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IMMUNOBIOLOGY
From the Division of Oncology, University of
Washington, and Immunex Corporation, Seattle, WA.
Dendritic cells (DCs) are potent antigen-presenting cells and have
shown promise to function as "natural" vaccine adjuvants. Currently, most cancer vaccine trials using DCs generate autologous DCs
ex vivo for each patient. Systemic treatment with Flt3 ligand (FL)
results in a marked increase of DCs in tissues such as spleen and lymph
nodes in mice and in the peripheral blood and skin of humans. In light
of these observations, we questioned whether FL could be used
systemically as a vaccine adjuvant to stimulate DC mobilization in
vivo, circumventing the need to generate DCs ex vivo. Ten patients with
HER-2/neu-overexpressing cancer were enrolled in a phase 1 study to
receive a HER-2/neu peptide-based vaccine targeting the intracellular
domain of the HER-2/neu protein. All patients received 20 µg/kg FL
per day subcutaneously for 14 days. Five patients received the
HER-2/neu peptide-based vaccine alone on day 7 of the 14-day cycle, and
5 patients received the vaccine admixed with 150 µg granulocyte
macrophage-colony-stimulating factor (GM-CSF) on day 7 of the FL
cycle. T-cell proliferative responses to HER-2/neu peptides and
intracellular domain protein suggest that vaccine regimens including FL
as an adjuvant were not effective in eliciting a significant HER-2/neu
protein-specific T-cell proliferative response. However, including FL
as a vaccine adjuvant was effective in boosting the precursor
frequency of interferon- Many newly defined tumor antigens are
self-proteins. The development of effective cancer vaccine strategies,
therefore, must focus on immunization methods that will effectively
circumvent tolerance. Dendritic cells (DCs) are the most potent human
antigen-presenting cells (APCs) and are uniquely qualified to act as a
cancer vaccine adjuvant designed to generate immunity to self-tumor
antigens. DCs have the ability to stimulate even a naive T-cell
population1 and have been shown to facilitate the
generation of immune responses directed against cryptic epitopes
because of more effective antigen processing and
presentation,2 a property critical to stimulating immunity
to the self.3 DCs, however, are rare cells in humans constituting less than 1% of circulating white blood cells. Methods to
expand DCs ex vivo to achieve large numbers of functional APCs are
under active investigation.4
Ex vivo generation of DCs and exogenous loading of antigen is the basis
of many human clinical trials designed to augment immunity to
self-tumor antigens.5,6 Some significant problems, however, are associated with the in vitro generation of DCs. First, tailor-making autologous DC vaccines for each individual will limit
widespread use of the vaccine. Second, the culture conditions used to
expand cells ex vivo may significantly affect their function and
antigen-processing capabilities. Finally, DCs generated in culture may
not traffic to draining lymph nodes in great numbers, thus limiting the
development of systemic immunity.7 Methods of stimulating
DC mobilization and trafficking in vivo, allowing the native immune
environment to naturally mature DCs, may overcome the functional
limitations imposed by ex vivo culture.
Flt3-ligand (FL) is a cytokine that, when administered systemically,
can increase numbers of circulating DCs more than
40-fold.8 Human DCs stimulated by the administration of FL
have been shown to be functional and can stimulate T cells in
vitro.8 Furthermore, the activation of DCs in vivo by FL
has been shown to be an effective way of circumventing tolerance during
active immunization in animal models.9 Studies have been
performed in the neu transgenic mouse, immunizing the animals to a
self-tumor antigen, neu, using FL as a vaccine adjuvant to mobilize DCs
in vivo.10 The timing of vaccine administration
corresponds to the kinetics of in vivo DC mobilization in
animals11,12 We questioned whether FL could be used as a vaccine adjuvant in vivo in
the human to stimulate immunity to HER-2/neu using a vaccine targeting
the ICD of the HER-2/neu protein. Previous studies, by our group, had
shown that granulocyte macrophage-colony-stimulating factor (GM-CSF)
applied locally by the intradermal route, along with an
antigen-specific vaccine, resulted in Langerhans cells, skin DCs,
mobilization, and recruitment.13 Furthermore, GM-CSF, as a
vaccine adjuvant, was effective in generating immunity when used with
an HER-2/neu peptide-based vaccine in humans.14 Systemic administration of FL in murine models results in markedly increased numbers of DCs in skin.15 Potentially, FL administration
could increase the number of DCs available for immune interaction
during intradermal immunization. Our strategy was to administer FL to patients with HER-2/neu-overexpressing tumors and to vaccinate intradermally with an HER-2/neu ICD peptide-based vaccine, with or
without concurrent local GM-SF administration, midpoint in the FL cycle.
Patient population
HER-2/neu peptide-based vaccine targeting the intercellular
domain
Detection of HER-2/neu peptide- and protein-specific proliferative T-cell responses by tritiated thymidine incorporation HER-2/neu-specific T-cell responses to peptide and protein were measured as previously described on freshly isolated peripheral blood mononuclear cells (PBMCs).17,18 PBMCs were analyzed for immune response at the end of a particular vaccine cycle, 14 days after the cessation of FL administration. Data are expressed as a standard stimulation index (SI) calculated from 24-well replicates. Phytohemagglutinin, incubated with patient T cells at a concentration of 5 µg/mL, was used as a positive control for the ability of T cells to respond to antigen and resulted in an SI greater than 2.0 in all patient assays reported (data not shown). PBMCs from 30 donors, all women without cancer ranging in age from 32 to 58 years, were evaluated in similar assays to establish baseline values. Means and 3 standard deviations of the T-cell response in the reference population to any of the HER-2/neu antigens tested was a maximum SI of 1.98; therefore, an SI greater than 2 was considered evidence of an immunized response. If subjects had an SI greater than 2.0 at baseline that is, pre-existent
immunity to HER-2/neu)19a postvaccination response was
defined as positive if it was greater than or equal to 2 times baseline.
FL may stimulate the autologous proliferation of T cells in culture by
increasing APC numbers in PBMCs. To verify that the use of FL in vivo
did not affect T-cell proliferation in vitro, we assessed whether the
mean cpm values of the 24 replicate resting T-cell wells Determination of delayed-type hypersensitivity responses Patients were skin-tested against their immunizing peptides 1 month after their last vaccination. Three hundred micrograms combined peptide solution, without any adjuvant, was injected intradermally on the patient's back, a site distant from the vaccine site. Induration was measured in 2 dimensions at 48 hours using calipers and was reported in cubic millimeters. All patients received 0.1 mL intradermal control test with sterile water, placed at the same time as the peptide test, with induration measured 48 hours later.Detection of HER-2/neu-specific T-cell responses by ELISPOT A 10-day ELISPOT assay was used to determine precursor frequencies of peptide- and protein-specific T lymphocytes as previously described on cryopreserved PBMC samples derived from patients before and after immunization.14 The assay was validated as linear and precise between 2.0 and 3.5 × 105 PBMCs per well. It had a detection limit of 1:100 000 and a detection efficiency of 93%. A positive response was defined as a precursor frequency that was significantly (P < .05) greater than the mean of control no antigen wells and detectable. The limit of detection of our assay is 1:100 000 precursors. PBMCs obtained before and after vaccination were analyzed simultaneously. Although the ELISPOT assay is sensitive and suitable for detecting low-level responses to vaccination,20-22 it is unknown whether the calculated precursor frequencies represent actual numbers of antigen-specific cytolytic T cells in the peripheral blood.
Patient characteristics Ten patients were enrolled. Nine patients had breast cancer with no evidence of disease. Five patients had stage III disease, 4 had stage IV disease, and 1 had ovarian cancer in first remission. Two patients, 1 with stage IV breast cancer and the patient with ovarian cancer, received only 2 vaccinations before having to withdraw from study to receive therapy for recurrent disease. Four of 5 subjects enrolled in the FL-alone arm completed 6 vaccines. The median age for those subjects was 46 years (range, 43-71 years), and the median time from chemotherapy was 5 months (range, 2-75 months). Four of 5 subjects enrolled in the FL and GM-CSF arm completed 6 vaccines. Their median age was 57 years (range, 46-67 years), and their median time from chemotherapy to the first vaccination was 6 months (range, 3-8 months). Immune response data are reported on the 4 patients from each treatment arm who completed 6 vaccinations. No patient enrolled had a personal or family history of autoimmune disease. Toxicity data are reported on all 10 enrolled patients.FL as a vaccine adjuvant was ineffective in generating detectable HER-2/neu protein-specific T-cell proliferative responses Figure 1 demonstrates T-cell responses developing to HER-2/neu peptides in the ICD vaccine and the ICD protein before and after immunization courses. None of the patients in the FL alone arm had a pre-existent immune response to p776, though one patient in the FL and GM-CSF arm had an SI of 5.0 to p776 before vaccination. After immunization, no patient in the FL-alone group developed immunity to p776 (Figure 1A). The P value comparing preimmunization and postimmunization responses for this group was P = .33. Three of 4 patients in the FL and GM-CSF arm did develop detectable immunity (mean SI, 5; range, 0.5-11.8; P = .09). Figure 1B demonstrates that no patients in the FL-alone arm had a pre-existent immune response to p927, and one patient in the FL and GM-CSF arm had an SI of 4 to p927 before vaccination. After immunization, no patient in the FL-alone group or the FL and GM-CSF group developed detectable immunity to p927 (P = .48 and .23, respectively). Preimmunization evaluation of the final peptide in the immunizing mix, p1166 (Figure 1C), shows no patients in the FL-alone group had evidence of pre-existent immunity to p1166 and that 1 patient in the FL and GM-CSF group had an SI of 8 before immunization. No patient in either FL arm developed detectable immunity to p1166 after completing all 6 immunizations (P = .28 [FL] and .24 [FL+GM-CSF]).
The demonstration of immunity to peptides developing after peptide immunization is a reflection of a patient's immune competence. However, we hypothesize that the in vitro surrogate of the ability to respond to HER-2/neu protein expressed endogenously in the major histocompatibility complex in vivo would be the detection of an immune response to ICD protein (Figure 1D). Two of 3 patients in the FL alone arm had a pre-existent immune response to ICD protein (SI 2.2 and 6.1), and 2 patients in the FL and GM-CSF arm had detectable pre-existent ICD protein-specific immune response (SI, 3 and 11.2). After completing all peptide immunizations, only 1 patient in the FL-alone arm had a detectable immune response to HER-2/neu ICD protein (SI, 4), and this patient had a pre-existent response (SI, 6.2) (P = .15). No patient in the FL and GM-CSF arm had detectable protein-specific immunity (P = .20). Thirty days after the last vaccination, patients were tested for a
delayed-type hypersensitivity (DTH) response to the pool of immunizing
peptides. Results are shown in Figure 2.
Previous studies from our group have demonstrated that HER-2/neu
peptide-specific DTH responses 10 mm2 or more correlate
significantly to measurable (ie, SI > 2.0) peripheral blood
HER-2/neu-specific T-cell responses.23 After immunization, all patients in the FL-alone arm (Figure 2A) had evidence
of some measurable DTH response, though none of them had a
postvaccination DTH of 10 mm2 or greater. Three of 4 patients in the FL and GM-CSF arm developed a postvaccination DTH 10 mm
or more, corresponding to those who generated measurable peripheral
blood T-cell responses (Figure 2B). No patient tested had a DTH to
sterile water negative control greater than 0.05 mm2.
Vaccine regimens including FL as a vaccine adjuvant were effective
in boosting the precursor frequency of IFN-  precursor frequency specific for immunizing peptides and
the HER-2/neu ICD protein was evaluated by ELISPOT. None of the
patients had detectable pre-existent IFN--secreting T cells specific for p776. After immunization, one patient in the FL-alone group developed an immune response to p776, frequency 1:5000 PBMCs (Figure 3A). The P comparing
preimmunization and postimmunization responses for this group was .20. Four of 4 patients in the FL and GM-CSF arm developed detectable
p776-specific T cells (mean frequency, 1:4000; range, 1:5800-1:3080;
P = .001). Evaluating the IFN--producing T-cell
response to p927, none of the patients had detectable pre-existent
IFN--secreting T cells specific for p927 (Figure 3B). After
immunization 3 of 4 patients in the FL-alone group developed immunity
to p927 (mean frequency, 1:4000 PBMCs; range, less than
1:100 000-1:1700; P = .10) (Figure 3B). All patients in
the FL and GM-CSF arm developed detectable IFN--producing T cells
(mean frequency, 1:3600; range, 1:8000-1:2400; P = .02). One patient in the FL-alone arm had a pre-existent, p1166-specific, IFN--producing response before immunization (1:2100) (Figure 3C).
After completing the vaccination cycle, 1 of 4 FL-alone patients developed a response (1:20 000). The P value comparing
preimmunization and postimmunization responses for this group was .35. The patient in the FL-alone arm had a pre-existent response that did
not increase to more than twice baseline and was, therefore, not
considered a positive responder. Two of 4 FL and GM-CSF patients
(1:30 000 and 1:7000) developed immunity specific for p1166.
Preimmunization and postimmunization responses for this group were
P = .07.
T-cell responses to HER-2/neu ICD protein were also assessed. One
patient in each arm had detectable precursor frequency to the intact
protein domain (FL alone, 1:4000; FL and GM-CSF, 1:3600) (Figure 3D).
After the completion of all immunizations, all 4 patients in each group
developed detectable IFN- Vaccine regimens including FL as a vaccine adjuvant were associated with the development of autoimmune phenomena in some patients In general, vaccine regimens including FL were well tolerated. Transient monocytosis developed in all patients who received FL as part of their regimen during periods of FL administration, consistent with the mobilization of DCs to the periphery (data not shown). Toxic reactions included a grade 1 rash in a patient taking FL and autoimmune serologic abnormalities in 2 patients taking FL and GM-CSF. One patient had grade 1 serologic abnormalities (ANA, anti-SSA, anti-dsDNA). After immunization she had a detectable IFN- peptide-specific T-cell
response to immunizing HER-2/neu peptides p927 (57 ± 12) and p1166
(80 ± 15), but not to the ICD protein. The second patient, who had
stage IV breast cancer, acquired grade 2 toxicity with serologic
abnormalities and Sicca syndrome, characterized by dry eyes and dry
mouth not requiring immunosuppressive treatment, 3 months after the
completion of the vaccine regimen (Figure
4). This patient did not develop any
detectable immunity to HER-2/neu peptide or protein after active
immunization. In addition, this patient had no personal or family
history of autoimmune disease, specifically collagen vascular,
rheumatoid arthritis, lupus, Sjögren syndrome, autoimmune thyroid
disease, or scleroderma. Clinical follow-up of this patient
demonstrated, 18 months after her final vaccination, no clinical
symptoms relating to Sicca syndrome, and her serology values became
negative except for a persistently elevated SSA. No patients had
evidence of autoimmune phenomena directed against tissues that
expressed basal levels of HER-2/neu.
In vivo mobilization of DCs for use in cancer vaccines offers an
attractive alternative to ex vivo DC generation. First, cytokines that
stimulate the proliferation, maturation, or migration of DCs in vivo
might allow universal application of DC-based vaccine strategies. It
may not be possible to generate DCs on all cancer patients in
vitro.24,25 Second, avoiding the manipulation of DCs ex
vivo removes the possibility of causing functional changes that occur
when DCs are cultured, such as early maturation and loss of phagocytic
and migratory capability before antigen loading. Third, stimulating DCs
or Langerhans cells in vivo may allow the natural processing necessary
to stimulate DC maturation and trafficking, which may improve antigen
presentation at the level of the draining lymph node.26,27
We hypothesized that increasing the number of circulating and skin DCs
with FL might result in a more robust immune response and a greater
percentage of patients immunized than what was seen using GM-CSF alone
as an adjuvant. Studies described here demonstrate that in immunizing
against the self-tumor antigen HER-2/neu, vaccine regimens that
included FL as a vaccine adjuvant may not be effective in generating
detectable HER-2/neu-specific T-cell proliferative responses; using FL
as a vaccine adjuvant resulted in boosting the precursor frequency of
IFN- FL and GM-CSF have been used as adjuvants in a variety of vaccines in
animal models. Local application of GM-CSF is associated with local
Langerhans cell recruitment and the up-regulation of major
histocompatibility class I and class II on APCs such as monocytes and
macrophage.13 FL has been used as a systemic adjuvant in
animal models. Indeed, even when administered by a route that is
classically associated with the generation of tolerance Recent investigations have demonstrated that FL and GM-CSF may
stimulate different subsets of DCs in vivo and that the cytokine microenvironment elicited, either type 1 or type 2, is markedly influenced by the particular DC subset generated. Evaluating a murine
model of cancer, using tumors engineered to express either GM-CSF or FL
demonstrated that GM-CSF-engineered cells were more potent in inducing
an antitumor response.30 GM-CSF elicited a diverse
cytokine environment consisting of Th1 and Th2 immune effectors. In
contrast, immune responses generated with FL-expressing tumor cells
were specifically restricted to a Th1 phenotypic
response.30 Our data support that FL is associated with
the development of a strong type 1 response. Significant HER-2/neu
antigen-specific precursor frequencies could be detected after
immunization only with assays specifically designed to evaluate
IFN- The association of autoimmune phenomena occurring in patients who received FL as a vaccine adjuvant suggests the APCs generated are functional. Autoimmune phenomena have been demonstrated with the use of DCs as a vaccine adjuvant in animal models.33 In vivo mobilization of DCs using FL will cause the peripheralization of DC precursors systemically. The antigen-presenting capabilities of these cells as they circulate to target organs is unknown. FL increases the number of circulating bone marrow-derived DCs. These DCs are, most likely, not fully matured and are capable of taking up antigen derived from the environment. It is possible that circulating DC precursors could respond to self-proteins in the environment and could elicit immunity to self. The in vivo kinetics or initiation process involved in generating this immune response is not defined. Although serologic abnormalities could be detected during active FL administration, the clinical syndrome associated with those abnormalities developed only after the study was completed, suggesting a more extended follow-up may be appropriate for evaluating the potential of an immune-mediated toxicity when evaluating immunotherapeutic strategies. Studies such as the one presented here demonstrate that FL, given
systemically, can influence immune responses generated in vivo. DC
precursors can be effectively mobilized by FL and are able to present
antigen, as evidenced by the demonstration not only of
HER-2/neu-specific T-cell responses inducing IFN-
We thank Chalie Livingston for assistance in manuscript preparation and Dr Kathleen Ruffner for assistance in the statistical analysis of laboratory data. Our heartfelt thanks go to all the patients who agreed to participate in this study.
Submitted May 21, 2001; accepted November 28, 2001.
Supported by grants from the National Institutes of Health/National Cancer Institute (K08 CA61834 and R01 CA75163), the Department of Defense (DOD) Breast Cancer Program, and the Cancer Research Treatment Foundation (M.L.D.). Patient care was conducted through the Clinical Research Center Facility at the University of Washington, which is supported by NIH grant MO1-RR-00037. Supported in part by a grant from the Immunex Corporation (M.L.D.). K.L.K. was supported by a DOD Breast Cancer Program Fellowship Award.
One of the authors (D.C.) is employed by Immunex Corporation, Seattle, WA.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mary L. Disis, Division of Oncology, University of Washington, Box 356527, Seattle, WA 98195-6527; e-mail: ndisis{at}u.washington.edu.
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M. E. Wolpoe, E. R. Lutz, A. M. Ercolini, S. Murata, S. E. Ivie, E. S. Garrett, L. A. Emens, E. M. Jaffee, and R. T. Reilly HER-2/neu-Specific Monoclonal Antibodies Collaborate with HER-2/neu-Targeted Granulocyte Macrophage Colony-Stimulating Factor Secreting Whole Cell Vaccination to Augment CD8+ T Cell Effector Function and Tumor-Free Survival in Her-2/neu-Transgenic Mice J. Immunol., August 15, 2003; 171(4): 2161 - 2169. [Abstract] [Full Text] [PDF]
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L. Hansson, H. Rabbani, J. Fagerberg, A. Osterborg, and H. Mellstedt T-cell epitopes within the complementarity-determining and framework regions of the tumor-derived immunoglobulin heavy chain in multiple myeloma Blood, June 15, 2003; 101(12): 4930 - 4936. [Abstract] [Full Text] [PDF]
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 T. E. Toliver-Kinsky, C. Y. Lin, D. N. Herndon, and E. R. Sherwood Stimulation of Hematopoiesis by the Fms-Like Tyrosine Kinase 3 Ligand Restores Bacterial Induction of Th1 Cytokines in Thermally Injured Mice Infect. Immun., June 1, 2003; 71(6): 3058 - 3067. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
; patients who received
FL and GM-CSF are shown as 
. (A) Responses against peptide p776. (B)
Responses against p927. (C) Responses against p1166. (D) Responses
against the HER-2/neu ICD protein. Data are expressed as a stimulation
index. Each symbol represents data on an individual patient. Bold bars
indicate the mean response for that group.
) are shown in one
patient plotted over time. This patient received FL and GM-CSF as a
vaccine adjuvant. Large arrows at the top of graph indicate the
development of nonspecific symptoms (Sx) of dry eyes, diagnosis of
Sicca syndrome (Sicca), and resolution of symptoms (No Sx).