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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2411-2419
By
From the Divisions of Oncology, Hematology, Bone Marrow
Transplantation and the Stanford Blood Center, Stanford University
Medical Center, Stanford, CA.
The idiotype (Id) determinant on the multiple myeloma (MM) protein
can be regarded as a tumor-specific marker. Immunotherapy directed at
the MM Id may stem the progression of this disease. We report here on
the first 12 MM patients treated at our institution with high-dose
therapy and peripheral blood stem cell transplantation (PBSCT) followed
by Id immunizations. MM patients received PBSCT to eradicate the
majority of the disease. PBSCT produced a complete response in 2 patients, a partial response in 9 patients and stable disease in 1 patient. Three to 7 months after high-dose therapy, patients received a
series of monthly immunizations that consisted of two intravenous
infusions of Id-pulsed autologous dendritic cells (DC) followed by five
subcutaneous boosts of Id/keyhole limpet hemocyanin (KLH) administered
with adjuvant. Between 1 and 11 × 106 DC were obtained by
leukapheresis in all patients even after PBSCT. The administration of
Id-pulsed DC and Id/KLH vaccines were well tolerated with patients
experiencing only minor and transient side effects. Two of 12 patients
developed an Id-specific, cellular proliferative immune response and
one of three patients studied developed a transient but Id-specific
cytotoxic T-cell (CTL) response. Eleven of the 12 patients generated
strong KLH-specific cellular proliferative immune responses showing the
patients' immunocompetence at the time of vaccination. The two
patients who developed a cellular Id-specific immune response remain in complete remission. Of the 12 treated patients, 9 are
currently alive after autologous transplantation with a minimum
follow-up of 16 months, 2 patients died because of recurrent MM and 1 patient succumbed to acute leukemia. These studies show that patients make strong anti-KLH responses despite recent high-dose therapy and
that DC-based Id vaccination is feasible after PBSCT and can induce
Id-specific T-cell responses. Further vaccine development is necessary
to increase the proportion of patients that make Id-specific immune
responses. The clinical benefits of Id vaccination in MM remain to be determined.
DESPITE THE AVAILABILITY of high-dose
therapy and peripheral blood stem cell transplantation (PBSCT) for the
treatment of multiple myeloma (MM), recurrent disease remains a major
problem for patients with MM. Immunotherapy may provide additional
control of residual disease. The unique biology of MM allows for the MM idiotypes (Id) and their peptide fragments to serve as tumor-specific antigens and potential targets for adjuvant immunotherapy. One of the
most potent means of stimulating immune responses is through the use of
dendritic cells (DC). Therefore, we have conducted a feasibility study
to evaluate a DC-based Id vaccination strategy for MM patients after PBSCT.
Prior studies by Eisen et al1,2 showed that myeloma-derived
Ig can serve as an immunogen in a murine mineral oil-induced plasmacytoma (MOPC) model. When the myeloma protein was used in a
vaccine it stimulated a tumor-specific protective immune
response.1,2 The specificity of the immune response was
restricted to unique determinants (idiotype) located on the
antigen-binding sites of the Ig molecule. Other investigators have
confirmed Eisen's findings in various myeloma model systems including
a more physiological model based on the spontaneously arising 5T2
murine myeloma.3,4 Studies of the immune mechanisms
contributing to Id-specific tumor protection have been discussed in
detail and have led to the assertion that Id-specific T cells are
important for tumor protection in MM.5 Bogen et
al6-8 have shown that Id-specific CD4+ T cells
are important for tumor protection in the MOPC 315 model. Others have
investigated Id-specific cytotoxic CD8+ T-cell clones, but
their role in tumor protection remains to be
determined.9,10 The ability to make these anti-Id immune responses, however, was impeded by the presence of circulating myeloma
protein.11
The importance of Id-specific T cells in myeloma is not well studied in
humans. A small number of Id-specific T cells has been reported in
patients with early stages of MM.12 Osterborg et
al13 reported on the Id immunization of five MM patients that stimulated T-cell immune responses in all five. Id presented by
monocyte-derived DC was able to stimulate a T-cell proliferative response to the MM protein.14 The immunogenicity of a human MM Id protein vaccine in a normal person and the subsequent transfer of
Id-specific T cells to a patient has been shown in the setting of
allogeneic bone marrow transplantation (BMT).15
Id vaccination for patients with B-cell non-Hodgkin's lymphoma has
succeeded in inducing specific immune responses in these patients.16,17 There was a strong correlation between the
presence of an anti-Id immune response and freedom from disease
progression as well as overall survival. Furthermore, the patients who
were in a complete remission at the time of their vaccination had a greater probability of making an immune response.17
A prospective randomized study has recently shown that high-dose
chemotherapy with autologous BMT is superior to standard chemotherapy
for the treatment of MM. High-dose therapy resulted in a higher
response rate, a prolonged event-free survival, and an increased
overall survival.18 Id vaccinations are, thus, likely to be
most efficacious in MM patients who have completed a PBSCT and have
achieved a status of minimal residual disease (MRD).5
Dendritic cells are capable of antigen uptake, processing, and
presentation on major histocompatibility complex (MHC) class I and II
molecules. They are efficient in inducing strong and protective immune
responses, especially T-cell immune responses.19 Immunization with antigen or tumor peptide pulsed autologous DC has
been successfully used in various animal models20,21 and has more recently been introduced into clinical trials for
lymphoma22 and melanoma.23 Based on these
observations we have conducted a study to evaluate the feasibility of
DC-based Id vaccination in MM patients after high-dose therapy and PBSCT.
Patients and PBSCT.
Fourteen eligible MM patients treated at our institution with PBSCT
were offered adjuvant immunotherapy on this phase I study. Two patients
declined to participate. All patients had laboratory, histopathologic,
and radiologic findings consistent with a diagnosis of MM.
Institutional review board-approved informed consent was obtained from
all study patients. Serum and BM samples were collected early in their
disease, preferably before initiation of chemotherapy. Patients
received high-dose therapy if they had chemotherapy-sensitive disease
(shown by a >50% decrease of monoclonal Ig and a decrease of BM
plasma cell involvement to <20% with initial chemotherapy), a
Karnofsky performance status of greater than 70%, and no renal, hepatic, cardiac, and pulmonary impairment. PBSC were collected after a
single treatment with cyclophosphamide (4 g/m2) followed
by granulocyte colony-stimulating factor (G-CSF) and stored as a backup
source of stem cells. Twenty-eight days later, a second PBSC collection
was obtained after a single treatment with etoposide (2 g/m2) followed by G-CSF. This second cell collection was
used for transplantation. The transplant conditioning regimen consisted of fractionated total body irradiation (FTBI) with a total dose of 12 Gy followed by intravenous (IV) administration of melphalan (MEL) at a
dose of 140 mg/m2. Patients who received prior ionizing
irradiation received carmustine (BCNU) at 550 mg/m2 in
place of FTBI and received MEL at a dose of 140 mg/m2 to
180 mg/m2 as part of a dose-escalation study. The patient
characteristics and response to high-dose therapy with PBSCT are
summarized in Table 1. Partial response
(PR) was defined as a reduction of the serum monoclonal spike by
greater than 50% of the pretransplant level, a lack of Bence Jones
proteinuria and less than 10% plasma cell involvement in BM biopsy and
aspirate specimens. Complete response (CR) was defined
as the repeated absence of the monoclonal Ig in immunofixation
electrophoresis and no evidence of clonal plasma cells in BM specimens.
Id purification.
Id proteins were isolated from serum samples obtained before or during
initial cytoreductive chemotherapy. Only those patients whose available
serum had an M-protein level greater than 0.4 g/dL were eligible for
the Id vaccination trial. The myeloma protein isotype and heavy-chain
subclass were determined with an enzyme-linked immunosorbent assay
(ELISA). Briefly, 96-well MaxiSorb immunosorbent plates (Nalgene Nunc,
Rochester, NY) were coated with goat anti-human kappa or lambda
antibody (Biosource, Camarillo, CA) in 0.1 mol/L carbonate buffer, pH
9.0, overnight. The plates were then washed several times in normal
saline containing 0.1% Triton-X 100 (Sigma, St Louis,
MO). Patient samples were serially diluted with
phosphate-buffered saline (PBS) containing 2% (wt/vol) bovine serum
albumin and incubated on the plates for more than 1 hour at room
temperature. The plates were washed again and the bound Ig was detected
with peroxidase-conjugated mouse monoclonal antibodies which were
specific for the different human heavy-chain isotypes (Southern
Biotechnology Assoc, Birmingham, AL).
DC vaccines.
The MM patients received their DC vaccines 3 to 6 months after
completing their PBSCT. Each patient was given two Id-pulsed DC
immunizations separated by 4 weeks. DC vaccines were prepared as
previously described.22 Briefly, mononuclear cells were
collected from peripheral blood by leukapheresis. The precursor DC were isolated from the mononuclear cells by a series of density gradient centrifugations. The DC preparations were cultured with purified Id for
36 to 48 hours, collected, washed free of Id protein, and administered
IV over 60 minutes in a volume of 100 mL. The composition of DC
preparations was monitored by light microscopy and flow cytometry.22 DC were identified by high MHC class II
expression and the absence of T- and B-cell markers. An average of 5.1 × 106 ± 2.9 × 106 DC were infused with
each immunization.
Id/keyhole limpet hemocyanin (KLH) vaccines.
Five Id/KLH vaccines were administered after the DC vaccines. The
Id/KLH vaccines were prepared as previously described.16,17 Briefly, purified Id proteins were conjugated with glutaraldehyde to
the immunologic carrier KLH at a 1:1 (wt:wt) ratio. The conjugates were
dialyzed against normal saline and then mixed with freshly prepared
ISAF adjuvant. ISAF consisted of 10% (vol:vol) squalane (Aldrich
Chemical, Milwaukee, WI), 5% (vol:vol) pluronic L121 (BASF,
Parsippany, NJ), and 0.4% (vol:vol) Tween 80 (Aldrich Chemical, Milwaukee, WI) in PBS.25,26 The Id/KLH vaccinations were
started 4 weeks after the second Id/DC vaccine and were administered at 4-week intervals.
Identification of MM Id encoding genes.
The myeloma-derived heavy- and light-chain Ig variable region genes
were isolated from BM aspirate specimens obtained before or in the
early part of induction chemotherapy. BM mononuclear cells (BM-MNC)
were isolated from the aspirate by Ficoll-Paque density centrifugation
(Pharmacia, Piscataway, NJ). RNA was isolated from 5 to 10 × 106 BM-MNC with RNAzol B (Tel-test, Friendswood, TX) or
with the RNeasy kit (Qiagen, Valencia, CA) according to the
manufacturers' protocols. First-strand cDNA was synthesized from the
RNA using random hexamer priming and Superscript RT (Life Technologies, Gaithersburg, MD) as described.27 The myeloma Id
heavy-chain variable region genes were amplified from the cDNA using
polymerase chain reactions (PCR). Six 5' primers corresponding to the
leader sequences of the six human heavy-chain variable region families (VHL1 to VHL6)28 were used in individual PCR. The 3'
primers used in the PCR corresponded to the appropriate gamma
(5'-CTTGACCAGG CAGCCCAGGGC-3') or alpha heavy-chain constant region
(5'-GAGGCTCAGC GGGAAGACCT-3'). PCR conditions were as
described,27 except for a few reactions where the annealing
temperature was reduced from 55°C to 50°C. In all cases,
electrophoretic analysis of the PCR reactions showed a predominant band
of the expected length in only one of the six VH leader primer
reactions. The PCR product was purified by electrophoresis through
agarose and extracting the DNA from the agarose using the QIAquick gel
extraction kit (Qiagen). The isolated PCR product was then ligated into
the vector, pCRII (Invitrogen, San Diego, CA) and transfected into
TOP10F' bacteria. Plasmid DNA was isolated from cultures derived from individual bacterial colonies and sequenced using dye termination PCR
(Perkin-Elmer, Norwalk, CT). A sequence for a heavy-chain variable
region was determined to be myeloma derived if it was identified in DNA
isolated from three bacterial colonies generated from the cloning of
two independent PCR products.
Generation of an Id-encoding recombinant adenovirus (IdAd).
A recombinant adenovirus containing a patient's MM-derived variable
region genes was made for selected cases. VH genes used for cloning
were obtained by reamplification of Id VH using Pfu DNA polymerase
(Stratagene, La Jolla, CA) and leader and constant region primers
containing unique restriction sites to aid in the cloning. The primers
used for identifying and isolating the VL genes contained restriction
sites so that the initial PCR product could be used for cloning into an
expression vector without reamplification. The patient's MM VH and VL
genes were cloned into the bicistronic Ig expression vector,
pTC.29 This vector allowed for the cloning of the heavy-
and light-chain variable region genes in frame with their respective
constant regions. Cytomegalovirus promoters were used
for each Ig gene. The expression cassette containing the Id genes was
then moved from pTC to the adenovirus transfer vector, pXCJL1. The
Id-gene-containing pXCJL1 plasmid was cotransfected with the
unpackageable adenovirus type V (AdV) plasmid pJM17 into 293 cells as
previously described.30 Intracellular recombination between
these two plasmids in this AdV permissive cell line resulted in the
generation of replication-deficient Ad. The recombinant virus was
plaque purified and then expanded on 293 cells. The titers of the
adenovirus stocks were determined by limiting dilution. Integrity of
the Ig genes was determined by assaying the supernatant of infected 293 cells for Ig expression by ELISA as described above.
T-cell assays.
Id-specific T-cell immune responses were monitored by a T-cell
proliferation assay as previously described17,22 except for
the use of the serum-free medium, AIM V (GIBCO-BRL, Grand Island, NY)
instead of an Iscove's modified Dulbecco's
medium-based culture medium. Briefly, heparinized
blood samples were obtained before each Id vaccination and 4 and 12 weeks after the last vaccination. Peripheral blood mononuclear cells
(PBMNC) were isolated from blood using Ficoll-Paque (Pharmacia) density
centrifugation. After washing, the PBMNC were plated at 5 × 105 cells per well in a 96-well "U"-bottom plate in
AIM V medium supplemented with titrating amounts of either Id protein,
an isotype-matched control Ig, or KLH. The PBMNC were cultured for 3 days and then split between two 96-well plates. Fresh medium containing
IL-2 was added to the wells such that the final concentration of IL-2 was 30 IU/mL. The plates were incubated for an additional 2 days. One
microcurie of 3H-thymidine was added to the wells and the
plate was incubated for an additional 12 to 18 hours. The cells were
then harvested onto glass filters, which were washed and counted in a
scintillation counter. All stimulations were done with triplicate or
quadruplicate wells for each protein concentration. T-cell
proliferation to the carrier protein KLH was used as an internal
control for the T-cellular immune response induced by the Id/KLH vaccination.
Patient description.
Twelve MM patients treated at our institution with a PBSCT were
enrolled in this study (Table 1). All of the patients were clinical
stage III before treatment.31 Only 2 of the 12 patients were female. The median age of the patients was 50 years. One quarter
of the patients had a myeloma protein isotype of IgA. Initial
chemotherapy was administered by their referring physician and
consisted of a variety of chemotherapy regimens and of varying duration. Most of the patients were treated with multiple chemotherapy regimens before transplant. All patients had chemotherapy-responsive disease before PBSCT. The pre-PBSCT conditioning regimen consisted of
high-dose melphalan with either BCNU (3 patients) or FTBI (9 patients).
After PBSCT, all but one patient (patient 6) achieved a PR or a CR
(patients 1 and 9).
Purification of Id protein.
MM idiotype protein was abundant and relatively easy to prepare from
pretreatment or early post initial treatment serum samples. More than
20 mg of Id protein was isolated from 10 mL or less of starting serum,
which was sufficient for DC vaccines, Id protein vaccines, and
immunological testing. Highly purified Id proteins were isolated using
methods optimized for isotype. The Id protein preparations were all
greater than 95% pure as determined by ELISA for isotype or light
chain restriction and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (data not shown).
Id vaccination.
The MM patients were immunized with a combination of Id-pulsed DC
vaccines and Id-KLH conjugate vaccines. Ten of the 12 patients completed their series of two Id-pulsed DC vaccinations plus 5 Id/KLH
booster injections. Patient 7 received two Id-pulsed DC and two Id/KLH
vaccines before developing acute leukemia. Patient 10 developed
progressive disease during his Id immunization and elected to withdraw
from the study after his second Id/KLH vaccine. Despite numerous cycles
of chemotherapy and high-dose chemotherapy with PBSCT, a sufficient
number of DC precursors could be routinely isolated from the
leukapheresis products (Table 2). The
immature DC precursors were enriched approximately 100-fold using a
series of density gradient centrifugations resulting in a cell
population consisting of 3% to 63% DC. The DC yield from a
leukapheresis collection ranged from 1 to 11 × 106 cells.
The purity of the DC preparations was somewhat lower than that seen
with lymphoma patients,22 which was likely due to increased
circulating immature cells which copurified with the precursor DC.
After isolation, the immature DC were incubated in medium containing
the patient's Id protein, washed, and administered IV. The patients
tolerated the DC infusions well with the vast majority of patients
experiencing no side effects or complications. Two of the patients
(patients 6 and 8) developed transient low-grade fevers and chills
while being infused with their first Id-pulsed DC vaccine and one
patient (patient 7) developed a postinfusion thrombophlebitis after his
second DC infusion.
T-cell proliferation.
Before immunization, none of the 12 MM patients had a measurable T-cell
proliferative response to either their Id protein or KLH. However, the
Id immunizations stimulated an Id-specific T-cell proliferative
response in two patients. Patient 1 developed Id-specific proliferation
that was first detectable after receiving two Id-pulsed DC vaccines and
five Id/KLH booster immunizations (Fig 1A).
The T-cell proliferation to an irrelevant isotype matched Ig that was
isolated in an identical manner was less than one half that seen with
the Id protein. The amount of T-cell proliferation measured depended on
the dose of stimulating Id. Patient 1 maintained a measurable T-cell
proliferative response for more than 3 months after completing the
immunizations. Patient 9 developed a T-cellular immune response after
two Id-pulsed DC vaccinations and a single Id/KLH boost (Fig 1B).
Similar to patient 1, the T-cell response was specific for the
patient's Id protein and dose-dependent. Similar Id-specific T-cell
proliferation could be measured after his second and third Id/KLH
immunizations.
Cytotoxic T-cell response.
Because MM cells were not available for use as target cells in a CTL
assay, we created surrogate target cells which expressed autologous MHC
class I and Id proteins. This was accomplished by transducing
autologous fibroblasts with IdAd. The Id heavy- and light-chain
variable region genes were isolated from BM samples using reverse
transcription (RT)-PCR. Two or more independent PCR reactions yielded
products with identical sequence, strongly suggesting that the
amplified DNA was derived from myeloma cells. The variable region genes
were then cloned into a bicistronic expression/adenovirus transfer
vector containing the heavy- and light-chain constant regions. This
transfer vector was used with an adenovirus plasmid to generate IdAd.
Fibroblast lines were established from skin punch biopsy samples
obtained from the patients. Sufficient numbers of fibroblasts necessary
for the CTL assay could be readily obtained after 3 to 4 weeks of
culture. The fibroblasts were infected with the adenovirus without
significant cytotoxicity. Approximately 60% to 80% of the fibroblasts
expressed the Id protein after adenovirus infection using an MOI of 100 to 1,000. Expression could be detected within 15 hours of infection
using immunohistochemistry of cytospin preparations. Infecting with a
lower MOI (10 to 100) resulted in fewer fibroblasts (20% to 35%)
expressing detectable Id; infecting with a higher MOI caused toxicity.
The FB cell lines infected with adenovirus still internalized
51Cr well with 5 × 103 cells routinely
incorporating 4,000 cpm to 8,000 cpm with a spontaneous release of less
than 20%.
Clinical observations.
The date of the last follow-up is May 31, 1998, with a minimum
follow-up of 16 months from PBSCT and 3 months from last Id-KLH injection. Of the 12 treated patients, 9 are alive 16 to 30 months after transplantation. Two patients have maintained a CR at 30 months
(patient 1) and 17 months (patient 9) after PBSCT. One patient (patient
6) had stable disease after PBSCT and is now 20 months after PBSCT.
Without any further chemotherapy treatment, this patient's M-spike has
gone from 2.3 g/dL before her Id vaccination to 1.8 g/dL after
completing her vaccinations. The myeloma progressed in 5 patients at 8 to 17 months after PBSCT. Two of these patients (patients 3 and 4) died
at 11 and 22 months after PBSCT. One patient (patient 7) died 10 months
after PBSCT of a secondary acute myeloid leukemia.
Compared to conventional chemotherapy, high-dose therapy with
autologous stem cell support offers improvements in CR rates, freedom
from disease progression, and overall survival,18,33 and is
emerging as the therapy of choice to achieve MRD. Unfortunately, the
majority of MM patients relapse after PBSCT, even those patients who
obtain CRs and good PRs. Additional chemotherapy dose intensification is unlikely to significantly improve the survival of MM patients. New
treatment approaches are needed for this disease. The rationale for
immunizing MM patients after PBSCT has been discussed5 and
is based on several premises. The Id protein is a unique immunologic marker for the malignant MM cells and an immune response directed against the Id protein may stem the regrowth of MM. However, the immune
system's ability to mount a response to the MM Id is likely greatest
when at a stage of minimal disease. In mouse MM models, the tumor
burden directly correlated to the development of Id-specific T-cell
unresponsiveness or anergy.11 Therefore, Id immunizations may be the most efficacious in the post-PBSCT period.
We thank Shoshana Levy, PhD, for scientific advice. We thank Anette
Grabski, RN, Ranjani Rajapaksa, BS, Debbie Czerwinski, BS, Adrienne van
Beckhoven, RN, Maria Fazio, RN, the nurses and physicians of the BMT
day hospital, the Stanford Medical School Blood Center, and the General
Clinical Research Center at the Stanford University Medical Center for
continuous support.
Submitted July 15, 1998; accepted November 19, 1998.
Supported in part by the National Institutes of Health (NIH) Program
Project Grant No. PO1 CA49605 "Bone Marrow Grafting for Leukemia and
Lymphoma" and NIH Grant No. CA33399. V.L.R. was supported by a grant
of Dr. Mildred Scheel Stiftung für Krebsforschung, C.Y.O. was
supported by a Howard Hughes Physician Fellowship, A.L. is a
postdoctoral fellow of the Department of Hematology, University of
Perugia, Italy, and R.L. is an American Cancer Society Clinical
Research Professor.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ronald Levy, MD, Division of Oncology,
Department of Medicine, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305.
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A Feasibility
Study
TOP
ABSTRACT
INTRODUCTION
L1,
5'-ATCACAGATC TCTCACCATG GTGTTGCAGA CCCAGGTC-3'; 
L1, 5'-ATCACAGATC TCTCACCATG GCCTGGGCTC TGCTGCTCC-3'; 
-galactosidase (AdLacZ) at equivalent MOIs. During the infection,
the fibroblasts were labeled by adding 4 µCi 51Cr to the
culture medium in each well. Fibroblast targets were carefully washed
four times and then coincubated with titered amounts of effector cells
(E) for 4 hours. Triplicate wells were used for each E:T ratio.
Spontaneous release was determined by the addition of medium and
maximal release by the addition of 0.5% Triton-X in medium. Specific
lysis was calculated as previously described.
.05 at an E:T ratio of
100:1, 33:1, and 11:1 as determined by the Mann-Whitney U test). The
requirement for Id stimulation and specific lysis of Id targets
strongly suggest that the cytolytic activity is I-specific and not due
to nonspecific cytolytic activity, such as that seen with natural
killer cells. The fibroblasts targets expressed class I but lacked
class II expression as determined by fluorescence-activated cell
sorting (data not shown) providing indirect evidence
for CD8+ class I- restricted CTLs as the effector cell
population. This Id-specific CTL activity was only measurable 1 month
after patient 1 received his second Id-pulsed DC vaccine. Because of
the complexity of the assay, only three patients were evaluated for
Id-specific CTL activity. The other two patients studied (patients 3 and 9) had no measurable Id-specific CTL activity.
Long-term results of a clinical trial.
Blood
89:3129, 1997