Post on 03-Apr-2023
Synthetic Tumor-Specific BreakpointPeptide Vaccine in Patients WithChronic Myeloid Leukemia andMinimal Residual Disease
A Phase 2 Trial
Nitin Jain, MD1; James M. Reuben, PhD2; Hagop Kantarjian, MD1; Changping Li, MD2; Hui Gao, PhD2;
Bang-Ning Lee, PhD2; Evan N. Cohen, BS2; Theresa Ebarb, RN1; David A. Scheinberg, MD, PhD3;
and Jorge Cortes, MD1
BACKGROUND: Imatinib is the current standard frontline therapy for chronic myelogenous leukemia (CML).
In the majority of patients, imatinib induces a complete cytogenetic response (CCyR); however, complete
molecular responses are infrequent. The Bcr-Abl fusion creates a unique sequence of amino acids that
could constitute a target for immunomodulation. METHODS: A mixture of heteroclitic and native peptides
derived from both b3a2 and b2a2 sequences was used to vaccinate patients with CML in CCyR who were
receiving imatinib therapy and who had stable Bcr-Abl transcript levels. RESULTS: Ten patients were en-
rolled, all with b2a2 transcripts (including 2 patients who had coexpression of b2a2 and b3a2). Patients
had received imatinib for a median of 62 months. Three of 10 patients achieved 1-log reduction in Bcr-Abl
transcript levels, including the 2 patients who had received previous interferon therapy, and 3 other
patients achieved a major molecular response. The vaccine was tolerated well, and there were no grade �3
adverse events. Vaccination did not affect the leukocyte profiles in peripheral blood except for regulatory T
cells, which were down-regulated briefly during the late stage of vaccination in patients who achieved
approximately 1-log reduction in Bcr-Abl transcript levels. CONCLUSIONS: The current data suggested that
vaccination-related transient disruption of immune tolerance may contribute to the reduction in Bcr-Abl
transcripts. Clinically, this Bcr-Abl peptide vaccine may transiently improve the molecular response in a
subset of patients with CML. Cancer 2009;115:3924–34. VC 2009 American Cancer Society.
KEY WORDS: peptide, vaccine, tyrosine kinase inhibitor, imatinib, chronic myeloid leukemia.
Chronic myeloid leukemia (CML) is a clonal disease characterized by a reciprocal translocation result-ing in a chimeric BCR-ABL gene.1,2 This fusion gene most commonly transcribes into an 8.5-kb messengerRNA (mRNA), which generates a chimeric protein with tyrosine kinase activity (p210 BCR-ABL).3 The
Received: December 16, 2008; Revised: January 29, 2009; Accepted: February 3, 2009
Published online June 17, 2009 in Wiley InterScience (www.interscience.wiley.com)
DOI: 10.1002/cncr.24468, www.interscience.wiley.com
Corresponding author: Jorge Cortes, MD, Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe
Boulevard, Box 428, Houston, TX 77030; Fax: (713) 794-4297; jcortes@mdanderson.org
1Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; 2Department of Hematopathology, The University
of Texas M. D. Anderson Cancer Center, Houston, Texas; 3Molecular Pharmacology and Chemistry Program and Leukemia Service, Memorial Sloan-
Kettering Cancer Center, New York, New York
3924 Cancer September 1, 2009
Original Article
breakpoint in the ABL gene usually occurs 50 of exon 2 ofABL (a2), whereas the breakpoint locations within BCR
vary. In most instances, this occurs either between exonsb2 (e13) and b3 (e14) (the b2a2 transcript) or betweenexons b3 (e14) and b4 (e15) (the b3a2 transcript).4 Theb3a2 rearrangement reportedly is more prevalent, repre-senting approximately 60% of all patients.5 Upon transla-tion of the chimeric gene, a new amino acid (lysine inb3a2 and glutamic acid in b2a2) is created at the fusionsite. This chimeric p210 protein provides a potential tar-get for immunologic approach, because the p210 proteinis expressed only on the CML cells.
Imatinib is standard therapy for patients with CML.
In the International Randomized Study of Interferon and
STI571 (IRIS) trial, 40% of patients who received front-
line imatinib achieved a major molecular response
(MMR) at 12 months, although only 4% of patients
achieved a complete molecular response (CMR) after a
median follow-up of 19 months.6 Although the molecular
response rate may continue to improve over time in some
patients, others achieve a plateau at which transcript levels
remain stable, and a third group of patients may have
increasing transcript levels, which may cause an eventual
relapse.7 The ability of imatinib to induce a complete
cytogenetic response (CCyR) in most patients, albeit with
few CMRs, provides an ideal opportunity to treat patients
with a vaccine strategy in a minimal residual disease state.
Fusion peptides from the junctional sequences prod-
uct of the BCR-ABL fusion have the ability to bind several
human leukocyte antigen (HLA) class I and II molecules
and to elicit peptide-specific T-cell responses.8-11 Such an
approach engendered interest in developing this peptide
as a possible strategy to induce tumor-specific immune
responses in patients who had received treatment with
imatinib. Native junction peptides, which are adminis-
tered in most trials to patients who have CML and mini-
mal residual disease, have induced a specific immune
response.12-15 To increase the immunogenicity of native
peptides, synthetic peptides can be generated through
selective mutations in their HLA-binding sequences (het-
eroclitic peptides).16-19
We conducted a pilot trial to evaluate the immuno-
genicity and antileukemic effects of vaccination with
CML breakpoint heteroclitic peptides as measured by a
decrease in BCR-ABL transcripts. We also investigated
the effect of vaccination on T-lymphocyte and B-lympho-
cyte subsets, natural killer (NK) cells, and dendritic cells
by flow cytometry as well as the proliferation of T
lymphocytes.
MATERIALS AND METHODS
Patients
Patients aged �18 years who had Philadelphia chromo-
some-positive, chronic phase CML were eligible if they
had received imatinib for at least 12 months and had been
on a stable imatinib dose for �6 months before starting
vaccination. There were no restrictions for HLA pheno-
type, and patients with either b3a2 or b2a2 transcripts
were eligible. All patients were required to be in CCyR
but without an MMR and had to have BCR-ABL tran-
script levels�0.5 log lower than the lowest value obtained
in the previous 6 months. To establish the baseline values
for reverse transcriptase-polymerase chain reaction (RT-
PCR) analysis for BCR-ABL, peripheral blood samples
were drawn 1 month before the first vaccination, 15 days
before the first vaccination, and on the day of the first vac-
cination. The average of these results was used as the base-
line transcript level. An MMR was defined as BCR-ABL/
ABL transcripts �0.05%. The protocol was approved by
the M. D. Anderson Cancer Center Institutional Review
Board, and all patients provided written informed
consent.
Peptides
The peptides were synthesized using standard technolo-
gies with 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase
synthesis and were purified by high-performance liquid
chromatography. All patients were screened to determine
the breakpoint junction that they expressed. The choice of
peptide vaccine administered depended on the breakpoint
expressed by each patient; patients who coexpressed both
breakpoints were vaccinated with the b3a2 cocktail (Table
1). Patients who had the b3a2 breakpoint were vaccinated
with a cocktail of 5 peptides, including 2 heteroclitic pep-
tides that differed from native peptides by 2 amino acids
and 3 peptides that had the native sequence. Two of the
native sequences were 9 amino acids long and had binding
affinities for HLA-A3 and HLA-B8, respectively, similar
to those used in previously reported trials. The third
Peptide Vaccine in CML/Jain et al
Cancer September 1, 2009 3925
peptide with the native sequence, which was 24 amino
acids long, was included because it could bind many dif-
ferent HLA-DR types and could be processed with other
peptides that may bind HLA class I or II types. Patients
who had the b2a2 breakpoint received a vaccine that con-
tained a cocktail of 2 peptides, 1 synthetic, heteroclitic
b2a2 peptide and 1 peptide that was 23 amino acids long
and was chosen using the same considerations that were
used for the long b3a2 peptide described above. The pep-
tide products were provided by Breakthrough Therapeu-
tics (Greenwich, Conn). Each vaccine was packaged as a
frozen, sterile liquid containing 100 lg of each of the 3
peptides in 0.5 mL phosphate-buffered saline and was
stored at �70�C. The material was manufactured under
good manufacturing practice (GMP) conditions at Amer-
ican Peptide, Inc. (Sunnyvale, Calif). The peptide product
was dispensed in vials and was stored frozen under GMP
conditions at BioServ Corporation (San Diego, Calif).
Treatment
The patients continued receiving imatinib at the same
dose they had been receiving for the previous 6 months.
Dose escalations were not allowed while patients were on
the study, and decreases in imatinib dose or treatment
interruptions for imatinib toxicity were allowed for imati-
nib-related adverse events. All patients received 70 lgof granulocyte-macrophage colony-stimulating factor
(GM-CSF) (sargramostim) subcutaneously at the same
anatomic site of vaccination 2 days before vaccination
(Day �2) and on the day of vaccination (Day 0) for each
administration. For each vaccination, patients received
100 lg of each of the peptides subcutaneously mixed with
the adjuvant montanide. Both GM-CSF and montanide
have been used in several clinical cancer vaccine trials to
enhance immunologic response.20-22 Thus, patients who
had the b3a2 transcript received the vaccine that con-
tained the 5 corresponding peptides (total peptide dose,
500 lg) with adjuvant, and patients who had the b2a2
transcript received the vaccine that contained the 2 corre-
sponding peptides (total peptide dose, 200 lg) with adju-vant (Table 1). Patients who had both b3a2 and b2a2
transcripts received the b3a2 cocktail.14,15 Vaccines were
given every 2 weeks �4 (Weeks 0, 2, 4, and 6), then on
Week 9, and monthly for an additional 10 months there-
after, for a total of 15 vaccinations over a 12-month
period.
Enumeration of Leukocyte Subsets
by Flow Cytometry
Immunophenotyping was performed on whole blood
samples at baseline and 3 months, 6 months, 9 months,
and 12 months after vaccination by using 4-color flow-
cytometric analysis to determine the changes in the per-
centages of T-cell subsets, including T-regulatory (TR)
cells, B cells, NK cells, natural killer T (NKT) cells, and
dendritic cells. Aliquots of fresh whole blood were reacted
with fluorochrome-conjugated mouse monoclonal anti-
bodies to detect total T cells (CD3), helper-T cells
(CD4), suppressor/cytotoxic-T cells (CD8), B cells
(CD19), and NK cells (CD56/16). For enumeration of
regulatory TR cells, frozen peripheral blood mononuclear
cells (PBMCs) were thawed and were assessed for the sur-
face expression of CD3, CD4, and CD25(high) and for
Table 1. Amino Acid Sequences
Amino Acid Sequence HLA Binding Characteristics
B3A2 transcript vaccineIVHSATGFKQSSKALQRPVASDFE Class II Native/long
KQSSKALQR A3 Native
GFKQSSKAL B8 Native
KLLQRPVAV A0201 Heteroclitic
YLKALQRPV A0201 Heteroclitic
B2A2 transcript vaccineVHSIPLTINKEEALQRPVASDFE Class II Native/long
YLINKEEAL A0201 Heteroclitic
HLA indicates human leukemic antigen.
Original Article
3926 Cancer September 1, 2009
the intracellular expression of transcription factor fork-
head box P3 (FoxP3) using a monoclonal antibody.23
An anchor population of lymphocytes was identified
by gating on cells that reacted with peridinin chlorophyll
protein-conjugated anti-CD4 with low side-scatter. By
using this reaction, we determined the percentage of
CD4-positive T cells that reacted with phycoerythrin-
conjugated anti-CD25 and fluorescein isothiocyanate-la-
beled anti-Foxp3 to define TR cells. In addition, using the
allophycocyanin channel, we assessed the expression of
programmed death-1 (PD-1), a member of the cytotoxic
T-lymphocyte (CTL) antigen 4 (CTLA4) family, and glu-
cocorticoid-induced tumor necrosis factor (TNF) recep-
tor (GITR), a member of the TNF receptor family that is
expressed preferentially at high levels on TR cells. Finally,
subsets of myeloid dendritic cells (mDC/DC1) and plas-
macytoid dendritic cells (pDC/DC2) were enumerated as
reported previously.24 Staining intensity was recorded as a
measure of fluorescence intensity using a FACSCalibur
flow cytometer, and the data were analyzed with Cell-
Quest Pro (BD Biosciences, San Jose, Calif).
The FoxP3 antibody was purchased from eBio-
science (San Diego, Calif), GITR was purchased from
R&D Systems (Minneapolis, Minn), blood dendritic cell
antigen 1 (BDCA-1) and BDCA-2 were purchased from
Miltenyi Biotec (Gladbach, Germany), and all other anti-
bodies were purchased from BD Biosciences. Whole
blood and PBMC samples from 8 control individuals
who, based on self-report, were in good health and were
not on any immunomodulatory medication were
obtained for the laboratory studies.
Proliferation of CD4-positive T cells
The proliferation of CD4-positive T cells was measured
before the first vaccination, 2 weeks after the fifth vaccina-
tion, and 2 weeks after the last vaccination. CD4-positive
T cells were isolated from PBMCs by positive selection
using immunomagnetic beads coated with anti-CD4 anti-
body and passage through a magnetic field in an Auto-
MACS cell separator (Miltenyi Biotec, Auburn, Calif).
To determine CD4-positive T-cell proliferation, 105
CD4-positive T cells per well were dispensed into quadru-
plicate wells of a 96-well microtiter plate and incubated
with a mixture of vaccine peptides at a concentration of
20 lg/mL, and the microtiter plate was incubated at 37�C
for 4 days in a humidity-controlled, 5% CO2 atmosphere.
On Day 4, 1 lCi of 3H-thymidine in 20 lL of tissue cul-
ture media was added to each well of the microtiter plate,
and the plate was incubated for an additional 24 hours. At
the end of this period, the cells were harvested onto mem-
brane filters, and the amount of 3H-thymidine that was
incorporated into DNA synthesis was measured using a
TopCount NXT scintillation and luminescence counter
(Packard Instrument Company, Inc., Brookfield, Ill).
Response Assessment
RT-PCR for BCR-ABL was performed in peripheral
blood every 3 months as described previously.25 Cytoge-
netic analysis in bone marrow metaphases was done every
6 months. A molecular response was defined as a 1-log
reduction of BCR-ABL transcripts or the achievement of
undetectable transcripts. Toxicities were graded according
to the Common Terminology Criteria, version 3.0
(National Cancer Institute).
RESULTS
Ten patients were treated, and all 10 completed the study
protocol with 15 vaccinations (Table 2). The median
patient age was 45 years (range, 28-63 years), and patients
had been on imatinib for a median of 62 months (range,
34-70 months). The daily dose of imatinib at the start of
vaccination was 800 mg in 2 patients, 600 mg in 5
patients, 400 mg in 2 patients, and 300 mg in 1 patient.
Other therapies before imatinib included interferon alpha
(IFN) (n¼ 2), cytarabine (n¼ 2), homoharringtonine (n
¼ 1), and hydroxyurea (n ¼ 8). All patients were in
CCyR at the start of the trial, and the median BCR-ABL/
ABL ratio was 0.238%. During the course of therapy,
none of the 10 patients required dose adjustments of
imatinib.
Three patients, including the 2 who had received
previous IFN therapy, achieved at least a 1-log reduction
in BCR-ABL transcript levels in at least 1 measurement
(Table 2). The 1-log reduction in BCR-ABL transcript
levels was achieved in all 3 patients after all vaccinations
had been completed 15 months after the start of vaccina-
tion; although, in all 3 patients, this response was tran-
sient. Five patients achieved an MMR (which was
sustained in 4 patients at the last follow-up). In addition,
Peptide Vaccine in CML/Jain et al
Cancer September 1, 2009 3927
1 of the 3 patients who had a 1-log reduction in BCR-
ABL transcripts transiently achieved a CMR (ie, unde-
tectable transcripts with a level of detection �4.5 logs)
15 months after the start of vaccination; however, 3
months later, he had detectable transcripts although still
with MMR. Both patients who had coexpression of
b3a2 and b2a2 achieved an MMR, but the improvement
represented a �1-log reduction in BCR-ABL transcript
levels for only 1 of them. One patient had a sustained
rise in BCR-ABL transcript levels throughout vaccina-
tion and lost CCyR by the time of the last vaccination
(10% Philadelphia chromosome-positive). This patient
was switched to dasatinib and eventually achieved
MMR.
Toxicities
Vaccination was well tolerated. The toxicities observed
included grade 1 or 2 injection site reaction (n ¼ 9); fa-
tigue (n ¼ 4); skin rash (n ¼ 3); diarrhea (n ¼ 3); bone
pain (n ¼ 2); nausea (n ¼ 2); and sinus drainage, head-
ache, leg pain, dizziness, tachycardia, myalgias, scalp itchi-
ness, and burning sensation in extremities (n ¼ 1 each).
No hematologic toxicity and no grade 3 or 4 adverse
events were noted. None of the patients required imatinib
dose reductions or treatment interruptions during the
treatment period.
Immunophenotype
We analyzed the differences in lymphocyte populations
between responders and nonresponders within the con-
straints of the small sample size. Because FoxP3 TR cells
could be responsible for maintaining peripheral immune
tolerance to BCR-ABL leukemic cells, TR cells were
enumerated before and at designated points after vaccina-
tion. Patients who achieved a 1-log reduction in BCR-
ABL transcripts (responders) had a reduction in their per-
centage of TR cells accompanied by a gradual increase in
the percentage of TR cells expressing GITR that peaked at
6 months after vaccination (Fig. 1). At 6 months, the me-
dian GITR expression by CD4-positive/CD25-positive/
Foxp3-positive TR cells was significantly higher in res-
ponders than in nonresponders (31.7% vs 4.5% of TR
cells, respectively; P ¼ .033; Mann-Whitney U test). At
baseline, CD4-positive/CD25-positive/Foxp3-positiveTable
2.PatientCharacteristics
HLAType
BCR-A
BL/A
BLRatio,%
No.
Age,y
Sex
Transcript
Type
AB
CDRB1
DQB1
Tim
eon
Imatinib,
mo
Imatinib
Dose,
mg
Previous
Therapies
Average
Baselin
e3 mo
6 mo
9 mo
12
mo
15
mo
18
mo
21
mo
24
mo
145
Woman
b2a2
1,30
7,53
6,7
9,15
2,6
64
600
Hydroxyurea
0.33
0.22
0.38
0.21
0.1
0.175
0.2
0.15
247
Man
b2a2
29,66
44,44
16,4
16,14
6,6
58
800
Interferon,hydroxyurea,
cytarabine
0.19
0.04
0.02
0.02
0.09
00.025
332
Woman
b2a2
2,24
13,38
3,7
8,12
4,3
35
800
Hydroxyurea
2.19
1.83
0.65
1.84
0.81
0.08
0.125
0.4
0.325
428
Man
Both
2,3
15,27
3,1
4,1
3,3
55
600
Hydroxyurea
0.13
0.15
NA
0.2
0.2
NA
0.025
544
Man
b2a2
2,32
18,18
7,7
11,11
3,3
60
400
None
2.74
13.4
11.6
13.4
21.05
631
Woman
Both
3,26
51,51
1,15
7,14
2,5
67
600
Interferon,hydroxyurea,
homoharringtonine,
cytarabine
0.083
0.26
0.14
0.28
0.24
0.001
0.025
752
Man
b2a2
2,3
39,62
3,7
1,8
4,5
63
600
Hydroxyurea
0.286
0.25
0.13
0.43
0.24
0.15
0.2
863
Man
b2a2
1,2
8,44
7,5
3,1
2,6
65
300
Hydroxyurea
1.665
0.73
1.74
0.78
0.725
0.925
1.9
940
Man
b2a2
1,24
40,40
15,15
15,15
6,6
36
600
None
0.12
0.16
0.11
0.02
0.05
0.125
10
51
Woman
b2a2
2,3
15,35
3,4
4,7
3,2
70
400
Hydroxyurea
0.085
0.04
0.1
0.03
0.025
BCR
indicatesbreakpointclusterregion;ABL,v-ablAbelsonmurineviraloncogene;HLA,humanleukocyte
antigen.
Original Article
3928 Cancer September 1, 2009
TR cells represented 3.7% of PBMCs in responders but
only 1.7% of PBMCs in nonresponders (P¼ .033). How-
ever, concurrent with the increased GITR expression
observed at 6 months, the responder TR percentage
dropped significantly to 1% (P ¼ .009). The only other
difference in leukocyte immunophenotype between res-
ponders and nonresponders was a decrease in CD4-posi-
tive T cells in nonresponders from baseline to 9 months
after vaccination (Fig. 2). No additional significant differ-
ences between responders and nonresponders were
observed in the percentages of any other leukocyte subsets,
including neutrophils, lymphocytes, monocytes, CD8 T
cells, central memory CD8 T cells, CD4 T cells, B cells,
NK cells, NKT cells, pDC/DC2, mDC/DC1, activated
pDCs, or activated mDCs.
Proliferation
There were increases in peptide-induced proliferation of
CD4-positive T cells at 3 months after vaccination (P ¼.05; Wilcoxon signed-rank test for paired samples), and
the trend was maintained at 12 months (Fig. 3). Never-
theless, there was no statistical difference in CD4-positive
T cell proliferation in response to CML peptides between
responders and nonresponders.
DISCUSSION
Immune-mediated events may participate in the suppres-
sion of CML. A prominent example is graft-versus-leuke-
mia, which is responsible for the eradication of CML after
allogeneic stem cell transplantation.26 Immune
FIGURE 1. Vaccination was associated with increased glucocorticoid-induced tumor necrosis factor receptor (GITR) expression
by regulatory T cells (TR) concurrent with a decrease in the percentage of TR. Peripheral blood mononuclear cells (PBMCs) were
collected from imatinib-treated patients with chronic myeloid leukemia (CML) before they were vaccinated with human leukocyte
antigen (HLA)-matched CML breakpoint heteroclitic peptides and at 3 months, 6 months, 9 months, and 12 months after vaccina-
tion. Patients who acquired a minimum 1-log reduction in BCR-ABL transcripts by Month 15 of follow-up were classified as res-
ponders (circles; n ¼ 3 patients) and nonresponders (triangles; n ¼ 7 patients). Staining and flow cytometric analysis for TR
markers was performed in batches according to the manufacturer’s protocol (eBioscience; San Diego, Calif) with surface marker
staining before permeabilization and staining with the forkhead box P3 (FoxP3) antibody (eBioscience). Responders had a signif-
icantly greater percentage of TR cells that expressed GITR at 6 months after vaccination (left plot) with a concurrent diminishing
percentage of CD4-positive/CD25(high)FoxP3-positive cells in PBMCs (right plot) (asterisk: P < .05; Mann-Whitney U test). Plots
show the mean � standard error of the mean. The table below the plots represents individual responses by responders and non-
responders at each point of assessment.
Peptide Vaccine in CML/Jain et al
Cancer September 1, 2009 3929
modulation also may be responsible in part for the thera-
peutic effect of IFN.27 Furthermore, CTLs that were spe-
cific for the PR1 leukemia-associated antigen were
identified in most patients with CML who responded to
IFN or allogeneic stem cell transplantation but not in
nonresponding patients or in patients who received chem-
otherapy.28 Thus, developing immunologic approaches
to treat CML is attractive. One approach is to use vaccines
to induce a tumor-specific immune response. Several of
these have been investigated, including PR1,29 the bcr-abl
junction peptide,13,15 and a heat-shock protein-based
vaccine.30
In this study, our objective was to improve the mo-
lecular responses of patients with CML treated with imati-
nib using a junction peptide vaccine. Only 3 of the 10
patients who were treated achieved the primary endpoint
of a 1-log reduction in BCR-ABL transcript levels, and all
3 responses were transient. These results are in contrast to
the more favorable results from previous trials using a
junction peptide vaccination (Table 3). Bocchia et al used
junction peptides with a native sequence to treat 16
patients who had CML, including 10 patients who had
received therapy with imatinib for a median of 16
months.15 In that study, 5 of 9 patients who were not in
CCyR at the start of vaccinations achieved a CCyR, and 3
of them achieved a CMR. The 1 patient who was treated
in CCyR (after 23 months of imatinib therapy) achieved a
half-log reduction in BCR-ABL transcripts. In another
study, Rojas et al14 reported on 19 patients who received a
junction peptide cocktail using native sequence peptides.
FIGURE 2. Vaccination had a minimal effect on leukocyte distribution. Leukocyte subsets were analyzed fresh by flow cytometry
before vaccination with human leukocyte antigen (HLA)-matched chronic myeloid leukemia (CML) breakpoint heteroclitic pep-
tides and at 3 months, 6 months, 9 months, and 12 months after vaccination. Patients who acquired a minimum of 1-log reduction
in BCR-ABL transcripts by Month 15 of follow-up were classified as responders (circles; n ¼ 3 patients) and nonresponders (trian-
gles; n ¼ 7 patients). A cross-sectional analysis indicated that CD3-positive/CD4-positive lymphocytes were significantly lower in
nonresponders than in responders at 9 months (P ¼ .033). Longitudinal analysis revealed a significant drop in the myeloid dendri-
tic cell (mDC) count among responders (P ¼ .009). No other significant differences or changes were observed. WBC indicates
white blood cells; CD3, T cells; CD19, B cells; NK, natural killer cells; NKT, natural killer T cells; þ, positive; CD8, suppressor/cyto-
toxic T cells; CD4, helper T cells; DC, dendritic cells; pDC, plasmacytoid dendritic cells.
Original Article
3930 Cancer September 1, 2009
None of the 5 patients who entered that study without a
major cytogenetic response (MCyR) to imatinib
responded to the peptide vaccine, whereas 13 of 14
patients who had a MCyR at the start of vaccinations
achieved a 1-log reduction in transcript levels.
There are several differences between those 2 reports
and our study, and these differences are summarized in
Table 3. One difference is that both studies used only pep-
tides with the native sequence; whereas, in the current
study, we used heteroclitic peptides aimed at increasing
the immunogenicity of the peptides. One other study
used the same heteroclitic peptides that we used in our
trial but with a different dose and schedule.19 In that
study, 2 of 3 patients who had low levels of fluorescent in
situ hybridization (FISH) positivity assessed before the
start of vaccination had negative FISH results during the
vaccinations. Molecular responses could not be assessed
by RT-PCR in that study, but all patients had detectable
transcript levels throughout the follow-up period.
Immune responses were observed in 4 of 7 patients with
the HLA-A02 phenotype and also in patients with the
HLA-A3 or HLA-B8 phenotypes but not in patients with
other phenotypes. Another important difference is that
the previous studies only used peptides that corresponded
to the B3A2 sequence, thus restricting eligibility to
patients who had this breakpoint. In the study by Maslak
et al19 and in our current study, the peptides that were
used corresponded to both sequences. In both studies, in
at least some instances, an immune and/or clinical
response could be triggered with b2a2 peptides. In this
regard, it is noteworthy that, in our study, all patients had
the b2a2 sequence (2 patients also had the b3a2
sequence). The study by Maslak et al using the same pep-
tides that we used appears to have induced more signifi-
cant immune responses. This may have been caused in
part because more patients expressed b3a2 in that study;
however, differences in the methodology used to investi-
gate immune response also may have been responsible for
these differences. Other studies used different combina-
tions of peptides, schedules of administration, and adju-
vant. Currently, the contribution of these factors to the
overall potential benefit of vaccination approaches cannot
be determined.
In evaluating the responses achieved in these trials, it
is important to consider that responses to imatinib
improve over time. Thus, the contribution of the vaccine
itself to any possible response is difficult to evaluate in
these single-arm studies. In the study by Bocchia et al,15
for example, patients were included after a median of 15.5
months on imatinib; whereas, in the study by Rojas
et al,14 evaluable information was provided for 11 of 19
patients, and they had been on imatinib for a median of
17 months. Similarly, in the study by Maslak et al using
same peptides that we used in our study, the median time
on imatinib was 27 months.19 According to the IRIS trial,
the projected rate of CCyR improved from 69% at 12
months to 87% at 60 months. Similarly, the 5-year fol-
low-up in the IRIS trial indicated that a 3-log reduction in
BCR-ABL transcript levels was observed in 53% of
patients at 1 year and in 80% of patients at 4 years (P <
.001).31 In a recent analysis from a subset of patients from
the IRIS trial, the incidence of CMR increased every year
for the first 6 years of imatinib treatment, and 7% of
patients achieved this response at 36 months compared
with 45% of patients at 81 months.32 Therefore, the pos-
sibility that the responses observed in these trials could
FIGURE 3. Proliferation of peptide-stimulated CD4-positive T
cells increased significantly 3 months after vaccination. CD4-
positive T cells were isolated from peripheral blood mononu-
clear cells by positive selection using an AutoMACS cell sepa-
rator (Miltenyi Biotec, Auburn, Calif). Then, 105 CD4-positive T
cells were incubated with a mixture of vaccine peptides in
quadruplicate wells for 4 days, and 1 lCi of 3H-thymidine was
added to each well and incubated for an additional 24 hours.
The amount of 3H-thymidine incorporation was measured on
Day 4 using a scintillation counter. The proliferation of CD4-
positive T cells, measured as the count per million (cpm),
increased significantly at 3 months after vaccination (asterisk:
P ¼ .05; Wilcoxon signed-rank test for paired samples), and
the trend was maintained at 12 months. Nevertheless, no stat-
istically significant difference was observed between res-
ponders and nonresponders at baseline, at 3 months after
vaccination, or at 12 months after vaccination.
Peptide Vaccine in CML/Jain et al
Cancer September 1, 2009 3931
Table
3.Summary
ofReportedChro
nic
Myeloid
Leukemia
VaccineTrials
Reference
Eligible
CML
Patients
HLATypes
Allowed
Peptides
SequencesUsed
Typeof
Adjuvant
Used
No.of
Patients
No.of
Patients
Receiving
Imatinib
Median
Tim
eReceiving
Imatinib,mo
CCyR
at
Baseline
Response
Pinilla-Ibarz
200012
PR
orCR
withWBC
<20,000;b3a2
transcript
All
Fivepeptides:HLA-A
3(KQSSKALQR),
HLA-A
11(ATGFKQSSK),HLA-A
3/11
(HSATGFKQSSK),HLA-B
8
(GFKQSSKAL)andclassIIpeptide
(IVHSATGFKQSSKALQRPVASDFEP)
QS-21
12
0NR
NR
OneCMR,1transient
cytogenetic
improve
ment
Cathcart200413
Measurable
disease
withPHR
orCHR
andWBC
<20,000;
b3a2transcript
All
Six
peptides:HLA-A
2(SSKALQRPV),
HLA-A
3(KQSSKALQR),HLA-A
11
(ATGFKQSSK),HLA-A
3/11
(HSATGFKQSSK),HLA-B
8
(GFKQSSKAL),andclassIIpeptide
(IVHSATGFKQSSKALQRPVASDFEP)
QS-21
14
2NR
Six
patients
(1on
imatinib)
Six
patients
withCCyRs
(3withtransientPCR
improve
ment);8
patients
withoutCCyR
(4withcytogenetic
improve
ment)
Bocchia
200515
SD
with�1
2moon
imatinib
or24mo
onIFN;b3a2
transcript
HLA restricted
Six
peptides:HLA-A
2(SSKALQRPV),
HLA-A
3(KQSSKALQR),HLA-A
11
(ATGFKQSSK),HLA-A
3/11
(HSATGFKQSSK),HLA-B
8
(GFKQSSKAL),andclassIIpeptide
IVHSATGFKQSSKALQRPVASDFEP)
QS-21
16
10
15.5
Onepatient(on
imatinib)
Nineim
atinib-treated
patients
withoutCCyR
(5achievedCCyR);1
imatinib-treatedpatient
inCCyR
(0.5
logtran-
scriptreduction)
Rojas200714
SD
withCHR
and�6
moofim
atinib;
b3a2transcript
All
Patients
withHLA-A
3andHLA-A
11,
KQSSKALQR
andKQSSKALQR-PA-
DRE;HLA-A
3-negativeandHLA-A
11-
negativepatients,GFKQSSKALand
GFKQSSKAL-PADRE;allpatients,
GFKQSSKALQRPV-PADRE
PADRE
19
19
17(Imatinib
responding
patents
only)
NR
Thirteenof14patients
in
MCyR
(1logreduction
inPCR);0/5
patients
notin
MCyR
responded
Maslak200819
MCyR
orCCyR
on
stable
imatinib
regi-
men;both
b3a2
andb2a2allo
wed
All
b3a2:Fivepeptides,including2hetero-
clitic
HLA-A
2bindingpeptides
(KLLQRPVAVandYLKALQRPV)and3
nativesequences(HLA-A
3,
KQSSKALQR;HLA-B
8,GFKQSSKAL;
andclassIIpeptide,IVHSATGFKQS-
SKALQRPVASDFE);b2a2:2peptides,
including1heteroclitic
HLA-A
2binding
peptide(YLINKEEAL)and1
classIIpeptide
(VHSIPLT
INKEEALQRPVASDFE)
Montanide,
GM-C
SF
13
13
27
10
Twoof3patients
without
CCyR
achievedCCyR;
inconsistentPCR
results
Currentstudy
CCyR
withim
atinib
�12mo;both
b3a2
andb2a2allo
wed
All
b3a2:Fivepeptides,including2hetero-
clitic
HLA-A
2–bindingpeptides
(KLLQRPVAVandYLKALQRPV)and3
nativesequences(HLA-A
3;
KQSSKALQR;HLA-B
8,GFKQSSKAL;
andclassIIpeptide,IVHSATGFKQS-
SKALQRPVASDFE);b2a2:2peptides,
including1heteroclitic
HLA-A
2–bind-
ingpeptide(YLINKEEAL)and1
classIIpeptide
(VHSIPLT
INKEEALQRPVASDFE)
Montanide,
GM-C
SF
10
10
62
10
Threeof10patients
hada
1-logreductionin
transcripts
CMLindicateschronic
myeloid
leukemia;HLA,humanleukocyte
antigen;CCyR,complete
cytogeneticresponse;PR,partialresponse;CR,complete
response;NR,notreported;CMR,complete
molecularresponse;PHR,par-
tialhematologic
response;CHR,complete
hematologic
response;WBC,whitebloodcells;PCR,polymerasechain
reactionanalysis;SD,stable
disease;IFN,interferonalpha;MCyR,majorcytogeneticresponse;GM-C
SF,
gran-
ulocyte-m
acrophagecolony-stimulatingfactor.
have occurred with continuation of imatinib alone cannot
be ruled out. In our study, the median time on imatinib
before the start of vaccination was 62 months, and the
minimum was 34 months. Responses after this time tend
to be more stable, thus making it more plausible that any
improvement observed after vaccination may have been
induced by the vaccine itself. Still, only a randomized trial
can address the extent to which vaccination may improve
the outcome of patients who have CML treated with
imatinib.
In the current study, T-cell proliferation upon acti-
vation with pool peptides produced a sustained increase
after 3 months of vaccination, suggesting a cross-reactive
T-cell response to the synthetic peptides (Fig. 3). How-
ever, reduced percentages of FoxP3-expressing TR cells
occurred only in patients who had achieved a 1-log reduc-
tion in Bcr-Abl transcript levels and may indicate disrup-
tion in immune tolerance leading to the elimination of
the leukemic cells by CML-specific cytotoxic T cells. The
higher percentage of GITR-expressing TR cells observed
only in responders further supports our suggestion that
the activation of CD4-positive/CD25-positive T cells
through GITR may help to break immunologic toler-
ance.33 However, it is noteworthy that there was a time
interval between the peak immune response (at approxi-
mately 6 months) and the time to best clinical response
(approximately 15 months). The reason for this discord-
ance is unclear.
In conclusion, heteroclitic peptides may transiently
improve the molecular response in a subset of patients who
have achieved a CCyR with imatinib. The approach pre-
sented here may extend the benefit to patients who have a
B2A2 breakpoint, a group that has a not previously been
targeted well. This approach may be combined with other
methods to impact the immune-mediated control of CML.
Conflict of Interest Disclosures
Supported by National Institutes of Health grant PO1 CA23766.
Memorial Sloan-Kettering Cancer Center owns patents forinventions of David A. Scheinberg, MD, PhD, related to severalof the peptides studied in this trial.
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Original Article
3934 Cancer September 1, 2009