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Departments of Medical Oncology [M. v. M., N. M., M. D., S. M., L. M. W.], Biostatistics [A. R.], and Pathology [H. S. C.], Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and Laboratory of Tumor Immunology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [P. A., K. Y. T., J. S.]
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ABSTRACT |
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. Therapy was well tolerated, without significant
toxicity attributable to vaccine. All patients had evidence of
leukocytic infiltration and CEA expression in vaccine biopsy sites. Six
patients with elevated serum CEA values at baseline had declines in
their levels lasting 4–12 weeks. These patients all had stable disease
after four vaccinations. After four vaccinations, patients who were
HLA-A-2-positive demonstrated increases in their CEA- specific
T-cell precursor frequencies to a CEA-A2-binding peptide from baseline.
The number of prior chemotherapy regimens was inversely correlated with
the ability to generate a T-cell response. ALVAC-CEA B7.1 is
safe in patients with advanced, recurrent adenocarcinomas that express
CEA, and it is associated with the induction of a CEA-specific T-cell
response. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenocarcinomas expressing this antigen do not stimulate a significant immune response against CEA, probably because of its expression during fetal development. Despite this, CEA has been evaluated as an immunogen for antitumor vaccines because there is minimal expression of CEA in adult tissues, limiting the possibility of immunization leading to a potentially harmful autoimmune response. Various vaccines that immunize against CEA have not caused toxicity to normal organs. Approaches have included vaccination with autologous tumor cells (10) , rCEA protein (11) , anti-idiotypic antibodies (12) , various viral constructs encoding the gene for CEA (13, 14, 15) , naked DNA (16) , and mRNA (17) .
When an antigen is presented to a naive T cell, two signals are
required to stimulate the T cell to divide and differentiate to produce
an immune response (18)
. First, an APC presents the
protein antigen as peptide fragments in the groove of the MHC class I
or MHC class II receptor to the TCR on CD8+ and
CD4+ T cells, respectively. The peptide is
recognized in a manner restricted by the MHC receptor. A second
costimulatory signal, which is antigen-independent and does not bind
the TCR, is also required (19)
. Without such a signal, the
T cell develops an anergic response to the antigen. One such signal can
be provided by B7 molecules. The two known B7 molecules, B7.1 and B7.2,
are also known as CD80 and CD86. Binding of these costimulatory
molecules to CD28 results in the production of multiple cytokines,
including IL-2, and IFN- by CD4+ and
CD8+ T cells.
The importance of B7 as a costimulatory molecule for the development of an effective antitumor immune response has been demonstrated by transfecting B7 into nonimmunogenic tumor cells. Tumor cells expressing B7.1 are rejected and also stimulate lasting immunity against wild-type tumor cells in murine models (20, 21, 22) . Analysis of response at the cellular level reveals production of various cytokines, but not IL-4. This suggests that a subclass of CD4+ cells, known as TH1, which are important in the generation of cytotoxic T-cell responses, have been stimulated. IL-4 production is characteristic of a TH2 response, which is responsible for the stimulation of humoral responses (23) . B7.1 has been reported by some groups to preferentially stimulate a TH1 response, whereas B7.2 stimulation induces a TH2 response (24 , 25) .
Vaccine strategies that result in the coordinated presentation of antigen with a costimulatory molecule may result in improved immunity. Hodge et al. (26) vaccinated tumor-bearing mice with varying ratios of vaccinia vectors containing CEA (rV-CEA) and B7.1 (rV-B7). They examined T-cell responses and found a dose-dependent T-cell response when mice were vaccinated with rV-CEA and control vector, and no response when the animals were vaccinated with rV-B7 and control vector. However, animals receiving rV-CEA and rV-B7 had increased T-cell lymphoproliferative responses, especially at a 3:1 ratio of rV-CEA:rV-B7. There were also similar trends with in vitro cytotoxicity assays using a murine adenocarcinoma cell line transfected with CEA. Splenocytes from animals vaccinated once with rV-CEA:rV-B7.1 had a 2.8-fold increase in cytotoxicity against the CEA-expressing tumor cells compared to splenocytes from animals vaccinated with rV-CEA alone. There was no cytotoxicity seen with the parent adenocarcinoma cell line, which did not express CEA irrespective of which vaccine the mice were immunized with.
ALVAC-CEA B7.1 (designated Avipox-CEA/B7.1) is a canary pox
vector encoding the gene for CEA and for B7.1. Canary pox is a member
of the Avipox genus, along with vaccinia, both of which have been used
for vaccine therapy. ALVAC has inherent characteristics that make it
more attractive than vaccinia. Vaccinia is very immunogenic, limiting
its serial use. Unlike vaccinia, canary pox can only replicate
productively in avian species, eliminating the rare risk of a vaccinia
infection (27
, 28)
. Although ALVAC does not replicate in
nonavian species, inoculations of recombinant Avipox into nonavian
cells result in the expression of the transgene(s). ALVAC vectors
expressing foreign antigens can elicit protective immune responses to
viral pathogens in nonavian species (29, 30, 31, 32)
. An ALVAC-CEA
vaccine has undergone Phase I testing in cancer patients and induced
CEA-specific CTL responses (15)
. In these studies
(15)
and others (14
, 17
, 33)
, CEA-specific
cytolytic T-cell responses were demonstrated against a 9-mer CEA
peptide designated CAP-1. These T cells were shown to be CEA-positive
and inhibited by anti-class I MAb. Recently, an enhancer agonist
epitope to CAP-1 has been identified (34)
and designated
CAP-1–6D (YLSGADLNL). This 9-mer peptide has now been shown to
(a) bind MHC class I, (b) induce
CD8+ cytolytic T cells, (c) induce
CTLs that are capable of lysing human carcinoma cells expressing CEA
and the MHC-A2 allele, and (d) induce
CD8+ T cells that produce enhanced levels of
TC1-type cytokines, INF-, and GM-CSF, but not IL-10 and IL-4
(34
, 35)
.
Here we report on the results of a pilot study treating 39 patients with advanced CEA-producing adenocarcinomas with the recombinant vaccine, ALVAC-CEA B7.1. Toxicity as well as clinical, serological, and immunological responses are presented.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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18 years of age,
have an Eastern Cooperative Oncology Group performance status of 0–1,
and adequate hematological, renal, and hepatic function. In patients
with known liver metastases, transaminase elevations up to 3 times the
upper limit of normal and total bilirubin up to 1.5 times the upper
limit of normal were permitted. All patients had anergy panels
performed to assess delayed-type hypersensitivity responses but were
not excluded from participation in the study if they were anergic. All
patients were HLA-typed to identify HLA-A2-positive patients, but
HLA-A2-negative patients were not excluded. A lapse of at least 4–6
weeks from all prior anticancer therapy was required. Measurable and
evaluable disease were allowed. Patients were excluded if they had
evidence of immunocompromise, such as a known history of HIV infection,
active eczema, atopic dermatitis, or any autoimmune disease, prior
radiation to >50% of all nodal groups, prior splenectomy, or
concurrent use of systemic steroids. Patients were also excluded if
there was a prior history of another malignant process active in the
last 2 years.
Treatment Schema
An initial dose escalation phase using 2.5 x
107 pfu (n = 3) and 1.0 x
108 pfu (n = 6)/injection was
followed by vaccination with the working dose of 4.5 x
108 pfu/injection (n = 30). The
vaccine was manufactured by Pasteur-Merieux Sera et Vaccins (Marcy,
France/Troy, New York). The vaccine was given as an intradermal
injection every 2 weeks for four injections. Patients were evaluated
for toxicity using National Cancer Institute Common Toxicity Criteria,
and for clinical response using standard response criteria
(36)
. Patients with evidence of objective response or
stable disease at 8 weeks were allowed to continue on study receiving
boost injections every 4 weeks, with re-evaluation every 8 weeks.
Patients were removed from study for disease progression. While on
study, patients were followed with routine laboratory testing as well
as serum CEA evaluations.
Correlative Studies
Biopsies.
Patients receiving the working dose of 4.5 x
108 pfu had 3-mm punch biopsies of the vaccine
site performed 48 h after vaccination. H&E-stained slides were
evaluated for evidence of necrosis, dermal leukocytic infiltration, and
perivascular inflammation and estimated using a scale of none, minimal
(1+), moderate (2+), or significant (3+). Sections were stained for CEA
with a 1:4000 dilution of a rabbit polyclonal antibody (DAKO Corp.,
Carpinteria, CA) using the Techmate 1000 automated stainer with its
protocol. Slides were pretreated with heat-induced epitope retrieval in
steamer with citrate buffer. Areas of CEA-positive staining were
determined by light microscopy. Negative control sections were stained
in the same manner with normal rabbit IgG, diluted to the same protein
concentration as the antirabbit CEA.
Anti-CEA Antibody ELISA Assay.
The presence of anti-CEA antibodies in patient serum before and after
vaccination was analyzed using an ELISA. Polyvinyl chloride 96-well
microtiter plates (Dynatech Laboratories, Chantilly, VA) were incubated
overnight at 4°C with purified preparations of nCEA and rCEA (both
from Vitro Diagnostics, Littleton, CO), as well as with BSA or human
serum albumin at 100 ng/well in 50 µl of PBS (pH 7.2). nCEA was
derived from biopsies of human colon adenocarcinoma from metastases to
the liver. rCEA was derived from human MCF-7 carcinoma cells
transfected with and expressing a glycosylated CEA. All CEA
preparations were negative for the presence of nonspecific
cross-reacting antigen (NCA), as determined using the
anti-NCA MAb B6.23 (37)
and an ELISA
(38)
. The wells were blocked for 1 h at 37°C with
PBS containing 5% BSA and then washed once with 1% BSA (assay
buffer). Patient serum and control pooled human serum (Gemini Bio
Products, Calabasas, CA) were diluted in assay buffer and added to
wells in triplicate in a volume of 50 µl/well. Purified IgG of
humanized COL-1, a complimentary determining region-grafted
antibody derived from the murine COL-1 anti-CEA MAb (39)
,
was used as a positive control antibody for CEA binding. After
incubation overnight at room temperature, the wells were washed four
times with assay buffer, and 50 µl of a 1:4000 dilution of
peroxidase-conjugated goat antihuman IgG (Kirkegaard and Perry
Laboratories, Gaithersburg, MD) was added to each well. After
incubation at 37°C for 1 h, wells were washed four times with
assay buffer, and 100 µl each of the chromogen
O-phenylenediamine dihydrochloride (Sigma, St. Louis, MO)
and hydrogen peroxide were added to each well. After a 10-min
incubation in the dark, the reaction was stopped with 25 µl of
4NH2SO4. The absorbance of
each well was measured at 490 nm using an ELISA microplate autoreader
(Bio-Tek Instruments, Winooski, VT).
ELISPOT Assay.
Assays were only done on samples from patients positive for the HLA-A2
allele. Samples were analyzed using a modification of the method
described by Scheibenbogen et al. (40)
.
Ficoll-purified mononuclear cells (PBMCs) were washed three times with
PBS, viably frozen at 
1 x 107 cells/ml
in 10% DMSO in heat-inactivated AB serum, and thawed just
before use (33)
. PBMCs obtained before vaccination and 2
weeks after the fourth vaccination were evaluated. Ninety-six-well
MultiScreen-HA plates (Millipore Corporation, Bedford, MA) were coated
with 100 µl of capture antibody against IFN- at a concentration of
10 µg/ml. After a 24-h incubation at room temperature, plates were
blocked for 30 min with RPMI 1640 containing 10% human pool AB
serum. Added to each well were 1 x 105
cells to be assayed. For each patient, between 5 x
105 and 5 x 106 total
cells were analyzed, and the results were expressed as number of spots
per 5 x 105. CIR-A2 cells pulsed with 25
µg/ml 9-mer CEA agonist peptide CAP1–6D (YLSGADLNL; Ref.
34
) were added to each well as APCs at an effector:APC
ratio of 1:3. Unpulsed CIR-A2 cells were used as a negative control.
HLA-A2-binding flu matrix peptide 58–66 (GILGFVFTL) was added to
identical wells at 5 µg/ml and was used as a peptide control. The
responding cells were determined by the use of a Domino Image Analyzer
(Optomax, Hollis, NH).
Statistical Methods.
Univariate paired t test was used to compare baseline
outcome measurements taken in the same patient at different time
intervals. Multivariate Hotelling’s T2 test was
used to compare differences between the treatment group (CEA) and the
control (flu). The critical significance level was set to 5%.
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RESULTS |
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.
The majority of the patients treated had metastatic colorectal cancer,
with a minority of patients having breast, lung, thyroid, gall bladder,
esophageal, pancreas, and appendix cancers. The majority of patients
had received prior therapy. All patients but two had received prior
chemotherapy, with a median number of three prior regimens, and a range
of one to six. Other prior therapies included radiation therapy,
immunotherapies, and hormonal therapy. All patients except for one had
measurable disease. The patient with nonmeasurable disease had colon
cancer with an elevated CEA with documented intra-abdominal disease at
laparotomy that was not visible by computed tomography scan. All
patients had metastatic disease. Sixteen of the 39 patients developed a
>2-cm reaction to one or more of the antigens of the CMI
Multi-test; all others were anergic. Twenty-six of the 39 patients were
HLA-A2-positive.
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. The most frequent toxicity
was local erythema and swelling at the vaccine site, associated with
myalgias and flu-like symptoms after vaccination; symptoms were worse
with the first vaccination and minimal with subsequent immunizations.
There were no hematological toxicities except for anemia in one
patient, which was deemed to be related to the underlying malignancy
rather than to vaccine therapy. Gastrointestinal side effects such as
nausea and vomiting were reported in eight patients. However, the
patients with these symptoms had colorectal cancer (n =
6), recurrent appendiceal carcinoma (n = 1), and
metastatic esophageal carcinoma to liver with celiac and retrogastric
adenopathy (n = 1). It was therefore difficult to
determine if these symptoms were attributable to the underlying disease
versus the therapy. Twenty-six % of patients had elevations
in transaminases as well as alkaline phosphatase. Again, the majority
of the patients (80%) had known metastatic liver disease; the other
two patients had bone (n = 1) and lung
(n = 1) metastases. Seven of these patients
exhibited increased liver function tests in association with disease
progression, whereas two patients had baseline elevations, which
remained unchanged throughout treatment, and another patient had
transient elevation of transaminases after the first injection only.
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Biopsy Data.
All but one patient treated with 4.5 x 108
pfu of ALVAC-CEA B7.1 had a punch biopsy of the vaccine site 48 h
after vaccination. All biopsies have shown leukocytic infiltrates in
the epidermis and perivascular regions, with 17 demonstrating an
infiltrate in the dermis (Fig. 1A)
. Fewer than 10% of
patient biopsies demonstrated necrosis within the dermis. All samples
stained by immunohistochemistry evaluating CEA expression demonstrated
CEA staining in the region of the inflammatory response in leukocytes,
in spindle-shaped cells suggestive of dendritic cells, and in
fibroblasts (Fig. 1B)
. Negative control sections using an
antirabbit IgG did not reveal any positive staining (data not shown).
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There was an increase in serum CEA between baseline and weeks 4 and 8,
and an increase between weeks 4 and 8. Six patients had declines in
their CEA levels while receiving vaccine therapy, as outlined in Table 3
, lasting a minimum of 4 weeks and as long as 12 weeks. The
differences between baseline and the lowest measurement were normalized
for each patient by the baseline. A mean decrease of 30.25% (SD =
16.59) was found significantly different than a mean decrease of 0% at
P = 0.0066 by a one-tailed t test. Patients
11 and 28 were HLA-A2-positive, and each had an increase in
CEA-specific T-cell precursor frequencies, as described below. All
patients with declines in their CEA had stable disease at initial
response evaluation and received vaccine boost injections after the
initial four immunizations. Patients 8, 9, 12, and 14 had negative
anergy panels at initiation of vaccine therapy.
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,
prevaccination sera from patient 16 (Fig. 2
, 
) showed no reactivity
to either native nCEA (Fig. 2A)
or recombinant rCEA (Fig. 2B)
. Little or no reactivity was also seen in sera obtained
after one vaccination. Sera obtained 2 weeks after the third
vaccination with ALVAC-CEA-B7.1, however, showed reactivity to both
nCEA and rCEA (Fig. 2, A and B)
but did not react
to the control BSA protein (Fig. 2C)
.
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, D, E, and F). Again, no
anti-CEA-specific IgG is observed before vaccination and after one
vaccination. However, anti-CEA-specific is seen after vaccination 2
(Fig. 2
, 
). This is potentiated after the third vaccination (Fig. 2
,
). Again, none of the sera reacted with the control BSA protein.
T-Cell Assays.
Nineteen of the 26 patients who were HLA-A2-positive received four
vaccinations and had baseline and post-vaccine 4 samples available for
T-cell assays. These samples were obtained from 1 patient receiving
2.5 x 107 pfu, 2 patients receiving 1 x 108 pfu, and 12 patients receiving 4.5 x
108 pfu. Lymphocytes from four patients were not
viable when thawed for evaluation; therefore, data are available for 15
of the HLA-A2-positive patients. T-cell assays using the HLA-A2 class I
allele 9-mer CEA peptide CAP1–6D and the HLA-A2 9-mer Flu matrix
peptide were used to investigate T-cell responses in patients positive
for the HLA-A2 allele. PBMCs were assayed after only 24 h in
culture in the presence of peptide to rule out effects of in
vitro selection of T-cell populations. The ELISPOT assay using CEA
and Flu peptides and PBMCs from each patient before and after
vaccination was always done simultaneously and coded to minimize
interpretive bias. Results are expressed as a precursor frequency of
IFN--secreting cells in response to the given peptide. A smaller
number in the denominator of the precursor frequency expresses a higher
number of precursors.
As seen in Table 4
, PBMCs from all 15
HLA-A2-positive patients showed a <2-fold difference in precursor
frequency to the Flu 9-mer peptide at baseline and after four
vaccinations with ALVAC-CEA-B7.1; this difference was not statistically
significant by a univariate paired t test (P = 0.123). In the same assays, however, these PBMCs showed a
statistically significant increase in the CEA-specific T-cell precursor
frequency after vaccination as compared to before vaccination
(P = 0.001). Twelve of 15 patients had at least a
2-fold increase in T-cell precursors specific to CEA peptide. Three of
these patients (4
, 17
, and 20) showed increases of at least 4-fold in
postvaccination precursor frequency, whereas patients 30 and 34 showed
increases in CEA-specific precursors of 9-fold and 14-fold,
respectively, after four vaccinations with ALVAC-CEA-B7.1. Patient 30
had only received concurrent chemoradiotherapy for rectal cancer, and
patient 34 received no prior chemotherapy for gallbladder cancer.
Patients 4, 10, and 11 had clinically stable disease for 8, 4, and 4
months, respectively; the other patients in this group progressed after
the initial four vaccinations. Patients 2, 6, 17, 18, and 30 had
negative anergy panels at baseline, whereas all other patients had
evidence of a delayed type hypersensitivity response. The hypothesis of
equality of mean differences between pre- and postvaccination T-cell
precursors for CEA and flu, respectively, was rejected in the
multivariate analysis (Hotelling’s T2 test,
P = 0.0035). Multiple regression analysis evaluated
whether the number of prior chemotherapy regimens, length of time with
cancer diagnosis, and prevaccine anergy status were associated with the
difference between initial and final CEA-specific T-cell responses in
HLA-A2-positive patients. The only significant predictor was the number
of prior chemotherapy regimens (P = 0.017), as shown in
Fig. 3
.
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DISCUSSION |
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All biopsies of vaccine sites in patients treated with 4.5 x 108 pfu ALVAC-CEA B7.1 showed evidence of CEA expression. This demonstrates that the ALVAC virus can infect cells of the dermis and epidermis and lead to the production of CEA protein that persists for at least 48 h. B7.1 expression was not evaluated because of the normal expression of B7.1 on APCs, which would be expected in the population of infiltrating leukocytes. Biopsies were performed only after the first vaccination. Clinically, patients had less erythema and swelling with subsequent vaccinations. It is possible that this decrease in local reaction was attributable to the development of an immune response against the viral vector, thus limiting infection by the virus, and subsequent production of CEA and B7.1. In other studies using ALVAC-CEA, multiple vaccinations lead to increasing numbers of CEA-specific T-cell precursors.4 Antibody assays against ALVAC using samples from this study are under way.
This Phase I study also shows that a dual-gene avipox vector containing
the CEA gene and the B7.1 gene can generate increases in CEA-specific
precursors from PBMCs before and after vaccination using an ELISPOT
assay in which cells were cultured less than 24 h. This short-term
assay was used to minimize potential artifacts that can be obtained by
culturing PBMCs several times in vitro in the presence of
peptide and IL-2. The use of another 9-mer peptide (Flu matrix) in each
assay, which was run simultaneously and coded, helped decrease bias and
assay artifacts. Moreover, PBMC ELISPOT assays from pre- and
postvaccination samples from the same patient were always thawed and
run simultaneously. The ratio of anti-Flu matrix T-cell precursors in
HLA-A2-positive patients ranged from 0.5–1.7, with a median of 1.2,
and did not statistically significantly differ from baseline to after
the vaccine (P = 0.123). However, against the CEA
peptide, the ratios ranged from 0.7 to 14.3, with a median of 3.2,
and were statistically different (P = 0.001). Patients
4, 10, and 11 had stable disease after the initial four vaccines, with
CEA ratios of pre- to postvaccine ratios of 4, 1.3, and 2.1.
The incorporation of the whole CEA gene within the vaccine has potential benefits over the use of peptides. Expression of the whole CEA protein in vivo allows for processing of the entire protein by each individual patient, leading to presentation of multiple CEA epitopes, including helper epitopes. This approach does not limit the patient population that can be treated, unlike approaches using HLA-restricted peptides. The immunological assessment of this clinical trial was limited to HLA-A2-expressing patients by the use of known HLA-restricted peptides in the ELISPOT assays. The development of an assay using whole CEA protein, which is under way in our laboratory, will allow for the evaluation of all immunological responses irrespective of HLA haplotype, and thus allow for testing of samples for which HLA specific peptides are not known.
There were no clinical responses in this Phase I study, and the
majority of patients had rises in the serum CEA values (Fig. 2)
.
However, 20% percent of all study participants and 27% of all
evaluable patients had stabilization of disease after four
immunizations. Eight patients continued therapy for 1
(n = 1), 2 (n = 3), 3
(n = 1), 4 (n = 1), and 6
(n = 2) months after the initial four immunizations.
Six patients also had declines in their serum CEA levels as outlined in
Table 3
. All patients with an initial decrease in serum CEA had stable
disease after four vaccines. Initial decreases in serum CEA may result
from the induction of an anti-CEA humoral response, resulting in
antibody-antigen complexes. Of the patients with CEA responses, two
also were HLA-A2-positive and had ratios of baseline to postvaccination
CEA-specific T cells of 2.1 and 2.3, respectively.
The difficulty in evaluating this and other anti-CEA vaccine clinical trials lies in the interpretation of the demonstrated immunological responses. As seen in this study, patients can develop anti-CEA peptide-specific T-cell responses as measured from PBMCs, but these responses are not clearly correlated with clinical response. T-cell responses in the regional lymph nodes and at tumor site(s) were not evaluated. The ELISPOT assay used in this study examines the response to one HLA-A2 peptide (CAP1–6D) and not to autologous tumor cells; it may be an inaccurate predictor of meaningful immunological responses. CAP1–6D, a modification of the CEA immunodominant epitope CAP1, has enhanced binding to CEA-specific TCRs (33) . The in vitro response thus may overestimate the in vivo ability of the T-cell precursor population to be stimulated; however, as discussed above, this method does diminish the risk of stimulating T-cell responses in vitro with rounds of antigen-pulsing and IL-2 exposure. In addition, the ELISPOT assay does not measure the binding avidity of the TCR for the CAP1–6D peptide presented in the groove of the HLA-A2 receptor. Binding avidity may be at least as important as precursor frequency for responses against CEA-expressing tumor cells. It has been demonstrated that T-cells stimulated with the CAP1–6D peptide have the ability to lyse human tumor cell expressing CEA in a MHC-restricted manner (33) . However, the T-cell precursor frequency alone may not be the appropriate laboratory correlate. A recent study in strains of T-cell transgenic mice whose CD8+ or CD4+ T cells had specificity for an influenza antigen demonstrated, even in the presence of 100% CD8+ T cells, that rejection of a murine tumor cell line transfected with influenza occurred only 60% of the time. Animals with both antigen-specific CD4+ and CD8+ T cells were completely protected from tumor growth (41) . Evidence of T-cell stimulation against autologous tumor cells may potentially be a more specific and valuable test; however, such information is difficult to obtain in the patient care setting.
It is unknown if the lack of clinical responses was attributable to an
insufficient stimulation of T-cell precursor numbers or to other
factors. Phase I studies traditionally include patients with
significant prior treatment and with metastatic disease, both of which
may impact on the ability of patients to mount effective antitumor
immunological responses. In the HLA-A2 patients studied, there was an
inverse correlation between their ability to increase their T-cell
precursor frequency and the number of prior chemotherapy regimens (Fig. 3)
. Others have demonstrated defective function of T cells
(42)
and dendritic cells (43)
in cancer
patients. Therefore, the lack of clinical response may reflect a
relatively immunocompromised patient population attributable to prior
therapy and advanced cancer, rather than inefficacy of an immunological
approach.
The copresentation of B7.1 with CEA was used to attempt to enhance the anti-CEA T-cell response. There have been no randomized trials comparing the two directly, but in studies using ALVAC-CEA alone using an i.m. injection given every 4 weeks, the increase in T-cell precursor frequency after three vaccinations is less than was observed in this study.4 Evaluation of adjuvants to enhance immunological response may allow the generation of a higher T-cell precursor frequency and suggest that a minimum threshold number of antitumor T-cell precursors is required for a clinical response. ALVAC-CEA B7.1 is presently being used with one such adjuvant, GM-CSF. GM-CSF has been shown to up-regulate MHC class II expression on macrophages, enhance the maturation of dendritic cells and stimulate their migration, produce a localized inflammatory response at the site of injection, as well as a systemic response in the bone marrow (44) . It stimulates the growth of APCs such as dendritic cells and macrophages. Irradiated tumor cells transfected with GM-CSF have been used as vaccines in murine models and have elicited enhanced immune responses against tumors, even upon rechallenge with nontransfected tumor cells (45) . Evidence of an increase in anti-CEA antibodies would indicate CD4+ T cells were also induced against CEA.
The clinical impact of a vaccine approach may not be observable until it is assessed in a patient population without immune compromise. Those patients with the most extensive chemotherapy pretreatment were least likely to have immunological response to vaccination. This supports testing vaccine strategies such as this one in the adjuvant setting with minimal disease. Future studies should prospectively assess the value of costimulation by comparing ALVAC-CEA with ALVAC-CEA B7.1-based strategies and should also formally assess the impact of chemotherapy on the induction of immune responses by vaccination.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants PO CA06927 and K12
CA01728, an appropriation from the Commonwealth of Pennsylvania,
and the Bernard A. and Rebecca S. Bernard Foundation. 
2 To whom requests for reprints should be
addressed, at Fox Chase Cancer Center, Department of Medical Oncology,
7701 Burholme Avenue, Philadelphia, PA 19111. E-mail: m_vonmehren{at}fccc.edu
3 The abbreviations used are: CEA,
carcinoembryonic antigen; APC, antigen-presenting cell; TCR, T-cell
receptor; IL, interleukin; MAb, monoclonal antibody; GM-CSF,
granulocyte macrophage colony-stimulating factor; pfu, plaque-forming
unit(s); nCEA, native CEA; rCEA, recombinant CEA; PBMC, peripheral
blood mononuclear cell; rV, recombinant vaccinia.
4 J. Schlom and J. Marshall, personal
communication.
Received 12/ 9/99; revised 3/ 1/00; accepted 3/ 2/00.
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 H. L. Kaufman, H.-J. Lenz, J. Marshall, D. Singh, C. Garett, C. Cripps, M. Moore, M. von Mehren, R. Dalfen, W. J. Heim, et al. Combination Chemotherapy and ALVAC-CEA/B7.1 Vaccine in Patients with Metastatic Colorectal Cancer Clin. Cancer Res., August 1, 2008; 14(15): 4843 - 4849. [Abstract] [Full Text] [PDF]
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 J. Schlom, J. L. Gulley, and P. M. Arlen Paradigm Shifts in Cancer Vaccine Therapy Experimental Biology and Medicine, May 1, 2008; 233(5): 522 - 534. [Abstract] [Full Text] [PDF]
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T. I. Naslund, C. Uyttenhove, E. K. L. Nordstrom, D. Colau, G. Warnier, M. Jondal, B. J. Van den Eynde, and P. Liljestrom Comparative Prime-Boost Vaccinations Using Semliki Forest Virus, Adenovirus, and ALVAC Vectors Demonstrate Differences in the Generation of a Protective Central Memory CTL Response against the P815 Tumor J. Immunol., June 1, 2007; 178(11): 6761 - 6769. [Abstract] [Full Text] [PDF]
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 R. H. Wilson Novel Therapeutic Developments Other Than EGFR and VEGF Inhibition in Colorectal Cancer Oncologist, October 1, 2006; 11(9): 1018 - 1024. [Abstract] [Full Text] [PDF]
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D. Nagorsen and E. Thiel Clinical and immunologic responses to active specific cancer vaccines in human colorectal cancer. Clin. Cancer Res., May 15, 2006; 12(10): 3064 - 3069. [Abstract] [Full Text] [PDF]
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S. Dai, T. Wan, B. Wang, X. Zhou, F. Xiu, T. Chen, Y. Wu, and X. Cao More Efficient Induction of HLA-A*0201-Restricted and Carcinoembryonic Antigen (CEA)-Specific CTL Response by Immunization with Exosomes Prepared from Heat-Stressed CEA-Positive Tumor Cells Clin. Cancer Res., October 15, 2005; 11(20): 7554 - 7563. [Abstract] [Full Text] [PDF]
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 S. Mosolits, G. Ullenhag, and H. Mellstedt Therapeutic vaccination in patients with gastrointestinal malignancies. A review of immunological and clinical results Ann. Onc., June 1, 2005; 16(6): 847 - 862. [Abstract] [Full Text] [PDF]
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M. G. Cusi, M. T. Del Vecchio, C. Terrosi, G. G. Savellini, G. Di Genova, M. La Placa, F. Fallarino, C. Moser, C. Cardone, G. Giorgi, et al. Immune-Reconstituted Influenza Virosome Containing CD40L Gene Enhances the Immunological and Protective Activity of a Carcinoembryonic Antigen Anticancer Vaccine J. Immunol., June 1, 2005; 174(11): 7210 - 7216. [Abstract] [Full Text] [PDF]
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 J. L. Marshall, J. L. Gulley, P. M. Arlen, P. K. Beetham, K.-Y. Tsang, R. Slack, J. W. Hodge, S. Doren, D. W. Grosenbach, J. Hwang, et al. Phase I Study of Sequential Vaccinations With Fowlpox-CEA(6D)-TRICOM Alone and Sequentially With Vaccinia-CEA(6D)-TRICOM, With and Without Granulocyte-Macrophage Colony-Stimulating Factor, in Patients With Carcinoembryonic Antigen-Expressing Carcinomas J. Clin. Oncol., February 1, 2005; 23(4): 720 - 731. [Abstract] [Full Text] [PDF]
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H. L. Kaufman Integrating Bench With Bedside: The Role of Vaccine Therapy in the Treatment of Solid Tumors J. Clin. Oncol., February 1, 2005; 23(4): 659 - 661. [Full Text] [PDF]
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 C. T. Garnett, C. Palena, M. Chakarborty, K.-Y. Tsang, J. Schlom, and J. W. Hodge Sublethal Irradiation of Human Tumor Cells Modulates Phenotype Resulting in Enhanced Killing by Cytotoxic T Lymphocytes Cancer Res., November 1, 2004; 64(21): 7985 - 7994. [Abstract] [Full Text] [PDF]
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L. E. Raez, P. A. Cassileth, J. J. Schlesselman, K. Sridhar, S. Padmanabhan, E. Z. Fisher, P. A. Baldie, and E. R. Podack Allogeneic Vaccination With a B7.1 HLA-A Gene-Modified Adenocarcinoma Cell Line in Patients With Advanced Non-Small-Cell Lung Cancer J. Clin. Oncol., July 15, 2004; 22(14): 2800 - 2807. [Abstract] [Full Text] [PDF]
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H. E. Zeytin, A. C. Patel, C. J. Rogers, D. Canter, S. D. Hursting, J. Schlom, and J. W. Greiner Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits Antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice Cancer Res., May 15, 2004; 64(10): 3668 - 3678. [Abstract] [Full Text] [PDF]
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G. J. Ullenhag, J.-E. Frodin, S. Mosolits, S. Kiaii, M. Hassan, M. C. Bonnet, P. Moingeon, H. Mellstedt, and H. Rabbani Immunization of Colorectal Carcinoma Patients with a Recombinant Canarypox Virus Expressing the Tumor Antigen Ep-CAM/KSA (ALVAC-KSA) and Granulocyte Macrophage Colony- stimulating Factor Induced a Tumor-specific Cellular Immune Response Clin. Cancer Res., July 1, 2003; 9(7): 2447 - 2456. [Abstract] [Full Text] [PDF]
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C. Palena, J. Schlom, and K.-Y. Tsang Differential Gene Expression Profiles in a Human T-cell Line Stimulated with a Tumor-associated Self-peptide versus an Enhancer Agonist Peptide Clin. Cancer Res., May 1, 2003; 9(5): 1616 - 1627. [Abstract] [Full Text] [PDF]
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J. W. Greiner, H. Zeytin, M. R. Anver, and J. Schlom Vaccine-based Therapy Directed against Carcinoembryonic Antigen Demonstrates Antitumor Activity on Spontaneous Intestinal Tumors in the Absence of Autoimmunity Cancer Res., December 1, 2002; 62(23): 6944 - 6951. [Abstract] [Full Text] [PDF]
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E. S. Kass, J. W. Greiner, J. A. Kantor, K. Y. Tsang, F. Guadagni, Z. Chen, B. Clark, R. D. Pascalis, J. Schlom, and C. Van Waes Carcinoembryonic Antigen as a Target for Specific Antitumor Immunotherapy of Head and Neck Cancer Cancer Res., September 1, 2002; 62(17): 5049 - 5057. [Abstract] [Full Text] [PDF]
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J. Schmitz, E. Reali, J. W. Hodge, A. Patel, G. Davis, J. Schlom, and J. W. Greiner Identification of an Interferon-{gamma}-inducible Carcinoembryonic Antigen (CEA) CD8+ T-Cell Epitope, Which Mediates Tumor Killing in CEA Transgenic Mice Cancer Res., September 1, 2002; 62(17): 5058 - 5064. [Abstract] [Full Text] [PDF]
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N. L. Berinstein Carcinoembryonic Antigen as a Target for Therapeutic Anticancer Vaccines: A Review J. Clin. Oncol., April 15, 2002; 20(8): 2197 - 2207. [Abstract] [Full Text] [PDF]
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H. Horig, A. Wainstein, L. Long, D. Kahn, S. Soni, A. Marcus, W. Edelmann, R. Kucherlapati, and H. L. Kaufman A New Mouse Model for Evaluating the Immunotherapy of Human Colorectal Cancer Cancer Res., December 1, 2001; 61(23): 8520 - 8526. [Abstract] [Full Text] [PDF]
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D. W. Grosenbach, J. C. Barrientos, J. Schlom, and J. W. Hodge Synergy of Vaccine Strategies to Amplify Antigen-specific Immune Responses and Antitumor Effects Cancer Res., June 1, 2001; 61(11): 4497 - 4505. [Abstract] [Full Text] [PDF]
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M. von Mehren, P. Arlen, J. Gulley, A. Rogatko, H. S. Cooper, N. J. Meropol, R. K. Alpaugh, M. Davey, S. McLaughlin, M. T. Beard, et al. The Influence of Granulocyte Macrophage Colony-Stimulating Factor and Prior Chemotherapy on the Immunological Response to a Vaccine (ALVAC-CEA B7.1) in Patients with Metastatic Carcinoma Clin. Cancer Res., May 1, 2001; 7(5): 1181 - 1191. [Abstract] [Full Text]
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