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Novel Vaccines for the Treatment of Gastrointestinal Cancers

Novel Vaccines for the Treatment of Gastrointestinal Cancers

ABSTRACT: Continuing advances in immunology and molecular biology during the past several decades have provided optimism that immunomodulatory strategies may be clinically useful in patients with cancer. Key advances have included: (1) recognition of the critical role of the antigen-presenting cell and greatly improved understanding of antigen processing and presentation, including the molecular interactions between HLA molecules and antigenic epitopes on the antigen-processing cell and the receptors on T cells, and (2) the roles of costimulatory molecules such as B7.1, ICAM-1, and LFA-3 in the induction and maintenance of an immune response. In addition, new techniques have allowed us to identify immunogenic antigenic determinants, alter their binding affinities, and evaluate the overall success of the intervention through both in vivo and in vitro assays. Carcinoembryonic antigen (CEA) is overexpressed in a large number of gastrointestinal, lung, and breast cancers. Clinical trials have established treatment protocols using viral vectors to immunize patients to CEA without producing deleterious autoimmune phenomena. By combining various vectors to include MUC-1 and/or CEA plus costimulatory molecules in a prime-and-boost regimen, we are beginning to see signs that this intervention can not only produce changes in immune function but also potentially improve clinical outcomes. Phase III studies to test these hypotheses are under way.

In the late 19th century, William
Coley at Memorial Sloan-Kettering
Cancer Center in New York
reported spontaneous regression of
sarcomas following severe bacterial
infections.[1] However, attempts to
reproduce these results with bacterial
extracts met with limited success.[2]
Subsequently, attention shifted from
infectious particles to antigens specific
for tumor cells recognized by
the immune system in a manner that
causes the rejection of the tumor as a
foreign tissue or non-self (tumorspecific
transplantation antigens). For
example, in mice, immunity to methylcholanthrene-
induced malignancies
could be produced by injecting genetically
identical animals with tumor
cell lysates.[3] This observation appeared
to reflect the tumor-specific
nature of the antigens induced by
chemical mutagens.

In the early 1960s, investigators
were encouraged by reports that malignant
cells from different tumors
share common tumor-associated antigens.[
4] The results of these studies,
coupled with advances in immunology,
led to the concept of immune surveillance-
a process in which
immunocompetent hosts are protected
from the emergence of malignant
clones, which are ostensibly identified
as non-self, by elements of the
innate and adaptive immune system.
[
5] These observations were exciting
and provided hope that stimulating
an active, specific immune
response might cure cancer, but subsequent
basic science and clinical studies
were disappointing. For example,
the concept of immunosurveillance
was questioned when athymic nude
mice were shown to have the same
incidence of tumors as wild-type animals,
and results of early clinical trials
with various approaches to stimulating
an active antitumor immune response
were not encouraging.[6]

More recent work has greatly expanded
our understanding of the interactions
between elements of the
immune system and cancer cells and
has renewed hope that therapeutic cancer
vaccines and other immunomodulatory
approaches may, in fact,
become valuable tools for clinical on-
cologists. Cytotoxic T lymphocytes
have been shown to kill HLA-matched
tumor cells in vitro, and immunogeninduced
upregulation of humoral
immunity has been shown to be associated
with improved outcomes in
patients with breast cancer or melanoma.[
7-9] The success of donor lymphocyte
infusions in patients with
hematologic malignancies has also
provided empiric evidence that immunomodulatory
therapy can be an
important clinical tool in patients with
advanced malignancies.[10,11]

Advances in Therapeutic
Cancer Vaccines

It is a key tenet of tumor immunology
that tumor cells express antigens
and the uniqueness of these antigens
makes them potential targets of innate
and adaptive immune elements.
Thus, it is no surprise that clinical
failures of therapeutic cancer vaccines
may be linked to a failure of antigen
recognition by the immune system.

With the advent of sensitive assay
systems, investigators have been able
to demonstrate the presence of naturally
occurring T cells directed against
tumor-associated antigens (TAAs).[12]
Gilboa has categorized TAAs that may
function in tumor rejection as two
types: (1) patient-specific, mutated
self-antigens; and (2) shared, nonmutated
self-antigens.[2]

Mutated self-antigens are the result
of the genetic instability of an
individual tumor and are generally
incidental to tumor pathophysiology.
While they would be anticipated to be
potent immunogens, identification and
isolation of these unique (private) antigenic
determinants would be impractical.
The significance of mutated
self-antigens to successful immunotherapy
was illustrated in a series of
experiments by Boon and colleagues.
These investigators were able to easily
transplant cell lines from teratocarcinoma
and Lewis lung carcinoma into
syngeneic mice. If, however, the malignant
cell line was first mutated by
exposure to N-methyl-N'-nitro-Nnitrosoguanidine,
development of cellline-
specific mutated self-antigens
elicited an immune response that protected
the mice from the transplanted
tumor cells.[13-15]

Unlike vaccines to private antigens,
vaccines to shared (public) antigens
as off-the-shelf therapeutics have the
potential to benefit many patients. In
most instances, these shared antigens
correspond to normal gene products
expressed on tumor cells, so patients
may be expected to be tolerant to many
of them. Other shared tumor antigens,
however, are fetal gene products not
expressed in adult tissues or are common
molecules related to malignancy
and found only in tumor cells. By
virtue of their limited distribution in
either time or space, oncofetal or sequestered
antigens, respectively, have
the potential to be targets for immunotherapy.
Recently reported examples
of oncofetal tumor antigens
include survivin, an inhibitor of
apoptosis expressed during fetal development
and in many types of cancer
cells,[13] and OFA, the precursor
to the mature laminin receptor found
in fetal cells.[16,17] In addition, some
proteins have been identified as common
to certain types of tumor cells
but absent from normal tissues. Human
telomerase reverse transcriptase
may be an example of a common tumor-
specific antigen since it is overexpressed
in many tumors but absent
from normal cells.[18]

Optimizing Antigenicity
The process of effective recognition
of antigens and the development
of an adaptive immune response is
complex. Tumor antigens must be
taken up by phagocytic cells such as
dendritic cells, processed into immunogenic
fragments of defined size, and
assembled into complexes with the
appropriate major histocompatability
complex (MHC) molecule-class I
for presentation to CD8 T cells and
class II for presentation to CD4
T cells. These complexes are then displayed
on the surface of the dendritic
cells.[19]

MHC molecules derive their name
from their role in transplant rejection.
In response to differentiating elements
released by inflammatory cells and
the pathologic process itself, dendritic
cells differentiate into potent antigen-
presenting cells and migrate to
lymphoid tissue, where they encounter
the T cells. T cells become activated
when their antigen-specific T-cell
receptor binds the antigen-MHC complex
presented on the surface of the
antigen-presenting cell. T cells only
bind an antigen-MHC complex if the
complexed antigen is recognized by
its T-cell receptor (cognate antigen).
The ability of an antigen to elicit an
immune response is partially a function
of the number of cognate reactive
(vs tolerant) T cells and the
binding affinity between the responsive
T cell and the antigen-presenting
cell.[2] However, the potency of an
immune response also depends on other
factors such as the interactions between
the MHC complex and CD4
and CD8 molecules on T cells and the
generation of costimulatory signals
between the antigen-presenting cell
and T cells.

Costimulatory molecules are expressed
on the surface of antigen-presenting
cells and are recognized by
corresponding receptors on T cells.
Tumor cells generally lack costimulatory
molecules and, therefore, function
very poorly as antigen-presenting
cells. Moreover, when antigen is presented
in the absence of costimulation,
T cells tend to become anergic,
which may be a mechanism for protection
of normal cells against an autoimmune
response and also is a
proposed mechanism of tumor escape.
The most widely studied costimulatory
interaction is through B7.1 (CD80) on
antigen-presenting cells and the molecule
CD28 on T cells (Figure 1).[2]
For the T cells to survive, proliferate,
and form memory T cells, additional
costimulatory interactions are required,
such as the interaction between
CD40 on the activated antigenpresenting
cell and CD40 ligand on
the T cell.

Thus, to optimize antigenicity, it is
reasonable for therapeutic cancer vaccine
developers to employ strategies
similar to those observed in vivo-ie,
to maximize the immunologic response
by presenting antigen in the
context of costimulatory molecules.
The importance of costimulatory molecular
signals may have been reflected
in the observations by Coley more
than a century ago that immune
responses
against weak antigens require
additional signals (eg, such as those
released during infection).[1]

In Vitro Quantification
of Immune Responses

While experiments can be conducted
in syngeneic animal models or with
standardized cell lines, it is difficult
to quantify responses of patients to
novel immunotherapies. Although two
patients may both have the same type,
grade, and stage of solid tumor, they
may differ in age, comorbidities, tumor
antigenicity, tumor suppressor
gene status, HLA type, dendritic cell
function, T- and B-cell numbers and
function, response to chemotherapy,
and so forth. Consequently, it is important
to develop quantitative methods
to evaluate the magnitude and
durability of immunologic responses
to cancer vaccines.[20] In vivo assays
have been available for some time
and include delayed-type hypersensitivity
skin reactions, depigmentation
of the tumor in the case of melanoma
vaccines, and quantification of tumor
infiltrating lymphocytes. In addition,
a variety of new in vitro analytic tools
have been developed.

These in vitro methods have been
important in the continuous improvement
of vaccine strategies and in the
selection of strategies to move to clinical
trials. Ultimately, an in vitro assay
may serve as a surrogate indicator
of therapeutic vaccine efficacy if the
measured immune responses can be
shown to correlate with clinical outcome.
These in vitro assay systems
include enzyme-linked immunospot
(ELISpot) for the measurement of antigen-
specific T-cell responses, flow
cytometric measurement of intracellular
cytokine production, and MHCpeptide
tetramer analysis. ELISpot
employs antibodies in a sandwich assay
with signal amplification to enumerate
cytokine-secreting cells. When
automated, the results are reliable and
reproducible. Cloned peptide-MHC
complexes can be stabilized and tetramerized
with streptavidin labeled
with phycoerythrin. When incubated
with mononuclear cells, the ability of
tetramers to stain and activate CD8
T cells is strongly dependent on binding
of CD8 to the same class I
molecule
engaged by the T-cell receptor.
Peptide-specific T cells can then be
enumerated in a flow cytometer.

All three methods are very sensitive,
but the peptide-MHC tetramer
analysis appears to be the most sensitive.
It is also the most technically
demanding assay and, at this time, is
not sufficiently robust for broad clinical
application.[21]

Development of CEA-Based
Vaccines in GI Cancers
CEA as a Target of
Immunotherapy

Carcinoembryonic antigen (CEA),
reported in 1965, is one of the first
oncofetal tumor antigens to be discovered.
It is normally expressed in
fetal colon. Although its expression is
restricted in adult life, CEA is present
in saliva, feces, serum, colonic mucosa,
and fluid from colonic lavages
in adults.[22] CEA is overexpressed
in a high percentage of adenocarcinomas,
particularly those of endodermal
origin (eg, non-small-cell lung,
stomach, colon, rectum, and pancreas).
As such, it is considered a shared (public),
nonmutated oncofetal self-antigen
and its concentration in the
circulation is widely used as a serologic
marker of malignancy. CEA is a
member of the immunoglobulin superfamily
with a molecular weight of
180 to 200 kDa.[23]

The gene for CEA codes for a
70-kDa protein and is located on chromosome
19. The difference in molecular
weight between the gene product
and the final antigen is secondary to
extensive post-translational glycosylation.[
24] In normal colonic epithelium,
CEA is localized to the luminal
surface-an arrangement that suggests
that it contributes to spatial orientation
of colonocytes and that it may
also function to preserve the adult
gut's mucosal barrier. In tumor cells,
however, CEA is irregularly distrib-
uted throughout the cell membrane.
As an intercellular adhesion molecule,
CEA may contribute to the formation
of metastasis.[22] There is a correlation
between serum CEA in patients
with cancer and the incidence of hepatic
metastases, but this could also
simply reflect tumor burden.[24]

Due to its association with malignancy
and wide distribution in human
tumors, CEA has the characteristics
of an ideal target for vaccine therapy.
However, there are several potential
problems that must be resolved. Since
CEA is also normally expressed in
adult life, the immune system is normally
tolerant to CEA.[22] The tolerance
develops in utero with deletion
of autoreactive clones of T cells
through a process of negative selection
within the thymus. Small numbers
of CEA-reactive T cells may still
reach the periphery. In general, tolerance
is induced in these clones by
some combination of clonal inactivation,
clonal deletion, and/or cytokinedependent
suppression and immune
deviation. Thus, strategies employed
to overcome tolerance to CEA might
overcome tolerance in these clones
and elicit an immune response that
would not only target the tumor but
could also induce autoimmune side
effects. Experimentally, tolerance to
CEA can be overcome without induction
of adverse autoimmune events.
Investigators have vaccinated mice
transgenic for human CEA and generated
anti-CEA antibodies, immunoglobulin
class switching, TH1-type
CEA-specific CD4+ cells, and CD8+
cellular cytotoxicity.[25,26]

CEA Vaccine Strategies:
Peptide Modification

As described above, T cells only
recognize antigen after it has been
processed into fragments (epitopes)
composed of 8 to 10 amino acids
(CD8+ cells) or longer (CD4+ cells)
and presented on the surface of the
antigen-presenting cell complexed
with MHC class I or II molecules,
respectively.[24] T-cell epitopes from
tumor antigens such as CEA have been
identified by either identifying the target
of an existing T-cell response or,
more commonly, using modeling to
predict the binding affinity of a particular
amino acid sequence to a specific
MHC class I or II molecule.[27]
At present, a number of CEA-associated
peptides have been identified and
have been used to generate T-cell responses.
The majority have been restricted
to the (MHC class I type)
HLA-A2, the supertype present in 40%
to 50% of humans.[28]. HLA-A3-
restricted peptides have also now been
identified and shown to be immunogenic.[
28] While less attention has been
focused on MHC class II-restricted
peptides for numerous reasons, evolving
understanding of the role of CD4+
cells in modulating antigen-specific
immune responses has led to increased
emphasis on these epitopes.[24]

As an antigen, CEA is weakly immunogenic.
It is reasonable to assume
that at least part of the explanation for
the low immunogenicity of native
CEA peptides is their low affinity for
MHC molecules. Therefore, to develop
a more effective therapeutic cancer
vaccine, strategies have been
employed to increase the immunogenicity
of CEA. If, in fact, binding affinity
is the problem, amino acid
substitutions of key residues that anchor
the peptide to the MHC molecule
might be used to increase the
peptide's affinity for MHC molecules.
This strategy has been employed effectively
to improve T-cell responses
to several melanoma antigens.[29]

Another successfully used approach
to enhance the T-cell response
is to modify the residues recognized
by the T-cell receptor. The HLA-A2-
restricted 9-amino acid peptide, CEAassociated
peptide 1 (CAP-1), was
modified by replacing the asparagine
at position 6 with an aspartic acid. In
in vitro assays, this modified peptide,
CAP-1-6D, was more effectively recognized
by T cells than the native
CAP-1 peptide[30] and significantly
upregulated the production of
granulocyte macrophage colony-stimulating
factor (GM-CSF) and interferon-
gamma by the target cell.[31] Gene
array studies demonstrated that, compared
with the native CAP-1 peptide,
CAP-1-6D stimulates the expression
of different genes and gene clusters in
CD8+ T cells than the native peptide.
Expression of lymphotactin and
granzyme B were increased many times
over the levels in CD8+ T cells stimulated
with CAP-1.[32] Lymphotactin
recruits T and NK cells into the region,
and granzyme B is transferred
from cytotoxic T lymphocytes to target
cells during the process of granule-
mediated cytotoxicity.[33,34]

Viral Vectors for Antigen Delivery
Recombinant viruses have been
used for both gene therapy and as
vectors for immunotherapy.[35] When
used as immunotherapeutics, genes for
a tumor antigen can be introduced
into a virus that directs the expression
of the antigen and stimulates a strong
immune response to the specific tumor
antigen. In the development of
vaccine therapy, the choice of a vector
is as important as the selection of
the tumor antigen. Potential viruses
include adenoviruses, retroviruses,
and poxviruses, and each has its advantages
and disadvantages. For example,
while the adenoviruses are
effective vectors and have been widely
used in experimental studies, they
infect nondividing cells and require
repetitive dosing because their genes
are only transiently expressed.[36] In
addition, adenoviruses generate an
immune response or enhance the immune
response in the approximately
80% of humans who already have neutralizing
antibodies to these viruses.[
37] Retroviruses integrate into the
host genomes and, therefore, bring
the risk of insertional mutagenesis.[36]

  • Poxviruses-
    Poxviruses such as
    the vaccinia virus are particularly well
    suited as vectors for therapeutic cancer
    vaccines. They are easily engineered,
    accommodate large inserts of
    foreign DNA, allow post-translational
    modification of expressed proteins,
    replicate accurately without helper
    viruses, stimulate potent immune responses,
    and have been extensively
    tested in humans as smallpox prophylaxis.[
    24] Recombinant vaccinia (rV)
    viruses that express human CEA have
    been developed.[38] Incorporating the
    gene for CEA into the virus involves
    inserting the nucleic acids for the
    gene's start site immediately adjacent
    to the transitional start site of a vaccinia
    gene promoter.[39]
  • Following transfection, the rV-
    CEA infected cells express a protein
    product recognized by monoclonal
    antibodies to CEA.[38] CEA transgenic
    mice, which express human
    CEA as a self-antigen and are immunologically
    unresponsive to CEA administered
    as a whole protein, respond
    to immunization with rV-CEA with
    TH1-type CEA-specific CD4+ responses
    and CEA peptide-specific cytotoxicity.
    Immunization with rV-CEA
    protected these mice from challenge
    with CEA-expressing tumor cells.[25]
    Vaccination with rV-CEA is also immunogenic
    in humans. Following vaccination
    with the viral construct,
    previously immuno-unreactive peripheral
    blood lymphocytes from patients
    with metastatic carcinoma were able to
    mount a cytolytic T-cell response to
    specific CEA epitopes in vitro.[40]

    Vaccinia virus has many properties
    desired in a vector. Although its
    intense immunogenicity potentially
    could limit its repeated use, this concern
    is not clinically significant since
    current strategies only employ it as a
    vector to prime the immune system.[
    41] Vaccinia proteins are highly
    immunogenic; consequently, it is likely
    that vaccinia can only be administered
    once or twice before a patient
    will develop high titers of neutralizing
    antibodies that will limit expression
    of recombinant genes. To
    circumvent this problem, researchers
    have studied potential alternative vectors
    such as attenuated vaccinia strains
    or avian pox viruses such as the canarypox
    virus.[42,43] An advantage
    of canarypox virus is that it replicates
    only in avian species. When administered
    to humans, it expresses its transgene
    products for 2 to 3 weeks in the
    infected mammalian cells but does
    not replicate and is unable to infect
    other cells, thus minimizing the likelihood
    that an enhanced immune response
    will neutralize the vaccine.[22]

    The protective and antitumor activity
    of canarypox virus has been
    demonstrated in preclinical models.
    [
    41] The first phase I trial of a
    fowlpox recombinant CEA vaccine
    was conducted in 1999 by Marshall et
    al.[44] Although no objective antitumor
    response was observed in patients
    with measurable disease during the
    trial, vaccination resulted in statistically
    significant increases in cytotoxic
    T-cell precursors specific for CEA.
    In preclinical studies, Hodge et al[35]
    showed that a combination vaccination
    strategy with rV-CEA administered
    first followed by fowlpox-CEA
    could generate a more vigorous Tcell
    response than either vaccine
    alone.[41] This approach was subsequently
    validated in a phase I study in
    patients with advanced CEA-positive
    malignancies.[45] The study confirmed
    that immunization was most
    effective when rV-CEA was the primer
    and the fowlpox-CEA used to boost
    the immune response. In addition, this
    study showed that further increases in
    cytotoxic T cells could be produced
    by administering GM-CSF (Leukine)
    with subsequent vaccinations.[46]

Dendritic Cells and CEA
Dendritic cells are the most potent
type of antigen-presenting cells and are
essential to prime the adaptive immune
response.[47] Although rare in the peripheral
blood, dendritic cells can be
harvested from the blood and expanded
in vitro. Alternatively, administration
of the hematopoietic growth factor
flt3 ligand can dramatically mobilize
these cells into the peripheral blood,
increasing numbers by as much as
20-fold.[48] When exposed for short
periods to high concentrations of antigen,
dendritic cells are said to be
"pulsed." Pulsing has been used to
generate more effective antigen-presenting
cells. Dendritic cells pulsed
with an HLA class I-restricted CEA
peptide epitope have been shown to
generate cytotoxic T lymphocytes specific
for CEA.[49,50] In a phase I study
of patients with advanced CEA-positive
malignancies, Morse et al administered
autologous dendritic cells pulsed
with CEA mRNA.[51] There were no
significant treatment-related toxicities.
Of 24 evaluable patients, 1 showed a
complete response, 2 had minor responses,
3 exhibited stable disease,
and 18 had progressive disease.

  • Role of GM-CSF-
    GM-CSF
    plays a role in the maturation and
    function of antigen-presenting cells
    and the resultant immune response.[
    52,53] The hematopoietic
    growth factor has also been shown to
    increase the immunogenicity of tumors.
    For example, tumor cells transfected
    with the GM-CSF gene produce
    the cytokine and have increased antigenicity.[
    54,55] In a study by Disis et
    al, a single dose of GM-CSF was shown
    to be as effective as complete Freund's
    adjuvant in generating specific cellular
    and humoral immunity.[52]
  • In patients with CEA-positive tumors,
    GM-CSF has been used to upregulate
    the activity of dendritic cells
    pulsed with CEA peptides. In a study
    of dendritic cells pulsed with CEA
    peptide plus GM-CSF and interleukin
    (IL)-4, although there was no identifiable
    tumor shrinkage, some patients
    developed long-term stable disease or
    decreases in serum CEA.[56]

    Patients immunized with recombinant
    CEA and then treated with
    GM-CSF at the time of each immunization
    demonstrated an augmented Tcell
    response and immunoglobulin
    (Ig)G titers at 12 months that persisted
    for 24 months after the last vaccination
    compared to controls.[57]
    Following immunization with fowlpox
    expressing GM-CSF, regional
    lymph nodes demonstrated significant
    and sustained increases in antigenpresenting
    cells. Coadministration of
    the avian poxvirus expressing either
    GM-CSF or CEA was found to generate
    antitumor immunity.[58]

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