non-immunostimulatory to an immunostimu-
The material presented here is a review of previously
published results. The approaches reviewed here focus on the
development of cancer vaccines that alter the context in
which TAAs are presented to the immune system, resulting
in greater activation of TAA-specific T-cells than that occur-
ring naturally in the host. Moreover, this review is on the
current state of the use of multiple costimulatory molecules
in vector based vaccines containing a TRIad of COstimula-
tory Molecules (designated TRICOM) as an active immu-
notherapy for a range of human cancers.
*Address correspondence to this author at the Laboratory of Tumor Immu-
nology and Biology, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, 10 Center Drive, Building 10, Room 8B09,
MSC 1750, Bethesda, MD 20892-1750, USA; E-mail: email@example.com
RECOMBINANT VACCINE VECTORS ENCODING
SIGNALS 1 AND 2
The primary strategy of many research groups has been
to explore modalities that will enhance the immunogenicity
of tumor antigens. We, and others, have explored this by
placing genes for different T-cell costimulatory molecules
into viral vectors along with the transgene for the TAA. Re-
combinant poxviruses represent an attractive delivery vehicle
for cancer vaccines for several reasons. In addition to being
inherently immunostimulatory, these viruses have a broad
host range, a high efficiency of infection, and can be engi-
neered to express multiple transgenes, which are efficiently
processed through the cytoplasm . We have employed
both orthopox (vaccinia and MVA) and avipox (fowlpox)
viruses as vectors to help define optimal cancer vaccine
strategies. Replication competent vaccinia viruses, as well as
replication defective MVA and avipox viruses, have been
utilized for this purpose.
In order for the immune system to mount an effective
antitumor T-cell response, an adequate number of T-cells
specific for the antigens expressed by the malignancy must
be activated. Both CD4+ and CD8+ T-cells have been shown
to play an important role in the development of immunity to
cancer and can lead to therapeutic immune responses in tu-
mor bearing animals. Initiation of an immune response re-
quires at least two signals for the activation of naïve T-cells
by antigen presenting cells (APCs). The first signal is anti-
gen specific and is mediated through a peptide:MHC class I
complex on the surface of an APC that directly interacts with
the T-cell receptor . The second signal is mediated
352 Current Pharmaceutical Design, 2006, Vol. 12, No. 3Garnett et al.
through the interaction of a costimulatory molecule(s) on the
surface of an APC with its ligand(s) on the T-cell surface
(Fig. 1). Although it is now recognized that T-cells receive
many signals during activation that are important for pheno-
type, proliferation, survival, and trafficking, this two-signal
hypothesis remains the framework for activation of naïve T-
cells. Recombinant poxviruses have been engineered to ex-
press both signal 1 (antigen) and signal 2 (costimulatory
molecules), with each transgene being driven by a different
Our laboratory has utilized three human TAAs as signal
1: carcinoembryonic antigen (CEA), mucin-1 (MUC-1), and
prostate-specific antigen (PSA). CEA and MUC-1 are over-
expressed in a spectrum of carcinomas versus adult normal
tissues, while PSA is characterized as a tissue-specific anti-
gen expressed in normal prostatic epithelium as well as in
prostatic carcinoma [4-6]. The majority of preclinical studies
have utilized CEA as a target antigen. Similar clinical studies
are ongoing employing vaccines encoding genes for CEA,
PSA and MUC-1 as vaccine targets. Preclinical studies have
demonstrated that when a TAA transgene is placed into a
poxvirus genome, its expression leads to a more vigorous
host T-cell immune response to the TAA than would be
achieved otherwise . This enhanced response is attributed
to the acute inflammatory response directed against the
highly immunogenic vaccinia proteins, which in turn creates
a pro-inflammatory environment.
The context of initial antigen exposure determines the
quality of the subsequent T-cell immune response . Out-
comes include proliferation , cytokine secretion , dif-
ferentiation, cell death or anergy  of responding T-cells.
The outcome of the immune response to the first signal (an-
tigen) is greatly dependent upon a second “costimulatory”
signal 2. The need for T-cell costimulation appears to be
especially important in the presence of weak signal 1 such as
TAAs . Stimulation of a T-cell without the appropriate
costimulation can result in unresponsive or non-functional T-
cells. To date, numerous T-cell costimulatory molecules
have been identified (reviewed in ). These costimulatory
proteins are normally present on APCs (Fig. 1) and include
genes within the B7 family, genes within the TNF-family
and molecules typically associated with adhesion, such as
ICAM-1 and LFA-3. Each costimulatory molecule has a
corresponding ligand on the T-cell as shown in Fig. 1. CD28
is the most extensively studied costimulatory molecule and
its ligands are B7-1 and B7-2 expressed on APCs. CD2 and
LFA-1 expressed on the surface of T-cells bind to LFA-3
and ICAM-1, respectively. These interactions are thought to
be primarily adhesive as they increase the interaction time
between T-cells and APCs. It remains an open question
whether these accessory interactions are either purely adhe-
sive or purely costimulatory or a combination of the two.
Fig. (1). Signal 1 and signal 2. Depicted are signal 1, a peptide presented in MHC class I to TCR, and signal 2, three costimulatory molecules
reported to have a positive influence on T-cell activity. Taken from .
TRICOM Vector Based Cancer VaccinesCurrent Pharmaceutical Design, 2006, Vol. 12, No. 3 353
MOLECULES IN VITRO
OF MULTIPLE COSTIMULATORY
T-cell costimulatory molecules are expressed on profes-
sional APCs, such as dendritic cells (DCs) and B cells, and
are not expressed on the vast majority of solid tumors. Be-
cause both signal 1 and signal 2 must be present on the same
cell for efficient T-cell activation, weak TAAs expressed on
the tumor in the absence of costimulation would not activate
T-cells. This interaction can lead to T-cell anergy or apopto-
sis of naïve T-cells . As a strategy aimed to circumvent
this negative outcome, a panel of viral vectors was engi-
neered to express both the tumor antigen and the necessary
costimulatory molecules . We have now developed and
evaluated immune responses to recombinant pox vectors
containing one, two and three different T-cell costimulatory
molecules, alone or in combination with a TAA.
Early in vitro studies demonstrated that weakly immuno-
genic murine carcinoma cells infected with pox vectors con-
taining the costimulatory molecule B7-1, and not control
vectors, were capable of inducing the activation of T-cells
directed against the tumor . Further study demonstrated
that the use of vectors containing three costimulatory mole-
cules (B7-1, ICAM-1, and LFA-3) was far better than vec-
tors containing two costimulatory molecules, which was only
moderately better than the use of a vector containing a single
costimulatory molecule. This TRIad of COstimulatory
Molecules (designated TRICOM) was selected because each
costimulatory molecule has an individual ligand on T-cells,
and each has previously been shown to prime a unique sig-
naling pathway in T-cells .
The level of activation of T-cells stimulated with
TRICOM infected cells has been shown to be synergistic and
not just simply additive between individual T-cell costimu-
latory molecules (Fig. 2). TRICOM-infected stimulator cells
greatly enhance T-cell proliferation in response to mitogen,
allo-antigens and peptide-pulsed targets . In addition,
this triad of costimulatory molecules stimulated IL-2 (inter-
leukin-2) production primarily from CD4+ T-cells and IFN-γ
(interferon-γ) production predominantly from CD8+ T-cells.
TRICOM-expressing vectors have also been successfully
used to infect murine DCs , murine DC precursors ,
human DCs , human B cells , and rhesus APCs .
Each of these model systems further confirmed the ability of
TRICOM-infected cells to significantly enhance immune
responses to mitogen, allo-antigens or specific peptides. In-
fection of these professional APC cell types with TRICOM
vectors consistently resulted in higher than normal expres-
sion levels of this triad of costimulatory molecules and en-
hanced capacity to stimulate naïve and effector T-cells .
MOLECULES IN VIVO
OF MULTIPLE COSTIMULATORY
Studies were conducted to determine whether an antigen-
specific immune response could be enhanced in vivo using
Fig. (2). T-cell activation using multiple costimulatory molecules. Left panel, three different T-cell costimulatory molecules (components of
TRICOM) and their respective ligands on T-cells. Right panel, Activation of naïve murine T-cells using Con A as signal 1, and none, one or
three (TRICOM) different costimulatory molecules as signal 2. APCs were infected with pox vectors containing individual costimulatory
molecules or TRICOM. Taken from reference .
354 Current Pharmaceutical Design, 2006, Vol. 12, No. 3Garnett et al.
recombinant vectors. Fig. 3 demonstrates the relative poten-
cies in the induction of T-cell proliferation in mice vacci-
nated with fowlpox vectors containing a TAA and either
none, one or three costimulatory molecules. Four vaccina-
tions with CEA-expressing viral vector induced a modest
CD4+ T-cell response against CEA protein. Four vaccina-
tions with CEA/B7-expressing vector induced an even
greater level of CEA-specific CD4+ T-cell responses. A sin-
gle vaccination with the CEA/TRICOM vector, however,
was capable of inducing the same level of CEA-specific T-
cells as four vaccinations with CEA/B7. T-cell responses
were further enhanced with continued vaccination with
CEA/TRICOM vector (Fig. 3). In addition to demonstrating
that a TRICOM vector is more effective than a vector con-
taining a single costimulatory molecule, these data suggest
that immune responses directed against the transgene were
further amplified following each vaccination .
DCs infected with vectors expressing costimulatory
molecules were used as a whole tumor cell vaccine in mice.
Wild type (WT)–, B7-1– and TRICOM-infected DCs were
pulsed with OVA peptide and administered i.v. to C57BL/6
mice to prime naïve T-cells in vivo . Splenocytes har-
vested 14 days after vaccination were then restimulated in
vitro and used in an antigen-specific CTL (cytotoxic T lym-
phocyte) assay against peptide-pulsed syngeneic target cells.
Mice vaccinated with B7-1–infected DCs exhibited a CTL
response that was significantly stronger than that of mice
vaccinated with uninfected DCs. However, the most potent
lytic activity was observed in mice vaccinated with DCs in-
fected with the TRICOM vector. Each of the groups men-
tioned above demonstrated higher CTL activity than control
mice vaccinated with peptide and adjuvant .
A series of studies conducted by Aarts et al. determined
the optimal way to utilize TRICOM based vectors .
These strategies involve the use of diversified vectors in the
priming and boosting vaccination of animals, as well as the
addition of exogenous cytokines at the time of vaccination.
Vaccinia viruses can efficiently infect mammalian cells and
replicate for 7 days before being eliminated by the host im-
mune system . Conversely, while fowlpox viruses can
replicate in avian cells, they are replication defective in
mammalian cells . As such, fowlpox vectors infect
mammalian cells and express their transgenes for approxi-
mately 14-21 days before the cell dies. Based on analysis of
antigen-specific immune responses, it was determined that
recombinant vaccinia vectors (rV) should be used once as a
priming vaccination and recombinant fowlpox vectors (rF)
should be used in the subsequent boosting vaccinations. Sec-
ondly, in this model the addition of cytokines, such as GM-
CSF (granulocyte macrophage–colony stimulating factor) or
IL-2, to vaccine was essential in inducing anti-tumor activity
as compared with the use of cytokines alone, or the use of
vaccines without cytokine. IL-2 has been found to enhance
T-cell proliferation and GM-CSF has been shown to recruit
APCs to regional lymph nodes. Initial studies revealed that
GM-CSF used with TRICOM vectors almost doubled the T-
Fig. (3). Comparison of strength immune response between vaccines encoding none, one or three costimulatory molecules. CEA-specific T-
cell responses following multiple vaccinations with rF-CEA, rF-CEA/B7-1, or rF-CEA/TRICOM. C57BL/6 mice were vaccinated s.c. one,
two, three, or four times at 2-week intervals with avipox wild-type (rF-WT) (closed square), rF-CEA (diamond), rF-CEA/B7-1 (triangles),
rF-CEA/TRICOM (circles), or HBSS buffer (asterisk). Two weeks following the last vaccination, purified splenic T-cells were tested for
reactivity to CEA protein, or negative control protein ovalbumin (all values negative) in a lymphoproliferation assay. Taken from reference
TRICOM Vector Based Cancer VaccinesCurrent Pharmaceutical Design, 2006, Vol. 12, No. 3 355
cell response when compared with the TRICOM vector used
alone . Utilization of these strategies served to further
amplify antigen-specific T-cell responses generated by
TRICOM vector vaccination.
Tumor therapy studies were conducted to determine the
therapeutic efficacy of vaccine regimes with different
costimulatory capacities, including vectors containing
CEA/TRICOM, CEA/B7-1 and CEA alone. In these studies
mice transgenic (tg) for CEA were utilized. These mice ex-
press CEA as a self-antigen in fetal and normal gastrointesti-
nal (GI) tissues at levels similar to those in humans. CEA-tg
mice have previously been shown to be tolerant to the in-
duction of CEA-specific T-cell or antibody responses fol-
lowing vaccination with CEA protein in adjuvant . CEA-
expressing murine tumor cells were inoculated intraspleni-
cally into CEA-tg mice followed by immediate removal of
spleens resulting in the development of peripancreatic tu-
mors expressing CEA . Fourteen days after tumor trans-
plant vaccination was initiated, with GM-CSF and IL-2 as
biological adjuvants with all vaccinations. Mice received
four weekly vaccinations. As can be seen in Fig. 4, this tu-
mor is lethal in 80-100% of mice by 10 weeks post-
transplant if left untreated. Only moderate anti-tumor effects
are seen using the CEA-based vaccine without the addition
of costimulation, and the use of CEA/B7 vaccines slightly
increased these anti-tumor effects. The use of CEA/
TRICOM vaccines, however, resulted in a statistically sig-
nificant increase in anti-tumor effect when compared with
the CEA, CEA/B7, TRICOM only or wild-type vectors.
Sixty percent of the mice receiving CEA/TRICOM vaccines
remained alive and apparently healthy through the 25-week
observation period, whereas only 25% of the mice receiving
CEA/B7-1 vaccines survived past 16 weeks (Fig. 4).
Fig. (4). Induction of anti-tumor responses in CEA transgenic (CEA-tg) mice by recombinant poxviral vectors encoding none, one or three
costimulatory molecules. CEA-tg mice bearing 14-day established peripancreatic metastases were divided into five treatment groups. Tumors
were transplanted by intrasplenic injection of MC-38 colon carcinoma cells that were transduced with CEA (open arrow, day 0). Vaccine
schedule is indicated by the closed arrows (weeks 2–5). Group 1 received an rV-CEA/TRICOM prime vaccination followed by three weekly
boosts with rF-CEA/TRICOM. Group 2 received an rV-CEA/B7-1 prime vaccination followed by three weekly boosts with rF-CEA/B7-1.
Group 3 received an rV-CEA prime vaccination followed by three weekly boosts with rF-CEA. Group 4 received an rV-TRICOM prime
vaccination followed by three weekly boosts with rF-TRICOM. Group 5 received rV-WT prime vaccination followed by three weekly boosts
with FP-WT. For groups 1-5, all prime vaccinations were administered with recombinant GM-CSF and low-dose IL-2, and all boost vacci-
nations were admixed with rF-GM-CSF and low-dose IL-2. Group 6 received only HBSS buffer injections. Taken from reference .
356 Current Pharmaceutical Design, 2006, Vol. 12, No. 3Garnett et al.
Studies were conducted to examine CEA-specific im-
mune responses generated in mice vaccinated as described
above. For these studies, a duplicate set of CEA-tg mice
were vaccinated but were not transplanted with peri-
pancreatic tumors . Twenty-one days after the last vacci-
nation, splenocytes were analyzed for CEA-specific immune
responses. Mice vaccinated with CEA/TRICOM vectors
exhibited significantly greater CEA-specific CD4+ T-cell
responses than mice receiving CEA/B7-1, CEA only,
TRICOM only or wild-type control vaccines as measured by
lymphoproliferation assay (Fig. 5A). In a similar manner,
IFN-γ production from CD8+ T-cells in response to CEA
peptide was the greatest in mice vaccinated with CEA/
TRICOM (Fig. 5B). Importantly, the generation of antigen-
specific T-cells increased in direct proportion with the num-
ber of costimulatory molecules inserted into the vector.
These results also demonstrate a concordance in the ability
of these vectors to mount CEA-specific CD4+ and CD8+ T-
cell responses, with anti-tumor responses measured by in-
creased survival . Serological studies revealed that there
was no evidence of neo-antibodies specific for B7-1, ICAM-
1, or LFA-3 in the mice successfully cured of tumor by the
CEA/TRICOM vaccination regimen. Moreover, no other
evidence of autoimmunity could be observed utilizing this
vaccine strategy .
TUMOR PREVENTION STUDIES
Early preclinical cancer studies utilized tumor prevention
models in mice to determine whether vaccination with
TRICOM vectors could induce long-term immunity. Normal
C57BL/6 mice were challenged with a high dose of CEA-
expressing murine tumor cells 100 days after a single vacci-
nation with rV-CEA/TRICOM . All mice vaccinated
with the TRICOM vector were alive 50 days after challenge,
whereas all mice vaccinated with rV-WT and rV-CEA vec-
tors succumbed to tumors in this simple model.
As noted previously, preclinical murine models express-
ing the complete human CEA gene as a transgene have been
generated with CEA expression occurring predominately
along the gastrointestinal tract, as it is in humans. To gener-
ate another clinically relevant model, Greiner et al. bred
these CEA-tg mice with MIN mice bearing a mutation in the
adenomatous polyposis coli (Apc) gene [26, 27]. MIN mice
carry a germ-line mutation of the murine Apc gene resulting
in the formation of multiple intestinal adenomas [27, 28].
Mice carrying both the MIN and human CEA genes (CEA-
tg/MIN) develop multiple intestinal neoplasms, which over-
express CEA to levels reminiscent of those reported for tu-
bulovillous intestinal adenomas from patients . Humans
with a homologous germ-line mutation in the Apc tumor
Fig. (5). Enhanced CEA-specific T-cell responses from CEA-tg mice vaccinated with TRICOM vectors. CEA-tg mice were divided into six
vaccination groups: Group 1 (closed circles) received an rV-CEA/TRICOM prime vaccination followed by three weekly boosts with rF-
CEA/TRICOM. Group 2 (closed diamonds) received an rV-CEA/B7-1 prime vaccination followed by three weekly boosts with rF-CEA/B7-
1. Group 3 (closed squares) received an rV-CEA prime vaccination followed by three weekly boosts with rF-CEA. Group 4 (open circles)
received an rV-TRICOM prime vaccination followed by three weekly boosts with rF-TRICOM. Group 5 (open diamonds) received an rV-
WT prime vaccination followed by three weekly boosts with FP-WT. For groups 1-5, all prime vaccinations were administered with recom-
binant GM-CSF and low-dose IL-2, and all boost vaccinations were admixed with rF-GM-CSF and low-dose IL-2. Group 6 (open squares)
received only HBSS buffer injections. In vitro assays were performed on splenic T-cells 3 weeks following the last vaccination. A, Lympho-
proliferation of splenic T-cells in response to CEA protein. Proliferation in response to the T-cell mitogen Con A (2.5 µg/ml) is shown in the
inset panel. B, IFN-γ production by T-cells in response to the 8-mer CEA526-533 peptide or control peptide VSV-N (open bars). Taken from
TRICOM Vector Based Cancer VaccinesCurrent Pharmaceutical Design, 2006, Vol. 12, No. 3 357
suppressor gene are predisposed to an inherited form of
colon cancer, familial adenomatous polyposis. This disease
is characterized by the early development of multiple colo-
rectal adenomas, which can subsequently form carcinomas
These CEA-tg/MIN mice have been utilized as a pre-
clinical model in which vaccine strategies can be tested for
the prevention of spontaneous intestinal tumorigenesis. As
such, CEA-tg/MIN were utilized in studies designed to
evaluate CEA-specific immune responses generated follow-
ing vaccine therapy of these mice. Animals received a pri-
mary vaccination with rV-CEA/TRICOM followed by
booster vaccinations with rF-CEA/TRICOM in combination
with GM-CSF as a biological adjuvant . The vaccine
regimen generated strong CEA-specific host immune re-
sponses in these mice, which resulted in significantly sup-
pressed intestinal tumor load when analyzed at necropsy as
well as improved long-term survival of mice . The ad-
ministration of the vaccine devoid of the CEA transgene,
however, did not suppress tumor formation. Forty weeks
post-vaccination, 50% of the mice that received CEA/
TRICOM vaccination remained alive, while none of the
control mice survived past week 27. In addition, no evidence
of autoimmunity directed against normal tissues was ob-
served in mice in which the CEA-based vaccine significantly
reduced intestinal tumor load .
A series of experiments by Kudo-Saito et al. sought to
determine if intratumoral (i.t.) vaccination with TRICOM
vectors could result in tumor eradication . CEA-tg mice
bearing day 8 CEA+ s.c. tumors were vaccinated with CEA/
TRICOM vectors and GM-CSF. Mice vaccinated s.c. with
rV-CEA/TRICOM and boosted s.c. with rF-CEA/TRICOM
on days 15, 22 and 29 showed no anti-tumor effects (Fig.
6D). However, mice vaccinated s.c. with rV-CEA/TRICOM
vectors on day 8 and boosted i.t. on days 15, 22, and 29 with
rF-CEA/TRICOM vectors had significantly inhibited tumor
growth, with 14 of 20 mice being cured (Fig. 6C). Moreover,
73% of mice cured of tumors subsequently rejected tumors
upon rechallenge, which demonstrated long-term immu-
nological memory. These data indicate that systemic priming
followed by i.t. boosting with TRICOM-based vectors is an
effective modality in the therapy of large aggressive tumors.
This vaccine strategy holds promise not only for the therapy
of s.c. tumors but also for other tumors accessible by surgery.
VACCINATION PLUS LOCAL TUMOR
The ability to combine TRICOM-based vaccines safely
with other modalities commonly used to treat tumors is im-
portant. Local radiation is an established therapy for human
tumors. Chakraborty et al. demonstrated a biological synergy
between local radiation of tumor and vaccine therapy with
TRICOM vectors . Tumor-bearing CEA-tg mice were
vaccinated s.c. with CEA/TRICOM vectors and GM-CSF in
combination with local tumor irradiation. Mice were vacci-
nated on days 8, 15, 22 and 29 post tumor transplant and
tumors received 2Gy/day of radiation for four days (days 11-
14). Subcutaneous vaccination alone and irradiation alone
did not significantly inhibit tumor growth (Fig. 7). However,
therapy of tumors with the combination of vaccine regimen
Fig. (6). Efficacy of intratumoral (i.t.) vaccination following subcutaneous priming with rV-CEA/TRICOM on advanced tumors in a self-
antigen system. CEA-tg mice were implanted s.c. with MC38-CEA+ tumors on day 0. Panel A: Control mice were administered PBS i.t. on
days 8, 15, 22 and 29. Panel B: Mice were vaccinated i.t. with rF-CEA/TRICOM on days 15, 22 and 29. Panel C: Mice were vaccinated s.c.
with rV-CEA/TRICOM on day 8. Mice were then boosted i.t. with rF-CEA/TRICOM on days 15, 22 and 29. Panel D: Mice were vaccinated
s.c. with rV-CEA/TRICOM on day 8. Mice were then boosted s.c. with rF-CEA/TRICOM on days 15, 22 and 29. Each virus was admixed
with rF-GM-CSF. Tumor volume was monitored 1-2 times a week. These data are the compilation of four separate experiments. Inset panels:
mean tumor volumes of mice responding to (heavy line) or failing (dotted line) vaccine therapy. Taken from reference .
358 Current Pharmaceutical Design, 2006, Vol. 12, No. 3Garnett et al.
and irradiation resulted in a marked and significant decrease
in tumor growth rate and volume (Fig. 7D); 55% of the mice
treated with the combination resolved their tumor mass and
remained tumor free for the duration of the experiment. This
synergy between the two modalities was shown to be due to
upregulation of surface Fas on the irradiated tumors and
massive infiltration of the tumor with T-cells that was not
seen when either modality was used alone .
QUALITY OF T-CELLS GENERATED BY TRICOM
While the number of antigen-specific CD8+ T-cells has
been shown to play a pivotal role in anti-tumor immunity, it
is thought that only high avidity CD8+ CTL can mediate
protective immunity. The affinity of antigen-specific CD8+
T-cells may ultimately be determined during the course of
initial contact with antigen and APCs. Previous studies
clearly demonstrated that the use of TRICOM vectors can
enhance the quantity of antigen-specific T-cells generated,
but did not address the quality of such T-cells. To address
this question, Oh et al. measured functional avidity of CD8+
CTL using a system in which signals from MHC Class I
(signal 1) and costimulatory signals (signal 2) were adjusted
by varying antigen dose and by the use of TRICOM poxvirus
vectors, respectively . A strong signal 1 resulted in an
increased frequency of CTL. However, strong signal 2, pro-
vided by infection of APCs with TRICOM vectors, was nec-
essary for the induction of high avidity CTL that killed target
cells more efficiently. In fact, only CTL induced with
TRICOM-infected cells were able to kill tumor cells
endogenously expressing low levels of antigen. Thus, al-
though TRICOM vectors have previously been shown to
increase the quantity of CTL responses, these studies dem-
onstrate that they can also improve the quality of CTL re-
sponse, especially those directed against a weak antigen such
as a TAA.
Fig. (7). Irradiation of tumor cells in vivo enhances efficacy of vaccine therapy. CEA-tg mice were injected with MC38-CEA+ tumor cells
s.c. Panel A: Mice received no treatment. Panel B: Mice were vaccinated with rV-CEA/TRICOM on day 8 (closed triangle), followed by
boosting with rF-CEA/TRICOM on days 15, 22, and 29 (gray triangles). Panel C: Tumors in mice were subjected to external beam irradia-
tion (2 Gy) in situ on days 11, 12, 13 and 14 (open inverted triangles). Panel D: Mice were vaccinated with rV-CEA/TRICOM on day 8
(closed triangle). Tumors were subjected to external beam irradiation (2 Gy) in situ on days 11, 12, 13 and 14 (open inverted triangles), fol-
lowed by boosting with rF-CEA/TRICOM on days 15, 22, and 29 (gray triangles). CEA-tg mice were injected with 3x105 MC38 tumor cells
(CEA-negative) s.c. Panel E: Mice received no treatment. Panel F: Mice were vaccinated with rV-CEA/TRICOM on day 8 (closed triangle),
followed by boosting with rF-CEA/TRICOM on days 15, 22 and 29 (gray triangles). Panel G: Tumors in mice were subjected to external
beam irradiation (8Gy) in situ on day 14 (open inverted triangle). Panel H: Mice were vaccinated with rV-CEA/TRICOM on day 8 (closed
triangle). Tumors were subjected to external beam irradiation (8Gy) in situ on day 14 (open inverted triangle), followed by boosting with rF-
CEA/TRICOM on days 15, 22, and 29 (gray triangles). In a subset of mice from each treatment group, tumors were surgically removed at
day 21 post-tumor transplant and co-stained with CEA and Fas antibodies. Inset panels: % Fas positive cells (mean fluorescent intensity). All
vaccines were co-administered with rF-GM-CSF. Tumor volume was monitored. Adapted from .
TRICOM Vector Based Cancer VaccinesCurrent Pharmaceutical Design, 2006, Vol. 12, No. 3 359
Another way to improve the quality of T-cells generated
with TRICOM-based vaccines is by enhancing the immune
response to the immunodominant TAA epitope via the use of
an agonist epitope. Tsang et al. have identified agonist epi-
topes for human CEA, PSA and MUC-1 [33-35]. These ago-
nist epitopes have the ability to activate T-cells to greater
levels than their native counterparts. Importantly, T-cells
generated utilizing these agonists can in turn kill tumor cells
expressing native epitope [33-35].
CLINICAL TRIALS EMPLOYING TRICOM BASED
To date, no vaccine has been approved by the FDA for
the treatment of cancer in the United States. Utilizing princi-
ples learned in hypothesis-driven preclinical animal model-
ing, TRICOM based vaccines are currently being investi-
gated in patients with advanced forms of cancer in a series of
clinical trials. A phase I trial using TRICOM constructs
given at monthly intervals has been completed . In this
trial, CEA/TRICOM vaccines modified to contain the im-
proved HLA-A2 motif within the CEA protein (CEA[6D])
were utilized . Both the vaccinia and fowlpox TRICOM
vaccines were well tolerated alone and in combination with
the cytokine GM-CSF. GM-CSF was given at the site of the
injection on days 1 through 4 of every 28-day treatment cy-
Fifty-eight patients with CEA positive advanced cancer
were treated in this trial. Twenty-three of 58 patients
achieved stable disease after four monthly vaccinations. In-
terestingly, 12 patients had progressive disease after their
second monthly vaccine before achieving stable disease after
their fourth vaccination. This need for multiple vaccinations
is not surprising since preclinical studies have shown that
with increasing numbers of CEA-TRICOM vaccinations, the
magnitude of the CEA-specific T-cell response also in-
creases. After the initial six vaccinations defined in the pro-
tocol, 12 patients who were still benefiting from the therapy
were shifted to an every-3-month schedule of boosting. In-
terestingly, all of these patients progressed on this admini-
stration schedule and when 11 of the 12 patients were re-
verted back to the monthly treatment schedule, six patients
restabilized. One patient with an advanced refractory small-
cell lung carcinoma on this trial had a complete pathologic
response at 15 months [36, 37].
This trial  was the first to incorporate all of the
strategies utilized in preclinical studies, including: (a) the use
of viral vector vaccines to enhance presentation of tumor
antigens to the immune system; (b) diversified prime and
boost strategies using both vaccinia and fowlpox vectors; (c)
the use of TRICOM costimulation to enhance T-cell re-
sponses; (d) altering the sequence of the tumor antigen CTL
epitope to enhance its immunogenicity; and (e) the use of
GM-CSF to enhance the recruitment of DCs to the vaccina-
tion site. Similar to preclinical studies, the immune responses
determined by ELISPOT assay in this clinical trial were
higher than those observed in previous clinical trials using
vaccinia and avipox-CEA vaccines with no costimulation
TRICOM based vaccines are currently being evaluated in
the clinical setting in both the NIH Clinical Center and at
numerous cancer centers throughout the country as part of a
collaborative tumor immunology program. Planned trials
will utilize various TRICOM vaccine vectors to treat a range
of human cancers including breast, lung, colorectal, mela-
noma, bladder and prostate. These trials will also evaluate
strategies such as intratumoral vaccination as well as combi-
nations of TRICOM vaccines with other therapeutic modali-
ties such as radiation and chemotherapy. CEA/TRICOM,
PSA/TRICOM, and CEA-MUC1/TRICOM vaccine vectors
are all under clinical investigation.
Five trials utilizing CEA/TRICOM vaccines are currently
recruiting patients for participation. First, a Phase I trial
treating non-small cell lung cancer (NCSLC) will evaluate
the safety and side effects of vaccine treatment with standard
radiation and chemotherapy treatment in patients with stage
III unresectable lung cancer . The second trial is a Phase
I study in patients with CEA-positive solid tumors that have
metastasized to the liver who are given vaccine with radia-
tion to the liver metastasis . A third Phase I trial is de-
signed to study the effectiveness of CEA/TRICOM vaccine
therapy in patients who have advanced or metastatic cancer
. In this study, the safety and feasibility of vaccinating
patients with autologous DCs infected with rF-CEA/
TRICOM vectors will be evaluated, as well as CEA-specific
immune response. The fourth trial is a Phase II trial designed
to evaluate the effectiveness of the combination of vaccines
and chemotherapy to treat metastatic breast cancer in pa-
tients whose tumors are CEA-positive . The final trial
utilizing CEA/TRICOM vaccines is a Phase II study de-
signed to compare the effectiveness of vaccine therapy with
and without docetaxel in patients who have metastatic lung
or colorectal cancer .
Patients are currently being recruited for participation in
four trials utilizing intratumoral vaccination. The first Phase
I trial is designed to examine the effectiveness of two differ-
ent vaccines, rF-TRICOM and rF-B7-1, administered di-
rectly into metastatic solid tumors . The second Phase I
trial is to study the effectiveness of neoadjuvant intravesical
vaccine therapy in treating patients who are undergoing cys-
tectomy for muscle-invasive bladder cancer . The third
trial is designed to evaluate the feasibility of an intra-
prostatic vaccination in prostate cancer patients with local
failure following radiotherapy . Finally, a Phase II trial
will utilize intratumoral vaccination to study the effective-
ness of vaccine therapy in treating patients who have metas-
tatic melanoma .
PSA/TRICOM vaccines are also being evaluated in the
clinic for the treatment of prostate cancer. This vaccine mo-
dality is currently being examined in both Phase I and II tri-
als. This vector is being utilized for the intraprostatic Phase I
trial mentioned previously . In an ongoing Phase I/II
study, this vaccine will be used to examine the safety and
immune response to sequential vaccinations in patients with
metastatic androgen insensitive prostate cancer ; thus far
objective radiographic responses and sustained PSA declines
have been seen (Todd, N., Gulley, J., Arlen, P., Society of
360 Current Pharmaceutical Design, 2006, Vol. 12, No. 3Garnett et al.
Urologic Oncology, 2004). In addition, a multi-center Phase
II trial will seek to evaluate the effectiveness of vaccine ther-
apy in treating patients who have metastatic androgen-
independent prostate cancer .
A second generation of TRICOM vaccines has been de-
veloped. Both the rV and rF vectors encode two tumor anti-
gens, both MUC-1 and CEA, in addition to TRICOM. This
vaccine strategy is being evaluated in both Phase I and Phase
II trials that will assess the immune response to vaccine in
patients with colorectal and other solid tumors [50, 51]. This
vaccine has already been tested in a Phase I clinical trial car-
ried out in pancreatic patients. This trial demonstrated high
levels of safety and preliminary evidence of increased sur-
vival . Based on these results, the first Phase III ran-
domized trial utilizing TRICOM vectors has been initiated in
second-line metastatic pancreatic cancer patients 1.
To move toward reducing the interval between disease
diagnosis and the initiation of vaccine therapy, major focus
will be on the use of vaccine regimens in combination with
front-line cancer therapies. This strategy would utilize cancer
vaccines in a minimal disease setting and likely optimize
vaccine efficacy in patients. Many of the trials mentioned
above will examine the combined use of TRICOM vaccines
in conjunction with other clinical therapies, such as local
external beam radiation of tumor and chemotherapy [39, 40,
The preclinical studies reviewed here demonstrate that
several different strategies can be used to enhance the immu-
nogenicity of a weak immunogen, such as a TAA. These
strategies include: (a) the use of viral vector vaccines to en-
hance presentation of tumor antigens to the immune system;
(b) diversified prime and boost strategies using both vaccinia
and fowlpox vectors; (c) the use of TRICOM vector based
costimulation to enhance T-cell responses; (d) altering the
sequence of the tumor antigen CTL epitope to enhance its
immunogenicity; and (e) the use of GM-CSF to enhance the
recruitment of DCs to the vaccination site. These preclinical
studies have formed the scientific basis for clinical trials for
further evaluation of these vaccines alone, or in combination
with conventional therapies for the treatment of a range of
We thank Debra Weingarten for her editorial assistance
in the preparation of this manuscript.
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