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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 673-683
Idiotype Vaccination in Human Myeloma: Generation of Tumor-Specific
Immune Responses After High-Dose Chemotherapy
By
Massimo Massaia,
Paolo Borrione,
Silvano Battaglio,
Sara Mariani,
Eloise Beggiato,
Patrizia Napoli,
Claudia Voena,
Alberto Bianchi,
Marta Coscia,
Barbara Besostri,
Silvia Peola,
Thomas Stiefel,
Jos Even,
Domenico Novero,
Mario Boccadoro, and
Alessandro Pileri
From the Divisione di Ematologia and II Servizio di Anatomia
Patologica dell'Universita' di Torino, Azienda Ospedaliera San
Giovanni Battista di Torino, Torino, Italy; Biosyn Arzeittemel,
Fellbach, Germany; and INSERM U277/Department d'Immunologie Institut
Pasteur, Paris, France.
 |
ABSTRACT |
Igs contain unique portions, collectively termed idiotypes (Id),
that can be recognized by the immune system. Id expressed by tumor
cells in B-cell malignancies can be regarded as tumor-specific antigens
and a target for vaccine immunotherapy. We have started a vaccination
trial in multiple myeloma (MM) using Id-specific proteins conjugated to
keyhole limpet hemocyanin (KLH) as immunogens and low doses of
subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF)
or interleukin-2 (IL-2) as immunoadjuvants. Twelve patients who had
previously been treated with high-dose chemotherapy followed by
peripheral blood progenitor cell (PBPC) transplantation entered this
study from August 1995 to January 1998. All patients were in first
remission at the time of vaccination. They received subcutaneous
injections of Id vaccines and immunoadjuvants in an outpatient setting.
The generation of Id-specific T-cell proliferative responses was
documented in 2 patients, whereas a positive Id-specific delayed-type
hypersensitivity (DTH) reaction was observed in 8 of the 10 patients
studied. DTH specificity was confirmed in 1 patient by investigating
the reactivity to synthetic peptides derived from the VDJ sequence of
the tumor-specific Ig heavy chain. None of the patients generated
soluble immune responses to Id, whereas the generation of soluble and
cellular immune responses to KLH was observed in 100% and 80%,
respectively. Eleven patients completed the treatment, whereas 1 patient failed to finish owing to progression of disease. Freedom from
disease progression (FFDP), measured from the date of first Id/KLH
injection to the date of first treatment after vaccination or last
follow-up, ranged from 9 to 36 months. These data indicate that the
immune competence status of MM patients is still susceptible to
specific immunization after high-dose chemotherapy and PBPC
transplantation. It remains to be determined whether generation of
Id-specific immune responses can reduce the relapse rate of patients
with minimal residual disease.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
MULTIPLE MYELOMA (MM) is still a fatal
B-cell neoplastic disease, with a median survival of less than 4 years.1,2 High-dose chemotherapy followed by autologous
bone marrow or peripheral blood progenitor cell (PBPC) transplantation
have recently increased the complete remission rate and remission
duration.3-5 However, overall survival has only been
slightly prolonged, and no evidence for a cure has been
obtained.6 All patients ultimately relapse even under
maintenance therapy with interferon- (IFN-) alone7 or
in combination with steroids.8,9 A possible strategy to improve the clinical outcome is to prolong the duration of the remission phase by inducing an active specific immune response. MM is
characterized by the clonal expansion of lymphoid cells with rearranged
Ig genes. Igs contain unique portions, collectively termed idiotype
(Id), that can be recognized by the immune system. Id expressed by
tumor cells in MM can be regarded as a tumor-specific antigen and a
target for active specific immunotherapy. MM has other immunologic
features that can be advantageously exploited in the setting up of
active specific immunotherapy. First, there is a T-cell population open
to exploitation as a source of specific antitumor effector
cells.10-14 Second, the T-cell effector mechanisms are not
exhausted by chronic tumor cell stimulation.15,16 However, despite the evidence of activation and immune recognition, it is clear
that T cells do not perform adequately in vivo and are unable to hold
the disease in check. Normal T cells can recognize and eliminate tumor
cells, but this ability is impaired in MM patients due to several
mechanisms, including inadequate tumor antigen presentation leading to
T-cell apoptosis.17 Immunotherapeutic strategies aimed at
conferring immunogenicity on autologous Id may achieve two goals: the
first is to rescue T cells from apoptosis; the second is to induce an
active specific immune response against tumor cells. Lynch and Eisen
were the first to prove in mice that Id can be rendered
immunogenic.18,19 In the light of these and other
experimental data, Id-specific proteins have come into medical use in
patients with follicular lymphoma.20,21 These pioneering
studies have provided the rationale for exploring the use of
autologous Id as a therapeutic vaccine in MM. Clinical results in the
allogeneic transplantation setting, including donor lymphocyte
infusions, have provided proof in principle that an adequate antitumor
immune response, unlike chemotherapy, may eradicate the
disease.22-25 We have therefore started a vaccination trial using Id-specific protein coupled to keyhole limpet hemocyanin (KLH)
and low doses of subcutaneous interleukin-2 (IL-2) or
granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of
an initial series of 8 MM patients in relapse or with resistant disease
has shown that Id vaccines are safe and can be administered in an
outpatient setting. Two patients had stable disease for 20 months with
no further chemotherapy. We now report the results of a subsequent study in which Id vaccines were administered in first remission as a
maintenance treatment after high-dose chemotherapy and PBPC transplantation.
| |
MATERIALS AND METHODS |
Patients.
Twelve MM patients (Table 1) entered this
study from from August 1995 to January 1998. Approval was obtained from
the Institutional Review Board for these studies. Informed consent was
provided according to the Declaration of Helsinki. MM was diagnosed as previously reported.26 According to the Durie and Salmon
staging system,27 9 patients were classified as stage III
and 3 were stage II; 1 was substage B. Seven were IgG, 4 were IgA, and
1 was Bence Jones myeloma. All patients had received previous high-dose chemotherapy followed by PBPC transplantation according to the Italian
Myeloma Study Group regimens3,28 and were in first remission after induction chemotherapy. Peripheral blood samples were
collected from age-matched normal donors (kindly provided by the local
Blood Bank) to set the reference values for some of the immunologic
analyses.
Vaccine preparation.
Id purification from serum and urine was accomplished by means of
precipitation and chromatography techniques that exploit Id-specific
molecular weight and isoelectric point. To reduce microbiological
contamination, chromatography separations were generally performed in
disposable conical tubes rather than in columns. Different strategies
were used to purify IgG, IgA, and light chains, as previously
reported.29 Purified IgG were obtained by ion exchange
chromatography followed by affinity chromatography. IgA were separated
by ammonium sulfate precipitation followed by gel filtration.  or
 light chains were isolated from urine by means of ammonium sulfate
precipitation. The precipitate was dissolved in physiologic saline and
dialyzed versus three changes of physiologic saline overnight. Further
purification was not required, because glomerular filtration itself
separates molecules on the basis of their size. When purified Id were
reanalyzed by high-resolution agarose gel electrophoresis, no
extra bands were observed above the nephelometry threshold. The median
recovery of IgG, IgA, and free light chain from 30 samples was 50%,
15%, and 16%, respectively. KLH (Biosyn, Arzneimittel GmbH, Fellbach, Germany), purified from the hemolymph of the keyhole limpet
(Megathura crenulata), was conjugated to Ids as previously
reported.20,21,29 Briefly, equal amounts of Id and 1 mg/mL
KLH in physiologic saline were mixed with 0.1% sterile glutaraldehyde
(Sigma, Milano, Italy) for 4 hours at room temperature. Final aliquots
of Id/KLH conjugates contained 0.5 mg of Id and KLH each per milliliter
of physiologic saline. Id/KLH conjugates were tested for Mycoplasma,
fungi, bacteria, and endotoxin contamination before vialing and storing
at  20°C.
On average, endotoxin contamination was about 3,000 USP-EU/mL, which is
approximately 10-fold higher than the European threshold for
parentalia. To decrease it, a final affinity chromatography was
performed by mixing 2 mL of conjugate with 2 mL of polymyxin (Bio-Rad
Laboratories, Irvine, CA) in conical tubes. After incubation at 4°C
for 12 hours, tubes were centrifuged for 5 minutes at 2,000 rpm and the
supernatant was carefully collected. This procedure was repeated twice
and yielded a mean concentration of 37 USP-EU/mL (range, l to 90 USP-EU/mL). Id/KLH conjugates were then sterilized by passage through a
0.2-µm filter and stored at 20°C until use. Id/KLH
conjugates were successfully manufactured in all 16 cases attempted. In
2 patients, vaccine treatment was not started because early relapse
occurred. Two patients refused to be enrolled in the study, after
initial informed consent and preparation of clinical grade Id vaccines
ready-for-use. Thus, the number of vaccines actually used versus the
number of preparations manufactured was 12 to 16.
Commercially available human polyclonal Igs (IgVena; Sclavo, Pisa,
Italy) were used as a control in delayed-type hypersensitivity (DTH)
skin tests. They were dialyzed versus physiologic saline, incubated
twice with polymyxin, passed through a 0.2-µm filter, and stored at
20°C until use.
Treatment schedule.
Patients received subcutaneous injections of 0.5 mg of Id-KLH
conjugates at time 0 and at 2, 6, 10, 14, 24, and 28 weeks. IL-2
(Proleukin; EuroCetus, Milano, Italy) at 1.5 IU/m2/d (2 patients) or GM-CSF (Leucomax; Sandoz, Milano, Italy) at 150 µg/m2/d (10 patients) was administered subcutaneously
close to the vaccine site for the next 5 days. The preferential use of
GM-CSF was based on both our initial series of 8 MM patients in relapse or with resistant disease and the first 4 patients of the present series, in whom GM-CSF proved to be a better immunoadjuvant than IL-2
in terms of anti-KLH antibody responses and DTH skin tests.
Clinical evaluation.
The serum level of the tumor-related heavy chain, the serum /
light chain ratio, and Bence Jones proteinuria were determined by
nephelometry (Sanofi Diagnostics Pasteur, Paris, France). Plasma cell
infiltration was determined by microscopic evaluation of bone marrow
aspirates after May-Grünwald-Giemsa staining. In patients with
normal high-resolution agarose gel electrophoresis and normal /
ratio, the disease was detected by immunofixation or by polymerase
chain reaction (PCR) using oligonucleotide primers and probes derived
from the tumor-specific Ig heavy-chain gene sequences.6,30,31
Freedom from disease progression (FFDP) was measured from the date of
first immunization to the date of progression or last follow-up.
Survival was measured from the date of first immunization to the date
of death or last follow-up.
Amplification and sequencing of the tumor-specific variable heavy
chain.
Bone marrow mononuclear cells were separated on a Ficoll-Hypaque
density gradient. RNA was isolated using the RNAzol B method (Biotech
Laboratories, Houston, TX), and total RNA (5 µg) was reverse-transcribed into Ig cDNA with an isotype-specific primer as
previously described.6 Amplification and sequencing of the tumor-specific VDJ were performed as previously
described.31 Briefly, 1 µL of Ig cDNA was amplified using
a VH3 consensus primer derived from the IgH framework region (FR1) and
an antisense primer derived from the 3' end of the JH region
(JH3). The reaction was performed for 33 cycles (denaturation at
94°C for 30 seconds, annealing at 65°C for 30 seconds, and
extension at 72°C for 30 seconds), with a final
extension at 72°C for 7 minutes. PCR products were run on a 2%
preparative agarose gel. The expected size was excised and
phenol-extracted. Direct sequencing of the amplified products was
performed using the Promega fmol system (Promega, Madison, WI)
according to the manufacturer's instructions. Reactions were performed
in a thermocycler at 68°C annealing temperature for 15 cycles.
Because the direct sequencing did not allow a complete reading of the
complementarity determining regions (CDRs), DNA was reamplified with
primers containing EcoRI and HindIII restriction sites
and cloned in a Bluescript SK vector (Stratagene, San Diego, CA).
Restriction enzyme analysis was performed on plasmid DNAs prepared by
the alkaline lysis method, and miniprep plasmid DNAs were then
sequenced. Sequence analysis was performed with the PC-GENE software
(Intelligenetics, Inc, Mountain View, CA).
Monitoring of minimal residual disease.
Bone marrow and peripheral blood were evaluated for the presence of
residual myeloma cells by PCR, using oligonucleotide primers and probes
derived from the tumor-specific Ig heavy-chain gene sequences, as
described above. Postswitch B cells were detected as previously
described.30 Briefly, 2 µL of 50 µL of total cDNA was
amplified with a 5' primer derived from the CDR2 and a 3' primer from the C or C first exon sequence. A nested-PCR strategy was used to detect preswitch B cells. The first amplification was
performed with a consensus primer for the variable region (VH.L or
VH.D) and a primer from the Cµ first exon (Cµ-5). Of this
amplification, 5 µL was reamplified using the internal primers CDR2
and Cµ-7. Twenty percent of the PCR product was analyzed by agarose
gel electrophoresis, blotted overnight, and hybridized to CDR3 probes
end-labeled with [-32P] (Amersham, Milano, Italy)
adenosine triphosphate (ATP). To avoid false-negatives, all of the cDNA
samples not producing PCR products were reamplified and the cDNA
quality was tested by amplifying the sequence of p53 exon 5 or n-ras
exon 2.
Peptide synthesis.
The VDJ sequence of the tumor-specific Ig heavy chain of patient 510 was translated into the amino acid sequence with the PC-GENE software
(Intelligenetics, Inc). The deduced amino acid sequence was analyzed
with a program predicting immunogenicity based on the potential beta
turn formation within the sequence. The sequences around the glycine
residues in position 17, 38 (part of CDR3), and 47 were identified as
the most potentially immunogenic regions. A sequence around glycine in
position 38 and including the CDR3 sequence (position 32-42) was
selected as the most tumor-specific sequence, whereas a sequence
corresponding to the FR3 region (position 22-31) was selected as an
internal control. The sequences in standard single-letter code with the
N-acetylated amino terminus on the left are Ac-ESRHAVYYCA-OH and
Ac-EAVGYGARFD-OH for the CDR3- and FR3-derived peptides, respectively.
Peptides were synthesized on polyethylene pins that had been radiation
grafted with hydroxyethylmethacrylic acid (HEMA).32 These
crowns were functionalized with Fmoc-protected amino acid esters of
4-hydroxymethylphenoxyacetic (HMP) that yield a free acid c-terminus on
cleavage. Amino acid coupling was performed in distilled
N1N-dimethylformamide (DMF) at 120 mmol/L by
means of HBTU/HoBT/NMM activation (120:120:180 mmol/L). After the
completion of the synthesis cycle, peptides were N-terminal-acetylated
with acetic anhydride. They were then simultaneously side chain
deprotected and cleaved using a mixture of 95% trifluoracetyl
(TFA)/2.5% anisole/2.5% ethanedithiol
(EDT). The peptide TFA solutions then were reduced under
vacuum and the cleaved peptide was precipitated with diethyl ether/petroleum ether at 40°C to 60°C (1:2
vol/vol). The precipitate was washed twice with the ether/petrol
mixture. The peptides were then air-dried, dissolved in a solution of
10% AcOH/MeCN, and sampled for analysis by mass spectroscopy and
high-performance liquid chromatography (HPLC). The
remaining solution was frozen and lyophilized.
Peptides were purified by reverse-phase chromatography with a Vydac RP
C18 250 × 10 mm column (Metachem, Torrence, CA)
installed in a Waters system (Cornerstone Water System, Phoenix,
AZ). The chromatogram was developed at a flow rate of 4 mL/min using 0.1% TFA in water and 0.1% TFA in MeCN as the limiting
solvent. Analytical HPLC was performed using a Merck
LiChrosphere (Merck, Whitehouse Station, NY) 100 RP C18
(250 × 4 mm; flow rate, 1.5 mL/min) installed in a Waters system.
Solvents used were similar to those used to purify the peptide. Ion
spray mass spectral analysis was performed on a Perkin Elmer Sciex API
111 biomolecular mass analyzer (Perkin-Elmer, Foster City,
CA). Purity was 96.5% and 98.4% for the CDR3- and the
FR3-derived peptide, respectively.
Dry peptides were solubilized in 10 mmol/L phosphate buffer and 100 mmol/L sodium chloride at pH 6.0 to give a final concentration of 1 mg/mL. A final affinity chromatography step was performed by mixing 2 mL of peptides with 2 mL of polymyxin (Affi-Prep; Bio-Rad Laboratories)
in conical tubes. After incubation at 4°C for 12 hours, tubes were
centrifuged for 5 minutes at 2,000 rpm and the supernatant was
carefully collected. This procedure was repeated twice and yielded an
endotoxin concentration of 0.2 USP-EU/mL (CDR3-derived peptide) and 0 USP-EU/mL (FR3-derived peptide). Final aliquots of unconjugated Id as
well as CDR3- and FR3-derived peptides were tested for Mycoplasma,
fungi, and bacteria before vialing and storing at 20°C.
Humoral responses.
Microtiter plates (Costar, Milano, Italy) were coated with 10 µg/mL
KLH, incubated for 12 hours at room temperature, and washed 3 times
with 0.05% Triton X-100 (Sigma). After incubation for 1 hour at room
temperature with phosphate-buffered saline (PBS) + 2% bovine serum
albumin (BSA), they were washed, blocked with 0.2% Tween-20 in PBS for
30 minutes, and washed. Preimmune and postimmune sera were diluted in
PBS + 5% fetal calf serum (FCS), dispensed into microwells, and
incubated for 1 hour at room temperature. After washing, horseradish
peroxidase (HRP)-conjugated-goat antihuman IgG or IgM (Cappel; Organon
Teknica Corp, West Chester, PA) was added and incubated for 1 hour at
room temperature. The enzyme substrate solution was added after
extensive washing and incubated for 10 minutes at 37°C. Absorption
was evaluated at an optical density of 405 nm with an enzyme-linked
immunosorbent assay (ELISA) microtiter plate reader (Titertek Multiskan
Plus; Flow Laboratories, Milano, Italy).
Anti-Id antibody responses were evaluated as follows. Microtiter plates
were coated with monoclonal antibodies (MoAbs) specific for the human
or chain present in the patient's Id. Plates were incubated
for 2 hours at room temperature and then overnight at 4°C. After
blocking with 0.2% Tween-20 in PBS for 30 minutes, 10 µg/mL purified
Id in PBS +2% FCS was added and incubated for 1 hour at room
temperature. Preimmune and postimmune sera were diluted in PBS + 2%
FCS, dispensed into microwells, and incubated for 1 hour at room
temperature. After washing, HRP-conjugated goat antihuman or chain (opposite to the light chain of the coating Id) was added and
incubated for 1 hour at room temperature. The enzyme substrate solution
was added after extensive washing and incubated for 10 minutes at
37°C. Absorption was evaluated at an optical density of 405 nm with
an ELISA microtiter plate reader.
Cellular proliferation assays.
Cellular proliferations to autologous Id, control Id (unrelated
isotype-matched Id), and KLH were evaluated at the end of the
vaccination procedure. Briefly, 5 × 106 PBMC/mL were
incubated in RPMI + 20% FCS at 37°C in a humidified atmosphere of
5% CO2 in air. After 2 hours of incubation, nonadherent cells were recovered and T cells were isolated by rosetting with sheep
red blood cells at 29°C for 1 hour to exclude the
majority of CD3 , CD2+ rosette-forming
cells.15 Adherent cells (AC) were gently recovered using a
cell scraper. AC were incubated overnight at 1 × 106/mL in RPMI + 10% pooled human AB serum supplemented
with 50 ng/mL GM-CSF alone (control culture) or with GM-CSF and 100 µg/mL autologous Id, 100 µg/mL control Id, or 100 µg/mL KLH
(experimental cultures). Tumor necrosis factor- (TNF-; Genzyme,
Cambridge, UK) at 10 ng/mL was added for the last 3 hours of
incubation. AC were then washed, irradiated, and cultured in
flat-bottomed microtiter plates with autologous purified T cells (1:5
ratio) for 5 days in RPMI + 10% FCS and 5 U/mL IL-2. Cell
proliferation was evaluated by pulsing 200 µL of cells with 5 µCi
3[H]TdR (47 MBq/mmol; Amersham, Milano, Italy) and
harvesting 4 hours later with a semiautomated sample harvester. The
filters were counted in a liquid scintillation counter. Controls were 14 normal blood donors (kindly provided by the local Blood Bank) and
were matched for sex and age. AC derived from normal donors were
incubated with GM-CSF alone or with GM-CSF and control Id or KLH.
Experiments were set up in such a way that T cells from at least one
normal donor were always studied simultaneously with T cells from the
patients. Results are expressed as the following stimulation index:
(3[H]TdR incorporation in the experimental
culture)/(3[H]TdR incorporation in the control culture) = Stimulation index.
The stimulation index to KLH was scored positive in MM when it was
above the mean plus 3 standard deviations (AVG + 3SD) observed in the
controls. The stimulation index to autologous Id was scored positive if
it was at least threefold higher than that observed to the control Id
in the same experiment.
DTH skin tests.
DTH skin tests were performed in 5 patients before vaccination and in
10 patients 1 month after the last injection of Id/KLH conjugates.
Unconjugated autologous Id was prepared as described above. At the KLH
conjugation step, one aliquot was left unconjugated and used for DTH
skin testing, whereas the remainder was used to prepare the vaccine.
Unconjugated autologous Id alone (0.5 mg) was injected intradermally
into the forearm. In parallel, an equivalent amount of human polyclonal
Ig (IgVena; Sclavo) was injected into the opposite forearm as a
control. DTH skin tests with the CDR3- and FR3-derived peptides were
performed by injecting intradermally 0.1 mg of each peptide
side-by-side on the same forearm. Final aliquots of unconjugated Id as
well as CDR3- and FR3-derived peptides were treated with polymyxin and
tested for Mycoplasma, fungi, bacteria, and endotoxin content before
vialing and storing at 20°C.
Skin biopsies were taken with a punch from selected patients at sites
of Id and/or control injections. Formalin-fixed, paraffin-embedded skin
biopsy sections were stained with the following antibodies: polyclonal
anti-CD3 (Dako, Milano, Italy; 1:100 final dilution), C8/144B MoAb
(CD8, IgG1-; Dako), OPD4 MoAb (CD4/CD45R0,
IgG1-; Dako; 1:50 final dilution), L-26 MoAb (CD20,
IgG2a-; Dako), and Leu7 MoAb (CD57, HNK1, IgM-;
Becton Dickinson, Milano, Italy; 1:20 final dilution). Staining was
performed after antigen retrieval with a microwave. Reactions were
shown by the avidin-biotin-peroxidase complex technique.
Analysis of TCRBV repertoire by molecular evaluation of CDR3 size
distribution.
RNA was extracted from 5 to 10 × 106 unfractionated
peripheral blood or bone marrow mononuclear cells using the TRIzol
Reagent (Life Technologies, S. Giuliano Milanese, Italy). RNA from skin biopsies was obtained by homogenization in the presence of guanidine isothiocyanate solution, followed by ultracentrifugation on a cesium
chloride discontinuous density gradient.33 Reverse
transcription of RNA into cDNA was performed at 42°C by AMV reverse
transcriptase (Promega kit). The CDR3 size distribution of the BV chain
was determined with a two-step PCR reaction.34 Briefly, the
first step consists of 24 reactions, each containing one specific human BV subfamily primer coupled with a consensus antisense BC primer. Sequences of BV and BC primers were derived from Genevée et
al,35 whereas numbering of BV subfamilies was obtained from
Wei et al.36 The PCR was performed in 96 polycarbonate
microwell plates (MJ Research, Watertown, MA) using a PTC-100 thermal
cycler (MJ Research). PCR conditions were as follows: denaturation at
94°C for 2 minutes; 40 cycles of amplification each consisting of
20 seconds of denaturation at 94°C, annealing at 60°C for 20 seconds, and extension at 72°C for 20 seconds; and a final
extension at 72 °C for 5 minutes. The second step, named run-off,
was performed to obtain a readable fluorescent product. Briefly, 2 µL
of the first PCR was further amplified for 5 cycles in the presence of
a nested consensus fluorescent BC primer or 13 BJ-specific fluorescent
primers. Sequences of BJ primers were derived from Pusieux et
al.37 The run-off reaction products were visualized on a
4.25% polyacrylamide sequencing gel in a 377 ABI DNA Sequencer
(Perkin-Elmer), and the size of the fragments obtained was compared
with a set of fluorescent size markers ranging from 35 to 350 nucleotides in length (Genescan 350 Tamra; Perkin-Elmer). Automatic
size analyses and CDR3 size distributions were determined with the
Immunoscope software package.38
| |
RESULTS |
Toxicity.
All courses were delivered on an outpatient basis without any acute
World Health Organization (WHO) grade III/IV toxicity. Local reactions
included erythema, induration, and local discomfort without any skin
breakdown at sites of injection. Mild axillary lymph node enlargement
occurred in 2 patients. Systemic toxicity was mostly associated with
the injection of cytokines and consisted in bone pain, myalgia, and
arthralgia in patients receiving GM-CSF (WHO grade I/II) and fever in
patients receiving IL-2 (WHO grade I/II). These symptoms were easily
controlled with oral acetaminophen.
Patients used a self-report diary to record side-effects and provide an
overall evaluation about toxicity, feasibility, and tolerability. Most
patients stated that their quality of life remained good or very good
during the vaccine treatment.
Clinical observations.
Eleven patients completed the treatment, whereas 1 patient failed to
finish owing to progression of disease. The clinical impact was
determined by evaluating the tumor mass, FFDP, and survival
(Table 2). The serum tumor-related heavy
chain level, the serum / ratio and Bence Jones proteinuria
(unique patient no. [UPN] 743), and bone marrow plasma
cell infiltration were used to determine the tumor mass before
vaccination and 1 month after the last immunization. Immunofixation was
used to detect the disease in 9 patients with normal serum M protein
level and normal serum / ratio (UPNs 14, 240, 453, 485, 510, 522, 535, 743, and 749). A PCR analysis was also used in 2 patients to
detect C- and Cµ-associated tumor-specific VDJ sequences in the
peripheral blood and bone marrow (UPNs 14 and 510; data not shown). Use
of the C region defines the cell differentiation stage and allows discrimination between clonally related preswitch B cells (which can be
regarded as potential myeloma cell precursors) and postswitch tumor B
cells.30 The vaccine treatment did not reduce the tumor mass, irrespective of its magnitude.
Five of the 11 patients who completed the treatment have maintained the
remission at 9 to 30 months after the first immunization, whereas 6 patients relapsed at 9 to 36 months (Table 2). Ten patients are alive.
At a median follow-up of 26 months, the median survival was not reached
and ranged from 11 to 39 months (Table 2).
Humoral responses.
Antibody responses to KLH were evaluated by comparing prevaccine with
postvaccine serum samples collected 1 month after the last injection of
Id/KLH conjugates (Table 3). Anti-KLH
responses were observed in all patients. On average, there was an
eightfold increase in anti-KLH antibody titer (8.12 ± 4.22; range,
3.2 to 15.7). The kinetics of anti-KLH antibody response was analyzed in 2 patients. IgM antibodies were detected after the first
immunization and boosted with the second and third, after which they
started to decline. The appearance of IgG antibodies was slightly
delayed; they were boosted by subsequent immunizations and remained
high up to the end of the vaccination procedure (data not shown).
Antibody responses to Id were evaluated by comparing the reactivity of
prevaccine and postvaccine serum samples with autologous and unrelated
isotype-matched Id. No increase was observed in the titers of specific
anti-Id antibodies (Table 3).
Cellular proliferation assays.
T-cell proliferative responses to KLH, control Id (unrelated
isotype-matched Id), and autologous Id were evaluated 1 month after the
last injection of Id/KLH conjugates (Table 3). Proliferations to KLH
and control Id were also determined in 14 age-matched normal donors and
were used as reference values. On average, the stimulation indexes to
KLH and control Id in the normal donors were 1.00 ± 0.15 and 0.75 ± 0.4, respectively. Nine of 11 patients tested showed a
positive response to KLH, whereas only 2 of 11 showed a positive
response to autologous Id (Table 3).
DTH skin tests.
DTH skin tests were performed in 10 patients 1 month after the last
injection of Id/KLH conjugates. A local reaction characterized by
erythema and induration was observed in 8 patients after 24 hours at
sites of Id injection only (Table 3). Two representative MM with a
positive and a negative reaction are shown in
Fig 1. Skin biopsies were performed in 6 patients: in 3, biopsies were taken from the Id sites only, whereas in
the other 3 they were taken from both Id and control sites. A
perivascular lymphocyte infiltration was observed at sites of Id
injection only (Fig 1C and D). Immunohistochemical characterization of
the lymphocytic infiltrates showed that most lymphocytes were
CD3+ in the Id challenge site with a slight prevalence of
CD8+ versus CD4+ cells
(Fig 2). The proportions of
CD56+ and CD20+ cells were negligible (data not
shown). The prevaccine DTH skin tests were negative (data not shown).
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| Fig 1.
DTH skin tests in MM receiving Id vaccines. Two
representative patients with a positive (A) and a negative (B) reaction
are shown. Unconjugated autologous Id alone (solid arrow) or an
equivalent amount of polyclonal human Ig as a control (open arrow) were
injected intradermally into the opposite forearms. Histologic
assessments (original magnification × 40) of skin biopsies taken at
sites of autologous Id challenge are shown in (C) and (D).
|
|
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| Fig 2.
Immunohistochemical characterization (original
magnification × 20) of lymphocytes infiltrating the skin at sites of
autologous Id challenge. A representative MM with a positive DTH
reaction is shown. Formalin-fixed, paraffin-embedded skin biopsy
sections were stained with the following antibodies: (A) polyclonal
anti-CD3; (B) OPD4 MoAb (CD4/CD45R0); and (C) C8/144B MoAb (CD8).
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In 3 patients, the DTH skin test remained positive up to 1 year after
the last immunization. In 1 of these patients (UPN 510), the VDJ
sequence of the tumor-specific Ig heavy chain was available. Thus,
synthetic peptides were derived from the CDR3 and FR3 sequences and
used for DTH skin testing. A positive reaction was observed at site of
challenge with the CDR3-derived peptide only
(Fig 3). The DTH skin test to KLH was
performed at the end of the vaccine treatment in 2 patients only. Both
showed a very strong local reaction and the test was not repeated (data
not shown).
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| Fig 3.
DTH skin tests with synthetic peptides derived from the
VDJ sequence of the tumor-specific Ig heavy chain: CDR3-derived peptide
(solid arrow) or an equivalent amount of FR3-derived peptide (open
arrow) were injected intradermally side-by-side into the forearm.
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TCRBV repertoire analysis.
The TCRBV repertoire expressed by T lymphocytes at sites of DTH skin
tests was analyzed in 2 patients, and a very high frequency of abnormal
CDR3 size distribution patterns was observed
(Fig 4). We distinguished 4 CDR3 size
distribution patterns: (1) normal, characterized by a Gaussian
distribution of BV-BC CDR3 fragments; (2) deteriorated, characterized
by one or more peaks above the normal Gaussian background; (3)
disrupted, in which the Gaussian distribution was replaced by 2 or more
predominant peaks; and (4) single-peak, characterized by a prominent
single-peak. The disrupted and single-peak patterns were indicative of
the presence of multiple clonal T-cell expansions, as shown by the
restricted usage of individual BJ gene segments in their predominant
peaks (data not shown and Fig 5). A
side-by-side comparison was performed with the TCRBV repertoires
expressed by lymphocytes derived from the peripheral blood and the bone
marrow. These preparations also showed a large frequency of disrupted
and single-peak patterns compared with age-matched normal donors
(manuscript in preparation). A restricted number of
abnormal prominent peaks of the same size within the same BV subfamily
were identified (Fig 5). Further analysis at the level of BV-BJ
transcripts showed that these peaks were almost identical in the usage
of individual BJ segments (Fig 5).
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| Fig 4.
Analysis of the TCR repertoire expressed by T cells at
sites of DTH skin tests with autologous Id. CDR3 size distribution
patterns at the level of BV-BC transcripts from a representative MM
(UPN 14) are shown. (X-axis) Sizes in amino acids of the CDR3 regions.
(Y-axis) Fluorescence intensities, reflecting the number of clones
using each CDR3 size combination. The high frequency of abnormal
patterns (ie, CDR3 profiles without a Gaussian-like distribution) is
indicative of the presence of multiple clonal T-cell populations.
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| Fig 5.
Identification of clonally related T-cell subsets in the
skin (A), peripheral blood (B), and bone marrow (C) from a
representative MM patient (UPN 14). (Upper panel) CDR3 size
distribution at the level of BV23-BC transcripts. (X-axis) Sizes in
amino acids of the CDR3 regions. (Y-axis) Fluorescence intensities,
reflecting the number of clones using each CDR3 size combination. Three
recurrent predominant peaks (7 aa, 9 aa, and 10 aa) are identified in
each sample. (Lower panel) CDR3 size distribution at the level of
BV23-BJ transcripts. The usage of individual BJ segments in the 7 aa, 9 aa, and 10 aa peaks from the skin (A1-A3), peripheral blood (B1-B3),
and bone marrow (C1-C3) samples are shown.
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DISCUSSION |
Id vaccination is a safe treatment in MM and can be delivered in an
outpatient setting. Local and systemic toxicities were mild (WHO grade
I/II) and more related to cytokines than to the Id/KLH conjugates
themselves. All patients rated the quality of life during the period of
vaccine treatment as good or very good. Failure to complete the
treatment owing to disease progression occurred in 1 patient only.
The tumor mass was not reduced by the vaccines, irrespective of its
pretreatment magnitude. A PCR analysis was used in 2 patients with
minimal residual disease (UPNs 14 and 510) to detect C- and
Cµ-associated tumor-specific VDJ sequences and evaluate the effect of
Id vaccines at the level of pre-B and B cells other than plasma cells.
These populations are clonally related to plasma cells30
and are optimal targets for vaccine treatment, because they carry
membrane-bound Id. The analysis was performed in the peripheral blood
and the bone marrow without detecting any difference before and after vaccination.
However, the lack of effects on the tumor mass was not associated with
a detrimental effect on the FFDP and survival. The date of the first
immunization was the reference time point used to calculate the FFDP.
Of the 11 patients who completed the treatment, 5 have maintained the
remission at 9 to 30 months after the first immunization, whereas 6 patients relapsed at 9 to 36 months. Current options for MM in first
remission are no treatment, IFN- alone, or IFN- and steroids.
Clinical results with IFN- alone are controversial, with some
studies showing improved FFDP7,39,40 and others no effect
whatsoever.8,41,42 The combination of IFN- and steroids
is more effective,9 but still unsatisfactory with a median
duration of 19 months. A major difference between Id vaccination and
other maintenance treatments is that the former was delivered as a
time-limited treatment (28 weeks), whereas the latter are delivered
indefinitely until relapse or toxicity. Patients maintained the
remission at 3 to 30 months after the last immunization without any
further maintenance treatment. It may be worth investigating whether
long-term delivery until relapse is more effective than a time-limited
schedule. Of the 12 patients treated, 10 are alive at 11 to 39 months
after the first immunization (4 to 32 months after the last immunization).
So far, there are only 2 studies limited to 5 patients, each about the
clinical use of Id vaccines in MM.43,44 Six patients had
early stage disease and were untreated, whereas the remainder had
received conventional chemotherapy from 2 to 5 years before vaccination. Vaccines consisted of autologous unconjugated Id precipitated in aluminium phosphate43 or administered in
the presence of GM-CSF.44 One patient experienced a 65%
decrease in the serum M protein level that lasted about 9 months, but
no other clinical data were reported.
Unlike chemotherapy, which requires tumor cytoreduction to be
effective, immunotherapy exploits more subtle mechanisms to achieve
tumor control in the absence of tumor mass reduction. In this regard,
immunologic monitoring is crucial to determine to what extent
immunotherapy has acted on the immune system and generated
tumor-specific responses, if any. We initially evaluated humoral and
cellular responses to KLH. KLH is commonly used as a protein carrier to
make Id immunogenic, but it can also be exploited as an internal
control to evaluate the efficacy of the immunization schedule. A
significant production of antibodies to KLH was observed in all
patients. Cellular responses to KLH were also generated and detected
using a cell proliferation assay or the DTH skin test. Thus, the immune
competence status of MM patients is not permanently impaired by
chemotherapy, even when multiple courses of high-dose chemotherapy
followed by autologous PBPC transplantation are delivered.
The detection of humoral and cellular responses to autologous Id was
more difficult. Id is a much weaker antigen than KLH: it belongs to
self and is under the protection of self-tolerance mechanisms. We could
not detect the presence of antibodies to Id with an ELISA assay. By
using an enzyme-linked immunospot assay (ELISPOT), Bergenbrant et
al43 have detected B cells producing anti-Id antibodies in
MM patients receiving autologous unconjugated Id. The number of these
cells increased after immunization and then diminished by the end of
the vaccine treatment. Conflicting results have also been reported in
follicular lymphomas. Antibodies to Id were documented in 17 of 41 patients receiving Id vaccines consisting of Id coupled to KLH and
emulsified in an immunologic adjuvant,21 but in none of 4 patients receiving a more effective vaccine formulation consisting of
dendritic cells pulsed with tumor-specific Id protein.45
Beside experimental variations, it is possible that we could not detect
antibodies to Id because they are bound by residual tumor cells or by
circulating Id that are not eradicated, even after multiple PBPC
transplantations.6
Cellular responses to Id were equally difficult to detect. A cell
proliferation assay detected Id-specific responses in 2 of 11 patients.
Similar data have recently been reported by Osterborg et
al,44 who detected Id-specific T-cell responses in 1 of 5 patients. Cell proliferation may not be the most appropriate readout to
investigate cell-mediated immunity. First, the generation of cytotoxic
T lymphocytes (which is the ultimate goal of vaccine treatment) may
occur in the absence of significant proliferation, but
rather in the presence of the appropriate cytokines. Second, the
frequency of Id-reactive T cells can be so low as to be undetectable in
a short-term cell proliferation assay.44 By using an
ELISPOT assay detecting cytokine production at the single-cell level, Osterborg et al44 showed that the frequency of Id-reactive
cells increased by about twofold to threefold in MM receiving
unconjugated Id vaccines and GM-CSF.
In our hands, the DTH skin test was the most reliable assay to
demonstrate the in vivo generation of Id-specific immune responses. This is based on the local recognition by CD4 and CD8 cells of antigens
against which the host has been immunized by prior exposure and is
gaining increasing attention as a convenient method to detect
antigen-specific immunity.46,47 Skin biopsies were
performed at sites of DTH skin tests, and the perivenular infiltration
of CD4+ and CD8+ lymphocytes, documented at
sites of Id challenge, was correlated with the degree of local
induration. This phenotype, in the absence of a granulocytic component,
is consistent with previous descriptions of antigen-specific DTH
reactions.48 The finding that both subsets were present
indicates that the immunization schedule was effective enough to
involve both arms of the immune system. The DTH skin tests were
negative in the study of Osterborg et al,44 in which MM
patients received autologous Id in the absence of KLH. This discrepancy
can be explained by a number of differences in the immunization
procedures, such as KLH conjugation, dose of GM-CSF, and number of immunizations.
The following findings strongly argue that DTH responses were
Id-specific and generated by the vaccine treatment. (1) The DTH skin
tests performed before vaccination were negative. The same aliquot of
unconjugated Id was used for DTH skin testing before and after
vaccination and to prepare the vaccine. Thus, the responses were not
triggered by the intradermal injection per se, and the prolonged Id
exposure did not generate any detectable reactivity a priori during the
course of the disease. (2) No reactivity was observed against an
equivalent amount of polyclonal human Ig. Our ethical committee did not
allow the use of allogeneic isotype-related Id as a control. To
minimize any bias in the preparation procedure, polyclonal Ig were
treated like unconjugated Id, ie, dialyzed versus physiologic saline,
incubated twice with polymyxin, passed through a 0.2-µm filter, and
stored at 20°C until use. Even if these preparations were
not the optimal control for IgA MM, they surely represented a very
reliable control to rule out any isotype-specific reactivity for IgG MM
patients. (3) We performed DTH skin tests using synthetic peptides of
clinical grade corresponding to the CDR3 and FR3 sequences of the
tumor-specific Ig heavy chain. The former only induced a detectable DTH
reaction. The CDR3 is the most variable and is therefore the region
most likely to be unique to the Ig produced by tumor cells. It has
recently been reported by Wen et al49,50 that synthetic
peptides derived from the CDR3 sequence of the tumor-specific Ig heavy
chain can induce T-cell proliferative and cytotoxic responses in either B-cell lymphoma or MM. These results have been obtained in vitro by
repeated rounds of appropriate stimulation using T cells isolated from
unvaccinated patients. We have detected in vivo a similar response
using the DTH skin test in a vaccinated patient 1 year after the last
immunization. So far, this is the most compelling in vivo demonstration
of the specificity of the immune response generated by Id vaccines in
MM. Thus, tumor-specific CDR3-reactive T cells have not been deleted
from the immune repertoire of MM patients and can be recruited by an
appropriate immunization procedure. Wen and Lim51 have very
recently reported a positive DTH reaction in a B-cell lymphoma patient
immunized with a naked CDR3-derived synthetic peptide in the presence
of KLH and GM-CSF.
We have performed a molecular characterization of the T cells
infiltrating the skin at sites of Id challenge by analyzing the
expression of their TCRBV repertoire. The high frequency of disrupted
and deteriorated patterns indicated the presence of multiple clonal
T-cell expansions.34,37,38 Such a multiple clonotypic
response strongly suggests that both CD4+ and
CD8+ cells are recruited by an antigen-driven process
rather than by a nonspecific inflammatory reaction.52-54
The high proportion of abnormal BV subfamilies involved in the anti-Id
response was expected, because it is well known that T-cell responses
to nominal antigens are oligoclonal and involve multiple TCRBV
subfamilies.53,54 Multiple clonal T-cell expansions have
also been detected in the peripheral blood and bone marrow of MM
patients compared with age-matched normal donors (S. Mariani,
manuscript in preparation). Most of the CDR3 distribution
patterns were different in the skin versus the bone marrow and the
peripheral blood (which, in turn, were similar), further evidence
against nonselective T-cell extravasation at sites of Id challenge. A
few abnormal BV subfamilies containing predominant peaks of the same
size were identified. Further analysis at the level of BV-BJ
transcripts showed that these peaks were almost identical in their
usage of individual BJ segments. Even though sequencing was not
performed, it is very likely that these are clonally related T cells
sharing the same antigen specificity.55 These data confirm
that the analysis of the TCRBV repertoire expressed by T cells elicited
at sites of DTH skin tests can be used as a tool with which to
"fish" for tumor-specific T-cell clones.55
In conclusion, we have been able to generate specific anti-Id immune
responses in MM patients in first remission after high-dose chemotherapy and PBPC transplantation. In our hands, DTH skin tests
were a convenient read-out to pick up the in vivo generation of anti-Id
immune responses. However, it is currently unknown whether
CD4+ and CD8+ cells specifically elicited in
the skin by Id challenge are indeed the same cells that can hold
myeloma cells in check in the bone marrow. Identification and
monitoring of clinically relevant tumor-specific immune responses
remains a major challenge in the setting of Id vaccination. Finally, it
remains to be determined whether the generation of such responses can
provide a better outcome in the setting of minimal residual disease
compared with other maintenance treatments.
| |
NOTE ADDED IN PROOF |
Subsequent to the submission of this work, Reichardt et
al56 have reported a series of 12 MM treated with Id-pulsed
autologous dendritic cells after high-dose chemotherapy and PBPC
transplantation. Eleven of 12 patients made strong anti-KLH cellular
responses and 2 of 12 developed cellular Id-specific proliferative responses.
| |
ACKNOWLEDGMENT |
The authors thank Prof John Iliffe for editorial assistance. They also
thank Drs Henri Gilbert and Gordon Tribbick (Chiron Technologies,
Clayton Victoria, Australia) for peptide synthesis.
| |
FOOTNOTES |
Submitted December 31, 1998; accepted March 10, 1999.
Supported by AIRC (Milano, Italy), MURST 60% (Roma, Italy), and
Compagnia San Paolo di Torino (Torino, Italy). Fellowship recipients
are S.P. (Comitato Gigi Ghirotti, Torino, Italy), S.M. (AIL, Torino,
Italy), and B.B. (Associazione Italiana Amici José Carreras,
Torino, Italy). The support of FIRC (Milano, Italy) to M.M. is also acknowledged.
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 Massimo Massaia, MD, Divisione
Universitaria di Ematologia, Via Genova 3, 10126 Torino, Italy; e-mail:
maxmass{at}iol.it.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION