Synthetic tumor‐specific breakpoint peptide vaccine in patients with chronic myeloid leukemia and...

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; [email protected]

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


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,



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


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.



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


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.



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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