Download Kinetics of tumor-specific T-cell response development after active

Clinical Immunology (2007) 125, 275–280
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Kinetics of tumor-specific T-cell response
development after active immunization in patients
with HER-2/neu overexpressing cancers
Lupe G. Salazar a,⁎, Andrew L. Coveler a , Ron E. Swensen a ,
Theodore A. Gooley b , Vivian Goodell a , Kathy Schiffman a , Mary L. Disis a
a
Tumor Vaccine Group, Division of Oncology, University of Washington, Box 358050, 815 Mercer Street,
Seattle, WA 98109, USA
b
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
Received 20 February 2007; accepted with revision 12 August 2007
Available online 29 October 2007
KEYWORDS
HER-2/neu;
Vaccination;
Immunity;
Kinetics
Abstract The ability of a cancer vaccine to elicit a specific measurable T-cell response is
increasingly being used to prioritize immunization strategies for therapeutic development.
Knowing the optimal time during a vaccine regimen to measure the development of tumorspecific immunity would greatly facilitate the assessment of T-cell responses. The purpose of this
study was to overview the kinetics of HER-2/neu-specific T-cell immunity evolution during and
after the administration of HER-2/neu peptide-based vaccination in the adjuvant setting.
Furthermore, we questioned whether the presence of preexistent HER-2/neu T-cell immunity or
the timing of immunity development over the course of active immunization influenced the
intensity of the elicited HER-2/neu-specific T-cell immunity. Our findings demonstrate that
maximal tumor-specific immune responses may occur toward the end of the vaccination regimen
or even after the scheduled vaccines have been completed. Additionally, the presence of tumor
antigen-specific immunity prior to vaccination is associated with greater magnitude immune
responses.
© 2007 Elsevier Inc. All rights reserved.
Introduction
The assessment of antigen-specific immune responses after
active immunization with cancer vaccines is one method to
prioritize vaccines for further development. As recently
recommended by The Cancer Vaccine Clinical Trial Working
Group [1], therapeutic cancer vaccines should be investi-
⁎ Corresponding author. Fax: +1 206 685 3128.
E-mail address: [email protected] (L.G. Salazar).
gated in two types of clinical studies: “proof-of-principle”
vaccine trials and efficacy trials. “Proof-of-principle” vaccine trials such as the one described here should be
conducted in the adjuvant setting in patients who do not
have rapidly progressing disease to allow for sufficient time
for biologic (i.e., generation of immune response) and
potential clinical activity to develop. Additionally, immune
responses should be evaluated sequentially with a minimum
of 3 time points (baseline and at least 2 follow-up time points)
and the frequency and magnitude of an immune response
should be defined for the population under study. Un-
1521-6616/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2007.08.006
276
fortunately, few vaccine studies to date have been conducted
in such a manner and as such the optimal timing for
evaluation of the immune response is not well defined.
Furthermore, very little data exist on the frequency and
magnitude of an immune response in the post-vaccination
setting once active immunization has ceased.
A more detailed understanding of T cell activation and
antigen processing has lead to a wealth of novel vaccination strategies that may significantly potentiate tumorspecific immunity. Thus, the identification of factors
associated with the development of T cell responsiveness
after vaccination remains critical. Murine studies would
suggest that the full potential of repeated vaccinations may
not be reached until late in the vaccine course or at a
distant time point after completing vaccination [2]. Moreover, the intensity or strength of a vaccine-induced
response appears to be vital to its efficacy in tumor
rejection [3]. Lastly, evidence indicates that serial tumor
peptide-based vaccination can successfully “boost” preexistent tumor-driven T cell responses [4]. Based on these
limited findings and recognizing that few studies have
explored the kinetics of the development of T cell
responses to vaccination with cancer antigens, we retrospectively evaluated the evolution of HER-2/neu-specific T
cell immunity over the course of active immunization and
at distant time points post-immunization in a large number
of cancer patients. We questioned whether there was an
optimal time to measure the development of T cell
immunity after a cancer vaccine as well as what role preexistent immunity had in the either augmenting or
inhibiting the vaccinated response.
Methods and materials
Patient population
Thirty-eight subjects with HER-2/neu overexpressing cancer
were enrolled in a Phase I HER-2/neu peptide vaccine study,
completed 6 vaccinations as previously described [5], and
were available for evaluation. Briefly, all subjects had
completed standard conventional therapy for their disease
so that they had no detectable disease or were stable on
hormonal or bisphosphonate therapy only. Additionally, all
subjects had been off and were able to remain off chemotherapy or other immune modulatory therapies for a
minimum of 30 days before enrollment and for the 7-month
duration of the study (6 monthly vaccines plus 1 month post6th vaccine follow-up). Subjects were vaccinated with one of
three vaccine formulations each containing three different
peptides derived from the HER-2/neu protein. All peptides
were potential helper epitopes of HER-2/neu predicted by
computer modeling, and empiric testing found them to be
immunogenic [6]. The HER-2/neu peptide-based vaccines
were admixed with GM-CSF and administered intradermally
once monthly for 6 months to the same regional draining
lymph node site [5].
Detection of peripheral blood T cell responses
HER-2/neu-specific T cell responses were measured at
baseline, 30 days after each vaccination, and then every
L.G. Salazar et al.
2 months for a period of 1 year from start of immunization.
To evaluate the effectiveness of this peptide-based vaccine
strategy in generating T-cell immunity to HER-2/neu protein,
[7] both HER-2/neu peptide-and protein-specific responses
were evaluated. In addition, T cell responses were continually measured every 1–2 months in several subjects for a
total period of 18 months from start of immunization. T cell
proliferation was assessed at different time points using a
modified limiting dilution assay as previously described [7].
While patient samples were assayed at different time
points, the modified limiting dilution assay is designed for
detecting low frequency lymphocyte precursors based on
Poisson distribution [8], and has recently been shown to
have adequate sensitivity and reproducibility in the ability
to measure a broad range of T cell responses; and was
determined to be a better discriminator of low-level
immune responses when compared to ELISPOT [9]. Results
were evaluated as a standard stimulation index (SI),
defined as the mean of all 24 experimental wells divided
by the mean of the control wells (no antigen). An SI N 2 was
considered evidence of an immunized response based on
analysis of a reference population [5]. If subjects had an
SI N 2 at baseline, i.e. preexistent immunity to HER-2/neu
[10], a post-vaccination response was defined as positive if
it was a minimum of 2 times baseline. Subjects were
considered to have developed an immune response if they
had a positive response to at least one peptide in the
immunizing mixture.
Statistical methods
Subjects who developed immunity by the 6th vaccine were
separated into 2 groups: those who achieved a positive
immune response early or late in the vaccine course. An early
response was defined as a positive response developing by
30 days after the third vaccine, i.e. during the first half of
the vaccine regimen. A late response was defined as a
positive response developing after the fourth vaccine, i.e.
during the second half of the vaccine regimen. The
probability of developing an immune response to vaccination
was summarized using cumulative incidence estimates,
where death without an immune response was regarded as
a competing risk [11]. The magnitude of both peptide and
protein immune response was evaluated as follows: the
maximum immune response (SI) for each patient was
selected regardless of whether it occurred before or after
the 3rd vaccine, and the mean maximum immune response
was calculated for each group. Two-sample t-tests were used
to test the difference between the maximum immune
response of the 2 groups. All reported p values are twosided.
Subjects who had a positive immune response at baseline
were compared to those who did not have immunity at
baseline. The magnitude of the peptide and protein immune
response was evaluated as follows: the maximum immune
response (SI) after vaccination for each patient was selected
regardless of whether it occurred before or after the 3rd
vaccine. The Mann–Whitney test was used to test the
difference between the protein responses. An unpaired
t-test with Welch’s correction was used to test the
difference between immune responses for the peptide
responses.
Kinetics of tumor specific immunity
277
Results
The majority of advanced stage cancer patients
develop HER-2/neu peptide-specific T cell immunity
early in the course of vaccination
Twenty subjects had complete evaluation of T cell responses
(baseline, then every 2 months for a period of 1 year from
start of immunization), 14 subjects had T cell evaluation at
baseline, then every 2 months until 30 days after the last
vaccine. Two subjects had T cell evaluation done at only 2
time points after baseline due to early study termination.
No patients received other interim treatments during the
1-year testing period. Fig. 1A shows the maximum SI to
Figure 2 Early immunity predicts the magnitude of HER-2/neu
peptide-specific T cell response. Shown are the mean maximum
immune responses for peptide T cell responses in subjects who
developed early immunity (defined as a positive response
developing by 30 days after the third vaccine, i.e. during the
first half of the vaccine regimen) or late immunity (defined as a
positive response developing after the fourth vaccine, i.e. during
the second half of the vaccine regimen) during the course of
vaccination.
immunizing HER-2/neu peptides at each time point for
each individual. Data suggest that peptide-specific immune
responses occur early in the course of vaccination. Fig. 1B
depicts the probability of developing a HER-2/neu peptidespecific Tcell response by a particular number of vaccinations
in the subjects who completed the entire course of
immunizations. The probability of developing Tcell immunity
by the 3rd vaccine, to at least one peptide in an individual’s
immunizing mix, was 82%. The probability of developing Tcell
immunity to at least one peptide in an individual’s mix after
the 6th vaccine was 95%. Thus, the majority of subjects with
advanced stage cancer were able to generate an immune
response to at least one of the HER-2/neu peptides in their
vaccine early, after only 3 vaccinations.
In addition, the magnitude of HER-2/neu peptide-specific
T cell responses was greater in subjects who demonstrated
detectable immunity by the 3rd vaccine. The average SI was
13.5 (range, 2.0–59) for subjects achieving a positive immune
response during the first half of the vaccine regimen and 4.6
(range, 1.2–15.5) for those achieving a positive immune
response during the second half of the vaccine regimen
(Fig. 2). The difference in mean maximum SI between the two
groups was significant (p = 0.004).
Figure 1 The majority of advanced stage cancer patients
develop HER-2/neu peptide-specific T cell immunity early in the
course of vaccination. (A) The maximum SI to HER-2/neu
peptides for each of the 38 subjects is shown at baseline, after
each vaccination, and during follow-up. Vaccination time points
correspond with evaluation of HER-2/neu-specific T-cell
responses which were measured 30 days after each vaccination,
before the next immunization as follows: time point 0 = 1st
vaccine; time point 1 = 30 days after 1st vaccine; time point
2 = 60 days after 1st vaccine; time point 3 = 90 days after 1st
vaccine; time point 4 = 120 days after 1st vaccine; time point
5 = 150 days after 1st vaccine; time point 6 = 180 days after 1st
vaccine. Dotted line depicts SI of 2.0. (B) The estimated
probability of developing a HER-2/neu peptide-specific T cell
immune response before and after each vaccination is shown.
The majority of advanced stage cancer patients
develop HER-2/neu protein-specific T cell immunity
later in the course of vaccination
Fig. 3A shows the SI to HER-2/neu protein at each time point
for each of the 38 subjects. As compared to the peptidespecific responses, more subjects developed protein-specific
immunity later in the course of vaccination, even after active
immunizations ended. Fig. 3B depicts the probability of
developing a HER-2/neu protein-specific T cell response by a
particular time point where the vaccination occurred during
months 1 through 6. The probability of developing a proteinspecific T cell response was 42% by the 3rd vaccine compared
to 62% by the 6th vaccine. An additional 13% of subjects
278
L.G. Salazar et al.
developed a protein-specific T cells response at a distant
time point after completion of all 6 vaccinations. All subjects that developed a protein-specific T cell response did
so during vaccination or within 6 months of completing all
6 vaccinations.
Preexisting immunity predicts magnitude of the
HER-2/neu protein-specific T cell response
Five of the 38 subjects had preexistent protein immunity. The
magnitude of protein-specific T cell response was greater in
subjects who did demonstrate a positive immune response at
Figure 4 Preexisting immunity predicts the magnitude of
HER-2/neu protein-specific T cell response. The median maximum protein immune response (SI) during vaccination in
subjects with (Yes) or without (No) pre-existent immunity is
shown. Dotted line at 2.0 represents baseline.
baseline as shown in Fig. 4. The median protein-specific Tcell
response after vaccination was significantly different
(p = 0.0089) between those with pre-existing immunity
(median SI 5.1 (range 3.8–32.1)) and those without preexisting immunity (median SI 2.2 (range 0.7–13.6)).
Discussion
Figure 3 The majority of advanced stage cancer patients
develop HER-2/neu protein specific T cell immunity later in the
course of vaccination. (A) The maximum SI to the HER-2/neu
protein for each of the 38 subjects is shown at baseline, after
each vaccination and during follow-up. Vaccination time points
correspond with evaluation of HER-2/neu-specific T-cell
responses which were measured 30 days after each vaccination,
before the next immunization as follows: time point 0 = 1st
vaccine; time point 1 = 30 days after 1st vaccine; time point
2 = 60 days after 1st vaccine; time point 3 = 90 days after 1st
vaccine; time point 4 = 120 days after 1st vaccine; time point
5 = 150 days after 1st vaccine; time point 6 = 180 days after 1st
vaccine. The asterisk (⁎) indicates the value of 32 achieved on one
subject after the second vaccination. (B) The estimated probability of developing a HER-2/neu protein specific Tcell immune
response before and after each vaccination and during follow-up
is shown.
There are many new vaccine strategies being developed to
target human self-tumor antigens [12]. Novel vaccine
approaches are designed to circumvent mechanisms by
which tumors escape immune surveillance. When vaccines
approach the transition from pre-clinical models to human
clinical trials much emphasis has been placed on the
quantitative measurement of the immune response, particularly a T cell response, as an initial assessment of vaccine
potency. As the use of cancer vaccines moves into the
adjuvant setting where evaluation of clinical response, i.e.
prevention of disease relapse, will require larger number of
patients, an indicator in Phase I studies of vaccine immunogenicity has increasing importance. The immunogenicity of a
novel vaccine may significantly impact whether that
approach progresses to later phase studies. There have
been few investigations attempting to determine the optimal
time during a vaccine regimen to measure the development
of tumor-specific immunity. Most studies focus on assessing
the development of T cell responses only during active
immunization. We overviewed the kinetics of the evolution of
T cell immunity during and after the administration of a HER2/neu peptide-based vaccine [5]. Data presented here
demonstrate that maximal tumor-specific immune responses
may occur towards the end of the vaccination regimen or
even after the scheduled vaccines have been completed. In
addition, the presence of tumor antigen-specific immunity
prior to vaccination is associated with greater magnitude
immune responses.
A major concern for the use of peptide-based vaccines is
the possibility of developing peptide-specific immunity with
no response to native protein. It is likely that peptide-based
vaccine strategies will be effective only if the peptide-specific responses generated allow recognition of native protein.
Unlike previous studies which have reported that T cells
Kinetics of tumor specific immunity
induced by peptides are unable to recognize the antigen
processed and presented naturally [13], the vaccination
strategy reported here was highly effective in generating
both HER-2/neu peptide- and protein-specific T cell immunity. While generation of peptide-specific immunity after
peptide immunization is a reflection of a patient’s immune
competence and their ability to be immunized, detection of
protein-specific response implies that the native protein was
taken up by APCs and processed so that the natural peptide
epitope presented by the APC class II MHC molecules was in
configuration similar to that of the immunizing peptides.
Additionally, the peptide was presented in a concentration
high enough to be recognized by immune T cells.
Findings in our study demonstrate that the majority of
subjects developed HER-2/neu peptide-specific T cell
responses early or by the 3rd vaccine. Furthermore, those
subjects who had a detectable peptide-specific immune
response by the third vaccine had very robust peptidespecific Tcell responses which were significantly greater than
those in subjects who demonstrated a detectable peptidespecific immune response later in the vaccine regimen (after
the 3rd vaccine). One may speculate that these early and
more robust peptide-specific immune responses were in part
due to effective “boosting” of preexistent immunity by
peptide vaccination resulting in rapid activation and expansion of T cells which had been spontaneously primed by
endogenous tumor antigen.
When is the optimal time to measure the development of
an immune response in relation to vaccination? Most studies
assess immunity during active immunization. Data presented
here would suggest that monitoring response at least a year
after vaccinations have ended would allow the identification
of all the subjects capable of responding. Unlike the early
HER-2/neu peptide-specific responses, the observed HER-2/
neu protein-specific T cell responses in this study primarily
occurred after completion of 6 vaccines with thirteen per
cent of these responses occurring up to 12 months after
starting vaccination. This may be due to the more involved
and complex process that measurable protein-specific T-cell
responses imply. Indeed, the generation of HER-2/neu
protein-specific responses was associated with epitope or
determinant spreading as previously reported [5] suggesting
that the immune repertoire to HER-2/neu evolves during the
course of vaccination. Thus, the detection of protein-specific
T cells after peptide immunization implies that the immunizing peptides represent natural epitopes of the HER-2/neu
protein, and this naturally expressed HER-2/neu protein is
being processed and presented in an augmented fashion. As
such, it is likely that protein responses to vaccination may
take longer to evolve and only by monitoring immunity long
term will detection of such responses be possible. While the
“optimal time for measurement of the development of
tumor-specific immunity” has not been well defined, recent
studies have indirectly provided information that would
argue for extended immunologic monitoring. Indeed, recent
findings by Monsurro and colleagues showed that protracted
exposure to antigen stimulation broadened and intensified
the extent of the immune response. However, the full potential of vaccinations may not be reached until late in the
vaccine course or until a distant time point after completing
vaccination [2]. These findings as well as data presented here
would suggest that ongoing assessment of immune responses
279
after active immunization has stopped is not only reasonable
but necessary.
Identification of factors associated with T cell responsiveness to vaccination remains critical. Data presented here
suggest that those subjects with preexistent immunity
potentially achieved the highest level of HER-2/neu-specific
T cell immunity after vaccination. Indeed, Speiser and
colleagues investigated whether pre-vaccine Tcell activation
differed between patients who responded versus those that
did not respond to a Melan-A peptide-based vaccine [14].
Peptide antigen-specific T cells were quantified and characterized ex vivo before and after vaccination and interestingly, several patients were found to have a relatively high
percentage of pre-activated Melan-A-specific T cells prior to
the initiation of vaccination. More importantly, positive
immune responses to vaccination occurred primarily in
those patients with preexistent immunity to Melan-A-specific
T cells prior to vaccination. These findings suggested that
vaccine responder patients might primarily be those with
spontaneous (i.e. tumor-driven) immune pre activation and
as such, significant activation and expansion of antigenspecific T cells may be the result of combined or sequential
stimulation by antigen provided by both the tumor and the
vaccine. Thus, developing vaccines that target antigens that
already demonstrate an increased incidence of endogenous
immune responses in cancer patients may improve the
vaccine potency and potentially, therapeutic efficacy.
In the clinical development paradigm for cancer vaccines,
the ability of a cancer vaccine to elicit a specific measurable
T cell response is increasingly being used to prioritize immunization strategies for therapeutic development. Data presented here would suggest that immunologic monitoring
should be performed for a more extended period of time than
just during active immunization. Assessing immune responses
for up to a year after the end of active immunization would be
a reasonable time frame to capture all patients capable of
responding to a particular vaccine. In addition, data
demonstrate that patients who have a preexistent antigenspecific immune response, detectable at the time of
initiating vaccination, achieve higher levels of tumor-specific
T cell immunity overall most likely due to the boosting of the
memory response.
Acknowledgments
This work was supported for MLD by the Cancer Research
Treatment Foundation and NIH grants R01CA7516 and
K24CA85218 and for LGS by NIH grant K23CA100691. Patient
care was performed in the University of Washington General
Clinical Research Center, NIH grant M01-RR-00037. We would
like to thank Sally Zebrick for assistance in manuscript
preparation.
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