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Blood, 1 March 2002, Vol. 99, No. 5, pp. 1517-1526
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma:
clinical and immune responses in 35 patients
John M. Timmerman,
Debra K. Czerwinski,
Thomas A. Davis,
Frank J. Hsu,
Claudia Benike,
Zheng Mei Hao,
Behnaz Taidi,
Ranjani Rajapaksa,
Clemens B. Caspar,
Craig Y. Okada,
Adrienne van Beckhoven,
Tina
Marie Liles,
Edgar G. Engleman, and
Ronald Levy
From the Division of Oncology, Department of Medicine,
and the Stanford Blood Center, Stanford University School of Medicine,
CA.
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Abstract |
Tumor-specific clonal immunoglobulin expressed by B-cell lymphomas
(idiotype [Id]) can serve as a target for active immunotherapy. We
have previously described the vaccination of 4 patients with follicular
lymphoma using dendritic cells (DCs) pulsed with tumor-derived Id
protein and now report on 35 patients treated using this approach. Among 10 initial patients with measurable lymphoma, 8 mounted T-cell
proliferative anti-Id responses, and 4 had clinical responses 2 complete responses (CRs) (progression-free [PF] for 44 and 57 months
after vaccination), 1 partial response (PR) (PF for 12 months), and 1 molecular response (PF for 75+ months). Subsequently, 25 additional
patients were vaccinated after first chemotherapy, and 15 of 23 (65%)
who completed the vaccination schedule mounted T-cell or humoral
anti-Id responses. Induction of high-titer immunoglobulin G anti-Id
antibodies required coupling of Id to the immunogenic carrier protein
keyhole limpet hemocyanin (Id-KLH). These antibodies could bind to and
induce tyrosine phosphorylation in autologous tumor cells. Among 18 patients with residual tumor at the time of vaccination, 4 (22%) had
tumor regression, and 16 of 23 patients (70%) remain without tumor
progression at a median of 43 months after chemotherapy. Six patients
with disease progression after primary DC vaccination received booster
injections of Id-KLH protein, and tumor regression was observed in 3 of
them (2 CRs and 1 PR). We conclude that Id-pulsed DC vaccination can
induce T-cell and humoral anti-Id immune responses and durable tumor
regression. Subsequent boosting with Id-KLH can lead to tumor
regression despite apparent resistance to the primary DC vaccine.
(Blood. 2002;99:1517-1526)
© 2002 by The American Society of Hematology.
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Introduction |
Low-grade follicular B-cell non-Hodgkin lymphoma
(NHL) is an incurable disease characterized by relatively slow growth
and excellent initial responsiveness to chemotherapy but also by a continuous pattern of relapse and death.1 Despite recent
advances in chemotherapy, radiation therapy, and supportive care,
overall survival for low-grade NHL has not significantly improved in
the past 3 decades. This fact, coupled with the substantial toxicities of standard treatments, has fueled the search for novel and more tumor-selective therapies.
Fortunately, there is a target expressed by B-cell lymphomas that can
be exploited using an active immunotherapeutic approach. This target is
the idiotype (Id), which is composed of the unique antigenic
determinants in the variable regions of the clonal immunoglobulin (Ig)
expressed by the tumor cells. Each normal B cell expresses an
immunoglobulin with unique variable region sequences in the heavy and
light chains that together form the antigen-binding site. When a B cell
undergoes malignant transformation, these sequences are maintained by
the malignant clone and can thus serve as a tumor-specific antigen. Id
proteins contain structures that can be recognized by
antibodies2-5 and by CD4+ T
cells5-8 and CD8+ T cells9-12 and
can be isolated from autologous tumor cells and formulated into a
custom-made vaccine.
In preclinical studies, vaccination of mice with Id protein coupled to
the highly immunogenic carrier protein keyhole limpet hemocyanin
(Id-KLH) has been shown to protect animals from subsequent tumor
challenge5,13-16 and to cure established lymphoma when combined with chemotherapy.16,17 In our previous clinical
studies, vaccination with Id-KLH plus a chemical adjuvant was found to induce anti-Id immune responses in approximately half the patients with
follicular NHL. These immune responses were correlated with improved
disease-free survival and overall survival.6,7
Subsequently, ways have been sought to improve the frequency and the
potency of anti-Id immune responses in patients with NHL.
The critical role played by dentritic cells (DCs) in the initiation of
immune responses makes them an attractive addition to cancer vaccine
strategies.18,19 When pulsed (cocultured) with
tumor-derived peptides or proteins or when transduced with tumor
antigen-encoding viruses or nucleic acids and administered as a
cellular vaccine, DCs have been shown to promote protective and even
therapeutic antitumor immunity in murine tumor models. These studies
have provided a compelling rationale for the clinical use of DCs in
active vaccination strategies against human cancer.20 Based on studies in a murine lymphoma model showing that vaccination with Id-pulsed DCs could induce protective antitumor
immunity,21 we initiated a pilot clinical trial in 1993 of
Id-pulsed DC vaccination for patients with follicular NHL. In the
preliminary report of our first 4 patients,22 we described
the induction of cellular anti-Id responses and evidence of antitumor
effects in 3 of 4 patients. Since our initial report, clinical activity
of DC-based vaccines has also been demonstrated in
melanoma,23,24 prostate carcinoma,25,26 renal
cell carcinoma,27 and carcinoembryonic antigen-expressing
cancers.28
We now report on our expanded experience and long-term follow-up of 35 patients with follicular NHL treated with Id-pulsed DC vaccination,
including a cohort of 25 patients treated in first remission after
their initial cytoreductive chemotherapy. Our observations include
durable, objective tumor regression in patients with measurable
disease, induction of T-cell anti-Id immune responses, and induction of
anti-Id antibodies capable of specifically recognizing and mediating
signal transduction in autologous tumor cells. In addition, we found
that patients who were resistant to or who had relapses after primary
Id-pulsed DC vaccination could achieve complete tumor regression after
the administration of booster vaccinations of Id-KLH protein. These
findings confirm the clinical activity of Id-pulsed DCs and Id-KLH
vaccination in follicular NHL and provide the rationale for applying
similar strategies to other B-cell malignancies.
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Patients, materials, and methods |
Patients
Patients were adults with stage III or IV follicular B-cell NHL
(follicular small-cleaved cell or follicular mixed small-cleaved and
large-cell by Working Formulation, follicle center cell grades I and II
by REAL classification). Each patient had a peripheral lymph node that
measured at least 2 × 2 cm and that was accessible to excisional
biopsy for diagnostic confirmation and vaccine production. Monoclonal
surface immunoglobulin expression was verified using flow cytometry.
The first 10 patients enrolled in the study (pilot phase) had
measurable relapsed or residual tumor after one or more previous
therapies (Table 1), and the remaining 25 patients were vaccinated after an attempt at remission induction with
chemotherapy (Table 2). Each patient's
physician determined the timing and choice of chemotherapy, and therapy
was continued until a maximal response was achieved. Clinical responses
were classified according to the standardized response criteria of
Cheson et al.29 Vaccinations were initiated at least 2 months after the completion of chemotherapy according to the schedule
below. Prevaccine patient evaluation included physical examination,
complete blood count, lymphocyte subset analysis, serum chemistry and
rheumatoid factor analyses, computed tomography (CT) of the chest,
abdomen, and pelvis, and bilateral iliac crest bone marrow biopsy.
These studies were repeated 1 month after the completion of
vaccination, and subsequent follow-up examinations performed every 3 months included physical examination, complete blood count, and serum
chemistry analysis. Repeat CT was performed every 6 months for the
first 2 years and annually thereafter, or as clinically indicated.
Prevaccine tumor status of patients was classified as either no
evidence of disease (NED), minimal residual disease (MRD), or residual
disease (RD), corresponding to the attainment and maintenance of a
complete response (CR), complete response unconfirmed (CRu), or partial
responses (PR), respectively, as specified in the Cheson criteria. The
study was approved by the Institutional Review Board of Stanford
University Medical Center, and all patients supplied written
informed consent.
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Table 2.
Characteristics and immune responses of patients vaccinated
in first remission after chemotherapy (n = 25)
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Vaccine production
In most cases, lymphoma Id proteins were isolated using the
rescue hybridoma method.6,22 This technique yields Id
proteins of either the IgG or the IgM isotype, depending on that
produced by tumor cells. An alternative strategy, molecular rescue, was used to prepare Id proteins for patients D17 and patients D21 to D26.
In this method, the genes encoding the heavy and light chain variable
regions from tumor Ig were amplified by polymerase chain reaction (PCR)
using family-specific immunoglobulin variable region
primers,30 cloned into plasmid vectors containing human IgG constant regions, and cotransfected into a mammalian cell line31 that then secreted the tumor-specific Ig product.
All tumor Id proteins were purified by affinity chromatography as previously described7 and, where indicated, were chemically conjugated with pyrogen-free KLH (Calbiochem, San Diego, CA) as previously described.6
We used peripheral blood DCs for our Id-pulsed DC vaccine approach
given the proven capacity of these cells to prime naive CD4+ and CD8+ T cells in
vitro.32,33 DCs were isolated from leukapheresis products
by a series of density gradient centrifugation steps, as previously
described.22 Peripheral blood mononuclear cells (PBMCs)
were obtained by leukapheresis using a COBE cell separator apparatus
followed by Ficoll-Hypaque sedimentation (Pharmacia, Uppsala, Sweden).
Monocytes were then removed by a discontinuous (50%) Percoll gradient
(Pharmacia). The resultant high-density fraction was cultured overnight
in RPMI 1640 plus 10% human AB serum in Teflon vessels (Savillex,
Minneapolis, MN) along with antigen at 2 µg/mL. During this period,
DC precursors spontaneously matured without exogenous cytokines and
acquired the low-density characteristic of DCs. The next day,
high-density lymphocytes were removed using a 15% metrizamide gradient
(Sigma, St Louis, MO), and the low-density, DC-enriched fraction was
then recultured overnight in antigen at 50 µg/mL and was harvested,
washed, and resuspended in sterile saline for re-infusion. The choice
of antigen concentrations for sequential overnight incubations with DCs
was based on our earlier in vitro studies using nominal
antigens32,33 and on the limited availability of Id
proteins. DCs were enumerated by flow cytometry as lineage (CD3, CD14,
CD19) negative and HLA-DR bright, and purity was confirmed by
morphology. For patients D1 to D22, DCs were divided and pulsed
separately with Id and KLH as in our original report,22
but patients D23 to D35 received DCs pulsed with Id-KLH conjugate as
indicated in Table 2.
Vaccine treatments
Antigen-pulsed DCs were administered intravenously after
premedication with acetaminophen and diphenhydramine. Patients received 3 monthly infusions, followed by a fourth given 2 to 6 months later.
Each infusion was followed 2 weeks later by subcutaneous injections of
0.5 mg soluble Id and KLH proteins (without DCs). In patients D1 to
D22, Id and KLH proteins were injected separately at different sites as
in our original report,22 whereas in patients D23 to D35,
Id-KLH conjugate was administered at a single site. Where indicated
(Table 3), certain patients received
further subcutaneous booster vaccinations consisting of 0.5 mg Id
protein coupled to 0.5 mg KLH and emulsified in the chemical adjuvant SAF1 as previously described.6,7 Five of these
vaccinations were administered at weeks 0, 4, 8, 12, and 20.
Cellular immune response assessments
T-cell proliferation assays were performed as reported
previously.6,7,22 Fresh PBMCs were cultured in
quadruplicate in media alone or with tumor Id, irrelevant Id proteins,
or KLH at 0.1, 1.0, 10, and 100 µg/mL. Incorporation of
[3H]-thymidine (Amersham Pharmacia Biotech, Piscataway,
NJ; 185 GBq/mmol) was measured after an overnight pulse (1 µCi [0.037 MBq]/well) on day 5. A response was interpreted as
positive when the incorporation of more than twice the background
(media alone) with at least one antigen concentration was observed on 2 or more occasions.
Tumor-specific cytotoxicity assays for patient D12 were adapted from
methods described by Schultze et al.34 Autologous
follicular lymphoma cells were activated by 3- to 5-day coculture with
irradiated (55 Gy) CD40L-expressing mouse L cells (a gift of Yong-Jun
Liu, DNAX, Palo Alto, CA). Activated tumor cells were then irradiated (64 Gy) and were used to restimulate cryopreserved prevaccine and
postvaccine (vaccine course 3) PBMCs for 2 weekly cycles. Interleukin-2
(Chiron, Emeryville, CA) was added at 30 IU/mL on the third day of
culture, with fresh medium added every 3 days thereafter. On day 14, cytotoxicity against unmodified, cryopreserved autologous follicular
lymphoma cells, irrelevant tumor cells (non-HLA matched follicular
lymphoma), and normal B cells was assessed in a standard 4-hour
chromium Cr 51 release assay.12 Normal autologous
peripheral blood B cells were isolated by negative selection using the
RosetteSep reagent (StemCell Technologies, Vancouver, BC, Canada), and
flow cytometry showed that they were more than 95% CD19+
and without evidence of light chain restriction (data not shown). Target cells were labeled with 51Cr (Amersham, 150 µCi
(555 MBq/mL) per 106 target cells) for 1.5 to 2 hours and
were washed 3 times, and 10 000 targets were incubated with effector
cells at the effector-target (E/T) ratios of 100, 30, 10, and 3. Unlabeled K562 erythroleukemia cells were added at a ratio of 50:1 to
labeled targets to quench natural killer cell-mediated lysis as
previously described.12 Spontaneous release of
51Cr was less than 20% for all target cell populations.
The percentage specific lysis is expressed as the mean of
triplicate values, determined using the equation: % Specific
lysis = (experimental cpm  spontaneous release cpm)/(maximum
release cpm spontaneous release cpm) × 100.
Humoral immune response assessments
Enzyme-linked immunosorbent assay was used to analyze sera of
all patients for anti-Id antibodies. Autologous IgM tumor Id proteins
or irrelevant IgM Id proteins were captured onto microtiter plates
coated with goat anti-human IgM (Biosource International, Camarillo,
CA). When the tumor Id was an IgG, the plates were coated directly
either with F(ab')2 fragments of the IgG Id produced by
digestion with immobilized pepsin (Pierce, Rockford, IL) or with whole
IgG Id. Prevaccine and postvaccine sera were serially diluted and
allowed to bind to the target Id proteins. Reagents used to detect
bound antibodies depended on the isotype of the target Id molecule.
When the Id protein was an IgM or a F(ab')2 fragment of
IgG, bound anti-Id antibodies of the IgG subclass were detected using
polyclonal goat anti-human IgG coupled to horseradish peroxidase (HRP;
Southern Biotech, Birmingham, AL). When the target Id protein was whole
IgG, bound anti-Id antibodies were detected with polyclonal anti-human
IgM HRP or with antibodies to the immunoglobulin light chain ( or
 ) opposite that of the tumor Id. A response was considered positive
when a 4-fold increase in anti-Id titer was found compared to the
prevaccine serum and the isotype-matched irrelevant Id proteins used as
specificity controls. Antibody responses to KLH were measured as
previously described.7 For tumor cell staining
experiments, cryopreserved IgM-positive tumor cells were incubated with
1% prevaccine or postvaccine serum overnight at 4°C, washed, and
incubated with goat anti-human IgG F(ab')2-phycoerythrin
(PE) followed by counter-staining with anti-human IgM
F(ab')2-fluorescein isothiocyanate (FITC; Biosource).
Stained cells were analyzed using a FACScan flow cytometer (Becton
Dickinson, San Jose, CA).
Analysis of signal transduction in tumor cells
Cryopreserved tumor cells (2 × 106/mL) were
incubated with 1% relevant or irrelevant prevaccine or postvaccine
serum for 3 hours on ice to allow the binding of anti-Id antibodies to
the cell surface, warmed to 37°C for 2 minutes, and immediately
chilled by the addition of ice-cold phosphate-buffered saline plus 1 mM Na3VO4 and washed once. Goat
F(ab')2 anti-µm antibodies (Biosource) served as a
positive control. Western blotting was performed as previously
described.35 Briefly, aliquots of cells treated as above
were lysed, and solubilized proteins were separated by 8% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing conditions and were transferred to nitrocellulose (Schleicher
and Schuell, Keene, NJ). The blot was then stained with
antiphosphotyrosine antibody 4G10 (Upstate Biotechnology, Lake Placid,
NY) followed by goat anti-mouse IgG-HRP (Southern Biotech) and was
developed using the enhanced chemiluminescence reagent (Amersham
Pharmacia Biotech). For the detection of tyrosine phosphorylation by
flow cytometry, cells treated as above were fixed and permeabilized
(FACS Lysing Solution; Becton Dickinson), then were stained with
antibody 4G10 coupled to FITC (Upstate Biotechnology). Aliquots of
cells counter-stained with phycoerythrin-labeled anti-CD20 or anti-CD3
antibodies were fixed in 1% paraformaldehyde, and phosphotyrosine
content was quantitated by FACScan.
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Results |
Pilot experience in patients with measurable follicular
lymphoma
Idiotype-pulsed DC vaccination was first applied in the pilot
phase of our study to 10 patients with measurable relapsed or residual
tumor after chemotherapy. Characteristics of these patients are shown
in Table 1. In this group, native, unmodified Id protein was used for
pulsing the DCs. Patients D1 to D4 were described in our initial
report.22 Eight of the 10 patients underwent 2 or more
chemotherapy regimens before vaccination, and all but one had multiple
sites of evaluable tumor. Total numbers of DCs administered over the 4 infusions were dictated by cell yield and ranged from 12 to
69 × 106 (median, 19 × 106; range,
2-32 × 106/infusion). Side effects (mild to moderate
chills, rigors, or fevers) were associated with less than 20% of
infusions and were self-limiting.
After vaccination, T-cell proliferative responses to Id protein
developed in 8 of the 10 patients. Serum anti-Id antibodies of the IgG
class were not found in any patient using our traditional anti-IgG
detection strategy,6,7 but 2 patients (D3 and D8) were
found to have serum anti-Id antibodies of the IgM class after vaccination. All 10 patients mounted humoral and cellular proliferative responses to KLH.
Evidence of antitumor activity was seen in 4 of the 10 patients,
including 2 who experienced CR. Complete regression of paracardiac and
mediastinal lymph nodes occurred in patient D1.22 This
patient's remission lasted 44 months, after which recurrent disease
was confined almost exclusively to bone, while all known prior sites of
disease were spared. Patient D9 experienced complete regression of
axillary (Figure 1), peri-aortic, and
iliac lymph nodes. This patient's CR lasted 57 months, when recurrent
disease was detected in a single previously uninvolved site (left
thyroid). Patient D2 had a PR of peri-aortic and para-iliac nodes
lasting 12 months. In patient D3, the only evidence of disease at the
time of vaccination was a tumor-specific PCR signal in the bone marrow
that became negative after vaccination,22 and she has
remained without clinical evidence of disease progression for more than
6 years. Of the 4 patients with clinical responses, 3 of 4 had T-cell
anti-Id immune responses, and patient D9 had no detectable in vitro
anti-Id response despite having achieved durable CR. There was no
correlation of immune or clinical responses to the number of DCs
infused. Follow-up status of the 10 patients is listed in Table 1. All 10 patients remain alive at a median follow-up of 64 months after vaccination (range, 47-94 months) and 103 months after diagnosis (range, 81-276 months). Patient D3 has not received any subsequent treatments, and the remaining patients have undergone additional treatments as indicated. Patient D8 remains in CR after booster vaccinations with Id-KLH protein (see below and Table 3).
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| Figure 1.
Complete regression of lymphoma after idiotype-pulsed
dendritic cell vaccination.
CT images of the chest of patient D9 show multiple enlarged left
axillary lymph nodes that normalized 11 months after vaccination.
Arrows indicate sites of disease before vaccination. Similarly sized
lymph nodes in the periaortic and iliac regions also completely
regressed.
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Vaccination of patients in first remission
Given the results of the pilot phase, we applied this treatment to
a group of 25 patients with advanced-stage follicular lymphoma after an
attempt at remission induction with chemotherapy. The characteristics
of these patients are shown in Table 2. Chemotherapy was administered
to achieve the best clinical response (response plateau with last 2 cycles). Patients were then observed until vaccine production was
completed (median, 5 months; range, 3-18 months). After chemotherapy
and at the time of vaccination, patients achieved and sustained the
following responses: CR in 5 patients, CRu in 11 patients, and PR in 7 patients (designated prevaccine tumor status of NED, MRD, and RD,
respectively). Two patients (patients D20 and D27) had progressive
disease (PD) at the time of vaccination (both at 9 months after
chemotherapy) and completed only 3 vaccinations before they needed
chemotherapy and had to be removed from the study. Numbers of DCs
administered (median, 16 × 106 over 4 infusions; range,
8-35 × 106 total DCs given,
0.7-12 × 106/infusion) and toxicities observed (less
than 20% incidence of transient flulike symptoms) were comparable to
those in the pilot phase of the study.
Immune responses of patients in first remission
Twenty-three patients completed the series of 4 vaccinations and
were considered evaluable for immune responses. All 23 patients mounted
humoral and cellular proliferative responses to KLH. Overall, 15 of the
23 (65%) patients mounted T-cell or humoral anti-Id responses. In
patients D11 to D22, native, unmodified Id protein was used for DC
pulsing as in the pilot study. Five of the 11 evaluable patients in
this subgroup mounted T-cell anti-Id responses, and anti-Id antibodies
of the IgM class, mirroring the low frequency of anti-Id antibodies
seen in the pilot phase, developed in one patient. To improve the
induction of Id-specific humoral immunity, 13 additional patients
(patients D23-D35) were immunized with DCs pulsed with the Id-KLH
conjugate. This modification was based on recent studies of Id-pulsed
DC vaccination in a murine lymphoma model, demonstrating that the use
of Id-KLH conjugate for DC pulsing led to improved induction of anti-Id
antibodies and tumor protection over native Id protein.36
Among 12 evaluable patients vaccinated in this manner, 4 mounted
cellular anti-Id responses, and 6 developed humoral anti-Id responses.
In 4 of these patients, high titers of anti-Id antibodies of the IgG
class were detectable. In 2 additional patients, anti-Id antibodies
were of the IgM class. The Id specificity of the humoral anti-Id
response evoked in 2 representative patients (D29 and D31) after
vaccination with Id-KLH-pulsed DCs is shown in Figure
2. These serum antibodies showed a high
level of binding to the relevant (autologous) tumor Id protein but a
low degree of binding to irrelevant Id proteins from other patients'
tumors. To further characterize these humoral anti-Id responses, we
next asked whether these sera could recognize tumor Id protein as
presented in its native conformation by tumor cells. Tumor cells were
incubated with either relevant or control prevaccine or postvaccine
serum and were stained for bound anti-Id antibodies. Figure
3 shows a flow cytometric analysis
demonstrating the ability of IgG antibodies in postvaccine serum to
specifically recognize autologous tumor cells but not control,
irrelevant tumor cells from another patient. Postvaccine sera could
specifically recognize tumor cells in 4 of 5 patients for whom tumor
cells were available for testing.
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| Figure 2.
Idiotype specificity of the
humoral anti-Id response after vaccination with Id-KLH-pulsed
dendritic cells.
Prevaccine or postvaccine sera from 2 patients (D31 in panel A, D29 in
panel B) were serially diluted and incubated on plates coated with the
patients' autologous tumor Id (relevant Id) or with Id proteins from 3 other patient's tumors (irrelevant Id). Binding of IgG anti-Id
antibodies was detected by anti-human IgG antibodies conjugated to
HRP, and expressed as optical density (O.D.). Panel A:  indicates
prevaccine sera on relevant Id (D31);  , postvaccine sera on relevant
Id (D31);  , postvaccine sera on irrelevant Id no. 1; ×, postvaccine
sera on irrelevant Id no. 2;
 , postvaccine sera on
irrelevant Id no. 3. Panel B: indicates prevaccine sera on relevant
Id (D29); , postvaccine sera on relevant Id (D29); , postvaccine
sera on irrelevant Id no. 1; ×, postvaccine sera on irrelevant Id no.
2; and , postvaccine sera
on irrelevant Id no. 3.
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| Figure 3.
Flow cytometric analysis of tumor-specific cell staining
by sera from patients vaccinated with Id-KLH-pulsed dendritic cells.
Tumor cells from patients D31 and D29 (upper and lower sets of panels,
respectively) were incubated with autologous postvaccine serum or that
of the other patient as a control. Bound anti-Id IgG antibodies were
detected by anti-IgG-PE (y-axis), and tumor B cells (IgM+) were
counterstained by anti-IgM-FITC (x-axis). Each patient's serum
specifically recognizes only the autologous tumor cells but not the
control, irrelevant tumor cells.
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Evoked antibodies induce signal transduction in tumor B
cells
We next investigated whether these serum anti-Id antibodies could
mediate signal transduction after binding to tumor cells. When B-cell
surface immunoglobulin is engaged and cross-linked by antigen, tyrosine
kinases are recruited to phosphorylate tyrosine residues in a number of
cellular proteins.37 Anti-Id antibodies, because they also
specifically cross-link surface immunoglobulin, can transmit a similar
signal. Previous studies have established that tumor regression induced
by passively administered anti-Id antibodies38 can be
correlated with ability of the antibodies to induce tyrosine
phosphorylation in tumor cells.35 However, such effects
have not been described using anti-Id antibodies from actively
vaccinated patients. Therefore, we treated tumor cells with
immune sera and assayed for the induction of tyrosine phosphorylation. As shown in the Western blot in Figure
4A, only cells treated with the relevant
postvaccine serum displayed the distinct pattern of signal intensities,
with more prominent bands at 140 and 75 kd. To verify and further
quantitate these effects on a per-cell basis, we used flow cytometric
analysis for cellular phosphotyrosine content. Using this method,
permeabilized tumor cells stained for total intracellular
phosphotyrosine could be counterstained for surface markers to
determine the population accounting for the increased signal. As shown
in Figure 4B, only treatment of cells with the relevant postimmune
serum results in a rightward shift in signal, indicating increased
phosphotyrosine content within the CD20+ tumor cell
population (left panel), whereas no such shift is seen in the
CD3+ tumor-infiltrating T cells (right panel). Mean
fluorescence intensities of the phosphotyrosine (4G10) signal induced
in the CD20+ cells by prevaccine serum, postvaccine serum,
or anti-µm antibodies (positive control) were 48, 103, and 110, respectively. These results indicate that serum anti-Id antibodies
induced by active vaccination can initiate signal transduction
specifically in tumor B cells. Such effects were observed in 2 of the 3 patients for whom tumor cells were available for testing.
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| Figure 4.
Idiotype-immune serum induces
tyrosine phosphorylation specifically in tumor B cells.
(A) Detection of tyrosine phosphorylation in tumor cells by Western
blotting. Tumor cells from patient D31 were incubated with the
indicated sera and then lysed. Solubilized proteins were separated by
SDS-PAGE and transferred to nitrocellulose. The blot was stained with
antiphosphotyrosine antibody 4G10. (B) Detection of tyrosine
phosphorylation in tumor cells by flow cytometry. Tumor cells from
patient D31 were treated with the indicated sera, permeabilized, and
stained with antibody 4G10 coupled to FITC. Phosphotyrosine content was
then quantitated in aliquots of cells counterstained with either
phycoerythrin-labeled anti-CD20 or anti-CD3.
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Clinical responses and follow-up of patients in first
remission
In evaluable patients with less than CR at the time of vaccination
(n = 18), sustained regression of residual tumor (in bone marrow,
lymph nodes, or pleura) was seen in 4 patients (22%; patients D13,
D15, D16, and D29). Patient D29, whose postvaccine sera specifically bound to tumor cells, had regression of lymphomatous pleural disease after vaccination (Figure 5). Pleural
effusion shown in the CT images of the chest was refractory to
cyclophosphamide, vincristine, and prednisone (CVP) chemotherapy and
remained cytologically positive before vaccination. However, 9 months
after vaccination with Id-KLH-pulsed DCs, it had almost completely
resolved. This patient also had a marked reduction in bone marrow
disease after vaccination and has remained without disease progression
for more than 21 months. Long-term follow-up of patients vaccinated
during first remission shows that 16 of 23 (70%) remain progression
free at a median of 43 months after the completion of chemotherapy
(range, 23-56 months). There has been a single death (patient D21)
associated with a transformation to aggressive diffuse large-cell
lymphoma. Six of 7 (86%) patients mounting humoral anti-Id responses
have remained progression free compared with 10 of 16 (63%) without antibody responses, though this difference is not statistically significant (P = .40, log-rank statistic). The proportion
of patients remaining progression free did not differ between patients
mounting T-cell or humoral responses (10 of 15, 67%) versus those
without immune responses (6 of 8, 75%).
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| Figure 5.
Regression of lymphomatous pleural effusion after
vaccination with Id-KLH-pulsed dendritic cells.
Despite chemotherapy, a left-sided pleural effusion persisted in
patient D29 and remained cytologically positive before vaccination.
Near-complete resolution is noted 9 months after completing vaccination
with Id-KLH-pulsed dendritic cells.
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Administration of booster vaccinations with Id-KLH protein plus
chemical adjuvant
Patients with persistent or relapsed tumors after DC vaccination
were offered booster vaccination with Id-KLH protein without DCs, as
performed in our previous vaccine trials.7 Biopsy samples were again taken from tumors of 4 patients before revaccination (patients D5, D6, D10, D12). Each was found to have retained expression of surface immunoglobulin heavy and light chains of the same classes found on the original tumor. Therapy consisted of 5 subcutaneous injections of the same Id protein used for DC vaccination yet was
coupled to KLH and was emulsified in a chemical adjuvant.7 Six patients were treated5 from the pilot study who had not fully responded and 1 who had a relapse after DC vaccination given during first remission (Table 3). The Id-KLH booster vaccinations were given
at different lengths of time after the original DC vaccination series
(range, 8-33 months), and all patients had multiple sites of
progressive, evaluable tumor. Three of the patients had immune responses to the booster vaccine series; 2 were T-cell responses and
one was a humoral response (Table 3). These responses were of the same
type (T cell, humoral) and of similar magnitude to those each patient
previously mounted to the DC vaccine, though the earlier responses had
waned in the interval before boosting. Each of the 3 immune responders
also had a corresponding clinical response; one PR lasting 14 months,
one CR ongoing at 48+ months, and one CRu lasting 16 months. The latter
patient (patient D12), treated 18 months after relapse following DC
vaccination given in first remission, had extensive lymphadenopathy
involving cervical, axillary, iliac, and inguinal lymph nodes,
including a pelvic mass measuring more than 4 × 5 × 6 cm. Bone
marrow was also involved. Within 2 weeks of the first injection of
Id-KLH, all palpable lymphadenopathy began to rapidly regress, and
physical examination results became normal in the next 4 weeks.
Normalization of all nodes was documented by CT at 1 and 4 months after
the completion of vaccination (Figure
6A-B), and the only evidence of tumor was equivocal findings in the bone marrow. At 16 months after vaccination, small recurrent lymph nodes were detected in the right side of the
neck. A repeat series of Id-KLH vaccinations (designated vaccine course
3) was then administered, and tumor regression occurred once again,
with CR documented 7 months later accompanied by an ongoing Id-specific
T-cell proliferative response (Table 3).
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| Figure 6.
Regression of tumors after booster vaccination with
Id-KLH + chemical adjuvant.
Patient D12 was given Id-pulsed DC vaccination during first
remission after chemotherapy but relapsed 15 months later. Subsequent
to the development of widespread disease 18 months later, she was given
injections of Id-KLH protein plus a chemical adjuvant. (A) CT images of
the pelvis show that bulky left-sided pelvic lymph nodes have
completely regressed 4 months after booster vaccinations. (B)
Regression of axillary (top panels) and inguinal (bottom panels) lymph
nodes after booster vaccinations. Arrows indicate sites of disease
before vaccination.
|
|
Given this patient's repeated tumor regression in response to
vaccination, we looked for evidence of antitumor cytolytic activity in
peripheral blood lymphocyte (PBL) effectors after restimulation with
CD40L-activated tumor cells.39 As shown in Figure
7, restimulated PBLs obtained after
vaccine course 3 exhibited specific cytotoxicity toward autologous
tumor cells. PBL obtained before vaccine course 3 had lower, but
measurable, cytolytic activity. There was no significant lysis of
purified autologous normal B cells (obtained after the attainment of
CR) or of follicular lymphoma cells from a patient of unknown HLA
haplotype (data not shown).
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| Figure 7.
Tumor-specific cytolysis by peripheral blood lymphocytes
derived from patient D12 before and after a repeat series of Id-KLH
vaccinations (third course of vaccinations).
Prevaccine and postvaccine PBMC effectors were restimulated using
2 weekly cycles of CD40L-activated autologous tumor cells plus
interleukin-2. After 14 days, the ability of effectors to lyse
unmodified, cryopreserved tumor cells was assessed in a 4-hour
51Cr release assay. Purified autologous normal peripheral
blood B cells serve as negative control targets. The percentage
specific lysis is expressed as the mean of triplicate values. Results
are representative of 3 independent experiments.  indicates
postvaccine course no. 3 versus tumor targets; indicates prevaccine
course no. 3 versus tumor targets; indicates postvaccine course no.
3 versus normal B cells; and  indicates prevaccine course no. 3 versus normal B cells.
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Discussion |
Our earliest studies of Id vaccination for NHL focused on
describing the induction of anti-Id immune responses6 and
their correlation with favorable clinical outcomes.7 With
our more recently developed DC vaccine approach, we directly assessed
antitumor effects by vaccination of larger numbers of patients with
measurable tumors. In the pilot phase of our DC study, we observed
antitumor activity in 4 of 10 patients consisting of 3 objective
radiographic responses and 1 molecular complete response. In 3 patients, these responses were complete and durable (44, 57, and 73+
months). Among patients with residual disease in first remission after chemotherapy, 4 of 18 also had objective reductions in measured tumor
burden. Two additional patients had tumor shrinkage after Id-KLH
booster vaccinations. Thus, 10 of 28 (36%) patients with evaluable
tumor had objective antitumor effects after our Id vaccine interventions. A novel finding in this study was the occurrence of
tumor regression in patients with resistant or relapsed disease after
retreatment with booster Id-KLH protein vaccinations. These observed
tumor regressions are instructive of several points. First,
revaccination using the same Id protein, reformulated as Id-KLH plus
chemical adjuvant, can lead to tumor regression despite the lack of a
clinical response to the initial Id-pulsed DC vaccine. Second, even
relatively large tumor burdens can undergo regression after Id-KLH
vaccination (Figure 6). Whether the prior immunization with Id-pulsed
DCs contributed to the observed antitumor responses to Id-KLH is
unknown. However, the rapid regression of tumor observed in patient
D12, beginning within 2 weeks of Id-KLH initiation, suggests
stimulation of a memory response, primed either through the previously
administered DC vaccine or through endogenous mechanisms. Taken
together, these observations provide unequivocal evidence of the
clinical activity of Id vaccination.
Induction of anti-Id immune responses was another important endpoint of
our study. The overall (humoral or T-cell) immune response rate of 65%
compared favorably with the 49% response rate seen in our earlier
study of Id-KLH vaccination.7 In keeping with the
principal role of DCs in priming T-cell responses, vaccination with
Id-pulsed DCs preferentially induced cellular responses over antibody
responses (Tables 1-2). Although 62% of patients given native
Id-pulsed DCs mounted cellular responses, only 14% produced anti-Id
antibodies. However, the ability of DCs to function as adjuvants for
the induction of a humoral anti-Id response varied according to the
form of Id used for DC pulsing, as previously observed in a murine
lymphoma model.36 Vaccination using DCs pulsed with native
Id induced detectable anti-Id antibodies, always of the IgM class, in
only 3 patients. Use of the Id-KLH conjugate for DC pulsing led to a
greater frequency of humoral anti-Id responses (6 of 12 patients; 50%)
including high-titer IgG antibodies, once again pointing to the
capacity of DCs to prime B-cell responses.18,36
Induction of a humoral anti-Id response is thought to be an important
goal of Id vaccination for several reasons. First, passively administered anti-Id murine monoclonal antibodies are able to induce
tumor regression in two thirds of patients.38 In addition, in murine lymphoma models of Id vaccination, anti-Id antibodies alone
can be sufficient to provide tumor protection.4,13,36,40 Thus, a broad anti-Id immune response, including anti-Id T cells and
antibodies, might have greater antitumor effects. Antibodies induced by
vaccination with Id-pulsed DCs were highly relevant to tumor cells
because they could bind selectively to autologous tumor cells in
addition to the isolated Id protein. Furthermore, these antibodies had
the capacity to stimulate signal transduction in tumor cells, as
indicated by increased tyrosine phosphorylation. Induction of tyrosine
phosphorylation by cross-linking surface immunoglobulin on B-lymphoma
cells has been shown to be an initial step in the cascade of events
leading to apoptosis,37,41,42 suggesting a direct
mechanism whereby anti-Id antibodies may exert antitumor effects. Vuist
et al35 previously demonstrated that in patients treated
with passive anti-Id monoclonal antibodies, achievement of a clinical
response was highly correlated with the ability of the antibodies to
stimulate tyrosine phosphorylation in tumor cells. Similarly, we have
found that among our treated patients, serum anti-Id antibodies capable
of binding to tumor cells could induce tyrosine phosphorylation in 2 of
3 patients. Whether this signal transduction response leads to
tumor cell apoptosis, correlates with clinical outcome, or can be
detected in patients treated with other types of Id vaccines is the
subject of ongoing studies.
Overall and progression-free survival of our selected group of 35 patients appears favorable in comparison to that historically observed
in follicular NHL,43 though a controlled trial would be
necessary to ascribe long-term outcomes to the vaccine interventions. Given the clinically significant tumor regressions observed in this
study, however, it is possible that the natural history of the disease
was altered in some patients. Bendandi et al39 have also
described excellent long-term disease-free survival in 18 of 20 patients (median since chemotherapy, 42 months) with follicular lymphoma vaccinated with Id-KLH plus granulocyte
macrophage-colony-stimulating factor. Because their study was limited
to patients achieving CR after chemotherapy, few comparisons can be
made with our current trial in which 20 of 25 patients had less than CR
at the time of vaccination. Nevertheless, all but 1 of our 5 patients
in CR has remained free of disease progression (Table 2).
Bendandi et al39 measured tumor-specific cytokine secretion
by patient PBL cocultured with autologous tumor cells in 19 of 20 patients, and they measured antitumor cytotoxic T-cell activity in a
subset of these patients. This 95% overall immune response rate is
higher than that observed in the current study. The difference may be
explained by the use of alternative assay techniques, their use of
Id-KLH plus granulocyte macrophage-colony-stimulating factor adjuvant,
or by selection of a more favorable first CR patient population. Indeed
it does appear from previous work7 and our current study
that immune responses after Id vaccination are more frequent in
patients in CR at the time of vaccination (4 of 5 immune
response-positive in the current study). Although we did not routinely
measure antitumor cytotoxic T cells in our patients, in the one patient
studied (patient D12) we did find considerable tumor-specific cytolytic
activity in PBLs obtained after repeat booster vaccinations with Id-KLH
and objective tumor regressions.
We believe that our findings have several implications for the design
of future lymphoma vaccine trials. Our results in NHL once again
confirm tumor antigen-pulsed DC vaccination as a promising platform for
cancer immunotherapy.20 This approach is more technically demanding than traditional protein (ie, Id-KLH) vaccination methods, and it remains to be proven superior in formal comparisons. Before embarking on such studies, it will be important to further refine this
technique, particularly with regard to the types and numbers of
antigen-pulsed DCs administered.19,20 Our use of
peripheral blood DCs isolated from leukapheresis product was based on
the demonstrated T-cell stimulatory capacity of this DC
fraction32,33,44 and its observed clinical activity early
in our pilot study.22 Numbers of DCs obtainable using this
method, approximately 5 to 10 million per infusion, do not allow for
dose-escalation studies. However, newer methodologies for propagating
DCs from monocytes45 or CD34+ hematopoietic
progenitor cells46 or from the mobilization of peripheral
blood DCs using Flt3 ligand28,47 now allow DC dose escalation to more than 108 cells. The use of greater
numbers of antigen-pulsed cells, cultured under conditions that promote
maturation and activation of DCs,48-50 may provide even
greater potency at inducing anti-Id immune and clinical responses.
Our observation of significant clinical activity in DC-vaccinated
patients after booster vaccination with Id-KLH suggests a potential new
strategy for the immunotherapy of follicular NHL, and further studies
using Id-KLH vaccines in tandem with Id-pulsed DCs are in
progress.51 These observations may also herald a shift in
our view of the optimal timing of Id vaccination. Earlier studies of Id
vaccination have been principally confined to patients in remission
after cytoreductive chemotherapy,6,7,39 based on the view
that vaccination might be more effective when the level of tumor is
minimal. Thus, little is known about the therapeutic effects of Id-KLH
vaccination in patients with progressive lymphoma. Indeed, in our
previous study, in vitro anti-Id immune responses were more frequently
detectable in patients in CR at the time of vaccination than in those
with residual disease,7 suggesting that the presence
of tumor might impair the host's ability to mount an effective
antitumor response. The finding that substantial tumor burdens may
regress after Id-KLH vaccination (Table 3; Figure 6) provides rationale
for further study of Id-KLH vaccination in patients with relapsed or
even untreated follicular NHL. In the latter case, avoidance of the
strongly immunosuppressive effects of chemotherapy before immunization
might prove to be an important advantage.
Given its validation as a susceptible target, lymphoma idiotype will
likely remain a highly relevant tumor antigen for NHL immunotherapy
despite the need to produce patient-specific material. In multiple
clinical trials, Id vaccine production has been shown to be feasible,
and evidence of clinical activity has been
obtained.6,22,39 Moreover, novel genetic approaches are
now yielding recombinant forms of Id and will continue to make
large-scale production of Id vaccines more practical.52-56
These technologies offer new sources of Id for loading into DCs. In the
future, as additional and possibly shared NHL-associated tumor antigens
are described, these too will be candidates for evaluation using
DC-based vaccine strategies.
| |
Acknowledgments |
We thank Hendrik Veelken for critical reading of the manuscript;
Dan Denney and Diane Ingolia for help in producing recombinant tumor Id
proteins; Yong-Jun Liu at DNAX for the gift of CD40 ligand-transfected L cells; and Patty Ciesla for assistance in preparing the manuscript.
| |
Footnotes |
Submitted June 22, 2001; accepted September 14, 2001.
Supported by National Institutes of Health grants HL57443 and CA33399.
J.M.T. and T.A.D. are recipients of Clinical Associate Physician awards
from the National Institutes of Health (RR-00070-CAP). R.L. is an
American Cancer Society Clinical Research Professor.
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: Ronald Levy, Dept of Medicine, Division of
Oncology, Stanford University Medical Center, CCSR Rm 1105, Stanford,
CA 94305-5151; e-mail: levy{at}stanford.edu.
| |
References |
1.
Horning SJ.
Natural history of and therapy for the indolent non-Hodgkin's lymphomas.
Semin Oncol.
1993;20:75-88[Medline] |
Top
Abstract
Introduction