Vaccines for haematological tumours
Review
Part II: Vaccines for haematological malignant
disorders
Simone Mocellin, Gianpietro Semenzato, Susanna Mandruzzato, and Carlo Riccardo Rossi
The search for new therapeutic approaches to haemato-
logical malignant disease involves exploitation of the
antitumour potential of adaptive immunity—the most
specific killing system against cancer currently known.
Here, we summarise immunological strategies behind
active specific immunotherapy, and describe the clinical
and immunological results from trials published to date.
Available data in humans support the hypothesis that
various vaccination regimens can polarise adaptive
immunity towards effective control of cancer-cell growth.
However, the exploratory nature of clinical studies done
thus far does not allow any cancer vaccine to be used as
standard treatment for haematological malignant
disorders. Because the cause of disease recurrence is the
presence of minimal residual disease after conventional
treatments, the adjuvant setting might be the most
appropriate therapeutic strategy for active specific
immunotherapy, when the immunosuppressive effects of
bulky disease are virtually absent and when the effector-
target ratio is favourable. In the near future, completion of
randomised phase III trials as well as clinical
implementation of the most recent insights into tumour
immunology that aim to overcome immune tolerance
towards malignant cells should allow investigators to
define the actual role of vaccines in the management of
haematological tumours.
Lancet Oncol 2004; 5: 727–37
The past two decades have seen substantial advances in
treatment of haematological malignant disorders (figure 1).
Present use of intensified radiotherapy and chemotherapy
protocols can lead to first remission in most patients, and
bone-marrow transplantation or stem-cell transplantation
are effective therapeutic options for those who have disease
recurrence.1,2 However, a substantial number of patients will
ultimately die of their disease.3 Therefore, new non-cross-
resistant treatment strategies that might improve outlook
for patients are awaited.
Emerging preclinical and clinical data suggest that
immune-cell mediators can recognise and kill malignant
cells in patients with haematological malignant disorders.
The lower rates of relapse in the setting of allogeneic
transplantation compared with those in autologous bone-
marrow transplantation;4 the striking clinical benefit of
donor-lymphocyte infusions;5 and the clinical effectiveness
of antibody-based therapies for treatment of non-Hodgkin
lymphomas6 as well as the finding that human T cells can
Oncology Vol 5 December 2004 http://oncology.thelancet.com
Bone-marrow Donor-lymphocyte
transplantation infusion
Chemotherapy Monoclonal
and radiotherapy Leukaemias antibodies
and
lymphomas
Kinase Active specific
inhibitors immunotherapy?
Others (eg,
interferon alfa
and tretinoin)
Figure 1. The arsenal of therapeutic weapons against leukaemias and
lymphomas is constantly growing.
destroy chemotherapy-resistant cell lines from chronic
myeloid leukaemia and multiple myeloma,7,8 have prompted
development of immunotherapeutic strategies against
haematological cancers.9 Among these approaches, active
specific immunisation or vaccination is emerging as a
valuable tool to polarise the adaptive immune system
against malignant cells.10 As is the case for infectious
diseases, anticancer vaccination is based on the assumption
that the immune system can recognise tumour-associated
antigens and destroy malignant cells that express them
(figure 2). Identification of leukaemia-associated or
lymphoma-associated antigens that are specifically targeted
by T-cell or B-cell responses has spurred development of
different strategies of active specific immunisation for
the treatment of haematological malignant disorders
(table 1).11
S Mocellin is a postdoctorate fellow, S Mandruzzato is an
immunologist, and CRR is Professor of Surgery; all at the
Department of Oncological and Surgical Sciences, University of
Padova, Italy. GS is Professor of Haematology in the Department of
Clinical and Experimental Medicine, University of Padova, Italy.
Correspondence: Dr Simone Mocellin, Dipartimento di Scienze
Oncologiche e Chirurgiche, Sezione di Clinica Chirurgica, Via
Giustiniani, 2, 35128 Padova, Italy. Tel: +39 049 8211851.
Fax: +39 049 651891. Email: mocellins@hotmail.com
727
 Review Vaccines for haematological tumours

Cytokines (eg, interleukin 2 and inter-

Naive helper
Naive cytotoxic T lymphocyte
leukin 12) can also cause paracrine
T-cell receptor
T lymphocyte costimulation. In the absence of such
secondary signals, T cells are rendered
anergic to the presented antigen.12 As
HLA class I and II,
and peptides
leukaemia or lymphoma cells generally
express high amounts of HLA class I
CD8 molecules, the decreased T-cell stimu-
CD4 latory activity of these cells is mainly
CD80 t(9;22) because of deficient costimulation. Active
CD86 t(8;14)
t(14;18) CD20
specific immunisation aims to compen-
sate for the decreased immunogenicity of
FAS Peptides
Tumour-
tumour cells. By comparison with solid
associated Haematological tumours, haematological malignant dis-
antigen tumour cell orders have favourable features that make
Proteasome them ideal targets for vaccine-based thera-
Ig
peutic interventions. Ease of tumour
Perforin accessibility, achievement of a state of
Granzyme CD28
FAS ligand CTLA4 minimal residual disease by current treat-
Th1-type cytokines ments, and the antigen-presenting-cell
(eg, interleukin 2
and interferon γ) properties of most lymphoid13 and
myeloid14–17 tumours facilitate an effective
Effector helper immunotherapeutic strategy. These fea-
T lymphocyte tures, coupled with the non-cross-reactive
Th2-type cytokines
nature of active specific immunotherapy
(eg, interleukin 4 and chemotherapy, have led to the
Effector cytotoxic and interleukin 10) integration of different immunothera-
T lymphocyte peutic strategies with current treatment
regimens in the clinical setting.

Antigen-specific vaccines
Antibodies Idiotype vaccines
B cell (IgM, IgG)
The specific antigenic determinants of Ig
variable regions, called the idiotype, are
produced by a single B-cell clone and are
Figure 2. Expression of unique tumour-specific antigens (eg, Ig idiotype, translocation-derived therefore unique tumour-associated
fusion proteins, or CD20) recognised by effector cells of adaptive immunity (ie, cytotoxic T antigens for B-cell malignant diseases. In
lymphocytes and B cells) make most haematological tumour cells ideal targets for active specific
animal studies of antitumour vaccination,
immunotherapy. In addition to conventional cross presentation of tumour-associated antigens
by professional antigen-presenting cells such as dendritic cells, several haematological these determinants serve as tumour-
malignant cells have antigen-presenting capabilities, which at the same time make them targets, associated antigens,18 and idiotypes have
and mediators, of the immune response after vaccination. been tested in non-randomised clinical
trials in patients with B-cell non-Hodgkin
Although results of active specific immunisation in lymphoma and multiple myeloma (table 2); randomised
humans for treatment of haematological cancers is phase III trials are also under way (table 3). Similar to other
substantially smaller than those recorded with solid tumours, vaccine formulations (eg, vaccines based on dendritic cells,
currently available results are encouraging and support heat-shock proteins, or on genetically engineered whole-cell
further investigation of the anticancer potential of the vaccines), they need to be custom-made for each patient.
immune system. We summarise current vaccination
strategies, and discuss clinical and immunological findings Multiple myeloma
from clinical trials. Tumour-specific idiotype protein secreted by multiple
myeloma cells can be followed easily in the blood of patients,
Vaccination strategies concentrations of which correlate with disease status. Unlike
Two signals are thought to be needed for effective T-cell B-cell non-Hodgkin lymphoma, the yield of idiotype protein
stimulation.10 The first signal is generated when the correct from patient’s serum is usually enough to prepare the
T-cell receptor recognises a peptide derived from tumour- vaccine.
associated antigen that is complexed with a HLA class I Although immune responses can be generated against
molecule. The second signal is delivered by costimulatory idiotype protein in multiple myeloma, the primary target of
molecules such as CD80 or CD86, which are expressed by such responses could be the circulating idiotype protein and
professional antigen-presenting cells such as dendritic cells. not the tumour cell. Moreover, animal studies have showed

728 Oncology Vol 5 December 2004 http://oncology.thelancet.com

 Vaccines for haematological tumours
Review

that free idiotype protein might inhibit idiotype-specific Table 1. Tumour-associated antigens expressed in
immunity.45 However, these concerns are more relevant to haematological malignant disease
antibodies, which can bind circulating idiotype and thus
might not reach the target tumour cells in sufficient Category Tumour-associated Tumour
concentration to be effective. Because T cells do not recognise antigen
intact protein, cell-mediated adaptive immune responses Unique BCR-ABL1* Chronic myeloid leukaemia
DEK-NUP214 Acute myeloid leukaemia
might be unaffected by circulating idiotype and could be AML1-CBFA2T1 Acute myeloid leukaemia
generated to recognise idiotype-specific peptides present on RAR␣ Promyelocytic leukaemia
the surface of plasma cells or their precursors. Ig idiotype* B-cell non-Hodgkin lymphoma
However, CD4-positive T-cell anergy has been described Complementarity B-cell non-Hodgkin lymphoma
as a consequence of high-dose administration of idiotype in determining region 3*
T-cell receptor idiotype T-cell lymphomas
animals.46 Although the effect on the cytotoxic T-cell response
P53 Miscellaneous
towards malignant cells in humans remains to be defined, RAS Miscellaneous
peripheral T-cell tolerance to high concentrations of idiotype Shared Melanoma antigen Multiple myeloma
could be a tumour-escape mechanism in patients with B-cell family
malignant disorders. Thus, idiotype vaccination should be B Melanoma antigen Multiple myeloma
family
reserved for eradication of minimal residual disease after
Cancer/testis antigen Multiple myeloma
high-dose chemotherapy. G antigen family Multiple myeloma
Bergenbrant and colleagues19 gave five patients injections Preferentially Acute myeloid leukaemia
of paraprotein taken from the patients, which had been expressed antigen
emulsified in aluminum phosphate. Despite some evidence of melanoma
for a transient increase in cellular and humoral immune Overexpressed Myeloblastin precursor Acute myeloid leukaemia
protein and chronic myeloid
responses and despite an association between B-cell response leukaemia
and decreased CD19-positive peripheral B cells, this regimen WT1* Acute myeloid leukaemia,
was insufficient to generate a sustained and clinically active chronic myeloid leukaemia,
anti-myeloma immunity. and acute lymphoblastic
leukaemia
In a further trial published by the same researchers,20 five MUC1 Multiple myeloma
patients were treated with idiotype combined with Viral LMP1, LMP2 (Epstein- Burkitt’s lymphoma
granulocyte-monocyte colony-stimulating factor (GM-CSF) Barr virus)
in the same site on subsequent days. All patients developed a Antigens from human Hodgkin’s lymphoma and
cellular immune response characterised in vitro mainly by an T-cell lymphotropic acute T-cell leukaemia
idiotype-specific increase in T cells that secreted interferon ␣ virus type I

and interleukin 2. This response was present in CD4-positive *Antigens used in the clinical setting.

and CD8-positive T-cell subsets, and could be inhibited by

the blockade of MHC class I molecules. Furthermore,
production of idiotype-specific IgM was induced in vivo.
However, delayed-type hypersensitivity to idiotype protein
was not found, and only one patient had a decrease in serum
paraprotein concentration.
Massaia and colleagues23 described a vaccine trial in which
idiotype was conjugated with the carrier protein keyhole
limpet haemocyanin (KLH) and administered with
interleukin 2 or GM-CSF.23 12 patients who had previously
undergone autologous stem-cell transplantation and who
were in complete clinical remission were subsequently
vaccinated at various times after transplantation. Nine of 11
patients tested had evidence of cellular response to KLH,
whereas only two of 11 patients had a substantial T-cell
response to idiotype protein. Interestingly, no humoral anti-
idiotype response was detected; however, development of
positive delayed-type hypersensitivity of the skin was
reported in eight of ten patients.
Rasmussen and co-workers21 reported on a study in which
six patients with IgG-producing multiple myeloma were
immunised with autologous purified M component, together
with interleukin 12 alone or combined with GM-CSF. This
active specific immunisation was the only treatment given to
these patients. The effect of idiotype vaccination on
circulating clonal tumour B cells was monitored by the
method of real-time allele-specific oligonucleotide PCR. Four
of six patients had a significant substantial decrease in
peripheral tumour burden. Furthermore, one patient had
complete molecular remission in the blood and three patients
had an idiotype-specific T-cell response. In the two patients
who did not respond to vaccination, one mounted a T-cell
response.
B-cell non-Hodgkin lymphoma
The first trial of idiotype vaccination in patients with
lymphoma was done by Kwak and colleagues.47 Patients
with follicular non-Hodgkin lymphoma were vaccinated with
idiotype–KLH conjugate while in clinical remission after
chemotherapy. To obtain sufficient amounts of idiotype
protein for vaccination, a somatic-cell hybrid was generated
by fusion of each patient’s tumour cells with a hybridoma cell
line, and the secreted idiotype was purified from bulk
supernatant. Complete tumour regression was recorded in
two patients who had measurable lymphoma lesions.
Subsequent analyses showed an increased frequency of T-cells
that had specific cytotoxic effects against primary lymphoma
cells in 11 of 16 vaccinated patients25—an increasing immune
response that correlated with disease-free survival. A final
analysis of 41 patients treated with this approach found that

Oncology Vol 5 December 2004 http://oncology.thelancet.com 729

 Review Vaccines for haematological tumours

Table 2. Vaccine trials for haematological malignant diseases

Cancer Clinical Vaccine Study design Toxic Response Immunological Ref

setting effects* findings
Multiple Stage I–III Idiotype Phase I Few OR, two of five T-cell response: 19
myeloma Immunological adjuvant: n=5 (decreased CD19- cytokine release, three
aluminium hydroxide positive B cells) of five
Route: intradermal OR better in immune B-cell response, three of
responders five
Multiple Stage II Idiotype Phase I Not OR, one of five (50% T-cell response: prolif- 20
myeloma Immunological adjuvant: n=5 reported decrease in serum eration, one of five;
aluminium hydroxide and paraprotein) cytokine release,
GM-CSF five of five
Route: intradermal B-cell response: five of five
Multiple Stage I Idiotype Phase I Few OR, five of six - T-cell response, 21
myeloma Immunological adjuvant: n=6 (molecular remission) four of six
aluminium hydroxide and
interleukin 12, with or
without GM-CSF
Route: intradermal
Multiple First remission Idiotype Phase II Few OR, none of 15- T-cell response: 22,23
myeloma Immunological n=15 (molecular remission) delayed-type hyper-
adjuvant: KLH and OS similar to sensitivity, 12 of 15
GM-CSF historical controls B-cell response,
Route: subcutaneous three of 15
B-cell Measurable Idiotype Phase II Few CR, two of 20 T-cell response: 24,25
non-Hodgkin disease (n=20) Immunological adjuvant: n=41 OS better in immune proliferation, seven
lymphoma or KLH and Thr-MDP responders of 41; frequency†,
(follicular) clinically Route: subcutaneous 11 of 16
complete B-cell response,
remission 17 of 41
B-cell Clinically Idiotype Phase II Few OR, eight of 11 T-cell response: 26
non-Hodgkin complete Immunological adjuvant: n=20 (molecular remission) cytokine release, 19
lymphoma remission KLH and GM-CSF of 20; cytotoxicity, six
(follicular) Route: subcutaneous of six
B-cell response, 15 of 20
B-cell Measurable Idiotype Phase I–II Few CR, two of four; B-cell response, eight 27
non-Hodgkin disease (n=4) Immunological adjuvant: n=9 molecular remission, of nine
lymphoma or clinical KLH three of five
(follicular) complete remission Route: subcutaneous
B-cell Residual DNA plasmid encoding Phase I–II Few CR, one of 12 T-cell response: delayed- 28
non-Hodgkin disease after idiotype dose escalation type hypersensitivity and
lymphoma conventional Immunological adjuvant: n=12 proliferation, six of 12
treatment none
Route: intramuscular with or
without intradermal injection
B-cell Stage IV Peptide: complementartity Pilot study Few OR, 0% T-cell response: prolif- 29
non-Hodgkin determining region 3 n=1 eration, cytokine release,
lymphoma Immunological adjuvant: and cytotoxicity, one
(follicular) KLH and GM-CSF of one
Route: subcutaneous
Chronic Partial or Peptide: BCR-ABL1 Phase I dose Few OR not reported T-cell response: delayed- 30
myeloid complete Immunological adjuvant: escalation type hypersensitivity, two
leukaemia remission QS-21 n=12 of 12; proliferation, three
Route: subcutaneous Restriction: HLA-A3, of 12; cytotoxicity, none of
HLA-A11, HLA-B8, 12
and HLA-DR
Chronic Chronic phase, Peptide: BCR-ABL1 Phase II Few OR, five of 14 T-cell response: CD4+ 31
myeloid measurable Immunological adjuvant: n=14 (molecular remission), T-cell proliferation and
leukaemia disease QS-21 Restriction: HLA delayed-type hyper-
Route: subcutaneous class I and class II sensitivity, 14 of 14; CD4+
T-cell cytokine release,
11 of 14; CD8+ T-cell
cytokine release four of 14
Acute Measurable Peptide: WT1 Pilot study Few CR, one of one T-cell response: 32
myeloid disease Immunological adjuvant: n=1 frequency and
leukaemia KLH and GM-CSF CK release, one of
Restriction: HLA-A2 one
Route intradermal and
subcutaneous Continued . . .

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 Vaccines for haematological tumours
Review

Table 2. Vaccine trials for haematological malignant diseases (continued)

Cancer Clinical Vaccine Study design Toxic Response Immunological Ref

setting effects* findings
B-cell chronic Progressive, Autologous tumour Phase I–II study Few OR, nine of 11 T-cell response: cytokine 33
lymphocytic intermediate, (engineered CD40 ligand) dose escalation (decreased release and proliferation,
leukaemia or high-risk Immunological adjuvant: n=11 blood-lymphocyte three of three
disease none count)
Route: intravenous
Multiple After high-dose Autologous tumour Phase I study Few OR, none of one T-cell response: cytokine 34
myeloma chemotherapy (engineered interleukin 2) n=8 (decreased serum release and cytotoxicity,
Immunological adjuvant: paraprotein) 0%
none
Route: subcutaneous
Low-grade Measurable Heat-shock protein (GP96) Phase II study Few PR, one of ten Not reported 35
non-Hodgkin disease Immunological adjuvant: n=10
lymphoma none
Route: intradermal
Multiple Advanced, Dendritic cells pulsed with Pilot study Few OR, transient minor T-cell response: 36
myeloma refractory idiotype n=1 decrease in proliferation,
disease Immunological adjuvant: serum paraprotein cytokine release and
none cytotoxicity, one of one
Route: intravenous
Multiple Advanced, Dendritic cells pulsed with Pilot study Few OR not reported T-cell response: 37
myeloma refractory idiotype n=2 proliferation and cytokine
disease Immunological adjuvant: release, two of two;
KLH and GM-CSF cytotoxicity, none of two
Route: intravenous B-cell response, one of two
Multiple IgG multiple Dendritic cells pulsed with Phase I Few OR, one of six T-cell response: prolifer- 38
myeloma myeloma idiotype n=6 (decreased serum ation, five of six; cytokine
Immunological adjuvant: paraprotein) release, two of six;
none cytotoxicity, three of six
Route: intravenous B-cell response, four of
five
Multiple Measurable Dendritic cells pulsed with Phase I–II Few OR, one of 12 T-cell response: 39
myeloma disease (n=10) idiotype (with subsequent n=12 (decreased serum proliferation, two of 12;
or clinically idiotype and KLH) paraprotein) cytotoxicity, one of three
complete Immunological adjuvant: Immune response
remission none correlated with clinical
Route: intravenous outcome
Multiple Advanced Dendritic cells pulsed with Phase I–II Few OR, one of 11 T-cell response: 40
myeloma disease idiotype (with subsequent n=11 (decreased bone- cytokine release,
idiotype and GM-CSF) marrow plasma cells) four of ten
Route: intravenous B-cell response, three of
ten
B-cell Measurable Dendritic cells pulsed with Phase II Few CR, four of 28 B-cell or T-cell response: 41,42
non-Hodgkin disease idiotype (with subsequent n=35 PR, six of 28 proliferation, 23 of 33
lymphoma (n=28) or idioytpe and KLH)
(follicular) clinically Immunological adjuvant:
complete none
remission Route: intravenous
Cutaneous Measurable Dendritic cells pulsed with Phase I–II Few CR, one of ten T-cell response: 43
T-cell non- refractory tumour n=10 PR, four of ten delayed-type
Hodgkin disease Immunological adjuvant: hypersensitivity, three of
lymphoma none eight; proliferation and
Route: intranodal cytokine release, three of
five
Chronic Interferon alfa- Autologous chronic Pilot study Skin OR, 0% T-cell response: delayed- 44
myeloid resistant myeloid leukaemic cells n=3 ulceration type hypersensitivity,
leukaemia disease and dendritic cells No HLA-matched (n=1) cytokine release, two of
Immunological adjuvant: donors available three; cytotoxicity, none
KLH of three
Route: intradermal
*Few toxic effects refers to no grade III–IV toxic effects. †Frequency refers to concentration of tumour-specific T cells in peripheral blood of patients. OR, overall response; CR,
complete response; PR, partial response; GM-CSF, granulocyte-monocyte colony-stimulating factor; KLH, keyhole limpet haemocyanin; Thr-MDP, threonyl-muramyl dipeptide.

Oncology Vol 5 December 2004 http://oncology.thelancet.com 731

 Review
20 patients developed an idiotype-specific humoral or cellular
immune response.24 Effective immunisation significantly
correlated with improved freedom from disease progression
compared with historical controls or with those who were
vaccinated but did not mount a detectable immune response.
Thus, these results suggest that patients might benefit from
such active specific immunisation if the vaccine is sufficiently
immunogenic to induce a measurable humoral or cellular
immune response.
DNA vaccines
DNA plasmids consist of a gene for a tumour-associated
antigen that is regulated by a promoter with constitutive
activity. Because of the striking advances in genetic
engineering, such vaccine formulation is especially suitable
for large-scale production. The protein antigen produced by
the target cells (usually myocytes or fibroblasts depending on
the injection route) is taken up by host antigen-presenting
cells, processed, and cross-presented to the immune system
in draining lymph nodes, although direct transfection of rare
antigen-presenting cells that reside at the injection site has
also been shown.48 A limitation of DNA vaccines common to
all antigen-specific immunotherapy is its restricted use in the
few tumours with molecularly defined tumour-associated
antigens. Although results from animal studies of
haematological malignant disease have been encouraging,49
clinical implementation of such active specific immunisation
is still in its early stages. Because idiotype protein is laborious
and time-consuming to produce, DNA vaccination is an
attractive alternative for delivery of idiotype vaccines, because
its DNA can be isolated rapidly by PCR.
Non-Hodgkin lymphoma
Only one clinical trial on a DNA vaccine for the treatment of
haematological malignant disorders has been reported to
date. Timmerman and co-workers28 did a phase I–II clinical
trial to study the safety and immunogenicity of naked DNA
idiotype vaccines in 12 patients with follicular B-cell
lymphoma.28 The DNA encoded a chimeric Ig molecule that
had variable heavy-chain and light-chain sequences derived
from each patient’s tumour, which were linked to the IgG2a
and ␬ mouse Ig heavy-chain and light-chain constant regions,
respectively. Patients received intramuscular injections of
DNA according to a dose-escalation regimen. After
vaccination, seven of 12 patients mounted either a humoral
or T-cell proliferative response to the mouse component of
the vaccine, and one patient had a measurable T-cell response
specific to autologous idiotype. Antibodies against the
idiotype were not detected in any patient. Patients then
received a second series of vaccinations to deliver the
maximum DNA dose of the escalation series both
intramuscularly and intradermally. Nine of 12 patients had a
humoral or T-cell response to mouse Ig, and six of 12 patients
showed an idiotype-specific humoral or T-cell response.
Subsequently, a third series of vaccinations mixed idiotype
DNA with DNA from GM-CSF. The number of patients who
responded to mouse Ig was about the same (eight of 12).
Seven of the nine patients who previously responded
continued to show humoral or T-cell reactivity toward mouse
732
Vaccines for haematological tumours
immunoglobulin,and there was one new responder. No other
patients showed idiotype-specific humoral or T-cell
reactivity.
Peptide vaccines
Peptide-based vaccines need knowledge, and matching, of a
patient’s HLA haplotype and expression profile of tumour-
associated antigens for appropriate vaccination of each
individual. Most known tumour-associated antigens are
presented in association with class I MHC molecules and are
recognised by the patient’s tumour-specific CD8-positive T
cells; a few tumour-associated-antigen epitopes are presented
in association with class II MHC molecules and are
recognised by CD4-positive T cells. Although many T-cell-
defined epitopes of tumour-associated antigens are now
available for potential clinical application as vaccines, most
are expressed by melanoma cells and few epitopes have been
characterised in other tumours, especially those that are
haematological.50 Although case reports of peptide-based
immunisation have been reported for other haematological
malignant disorders (table 2), the largest (although still
limited) clinical experience has been described in patients
with chronic myeloid leukaemia. One possible tumour-
associated antigen for this disease is the BCR–ABL1 chimeric
fusion protein, which is uniquely expressed by chronic
myeloid leukaemic cells. Preclinical studies51,52 found that this
protein has immunogenic peptides, and two trials have been
done in humans (table 2), with others under way (table 3).
Other tumour-associated antigens specific to leukaemia are
under assessment as a source of immunogenic peptides. For
example, myeloblastin precursor protein (previously called
leucocyte proteinase 3) is a myeloid-tissue-restricted 26 kDa
serine protease that is abundantly expressed in azurophil
granules in healthy myeloid cells, and is overexpressed by
between two fold and five fold in some leukaemia cells where
it could be important for maintenance of leukaemic
phenotype. Cytotoxic-T-lymphocyte cell lines that recognise
a HLA-A2.1-restricted nonapeptide derived from
myeloblastin precursor preferentially lyses progenitor and
malignant cells from chronic myeloid leukaemia over healthy
bone-marrow cells.53 Active specific immunisation with
myeloblastin precursor protein is under assessment in a
clinical trial (table 3).
Chronic myeloid leukaemia
Pinilla-Ibarz and colleagues30 did a dose-escalation study of a
vaccine made from multivalent peptides (ie, five peptides
restricted by HLA class I and II) that spanned the b3a2
breakpoint of the BCR–ABL1 fusion protein.30 The
researchers found peptide-specific delayed-type-
hypersensitivity reactions in vivo and T-cell proliferation in
vitro, but no peptide-specific cytotoxic-T-cell responses in
vitro.
Polyvalent vaccines
A limitation of antigen-specific vaccines is that immune
responses will be restricted to the single tumour-associated
antigen targeted by the vaccine. Consequently, although this
strategy might initially eliminate tumours that express the
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 Vaccines for haematological tumours
Review

Table 3. Vaccine trials in progress for haematological malignant diseases

Trial Cancer Clinical setting Vaccine Study design

UNMC-260-00 B-cell non-Hodgkin After autologous transplantation Idiotype Phase II
lymphoma (follicular) Immunological adjuvant: n=20
KLH and GM-CSF
NCI-00-C-0050 B-cell non-Hodgkin Stage III–IV responsive to conventional Idiotype Phase III
lymphoma (follicular) treatment Immunological adjuvant: randomised trial
KLH and GM-CSF n=563
CUMC-0101-142 B-cell non-Hodgkin Stages III and IV responsive Idiotype Phase III
lymphoma (follicular) to conventional treatment Immunological adjuvant: randomised trial
KLH and GM-CSF n=360
NCI-00-C-0133 Mantle-cell non-Hodgkin All stages of de novo disease Idiotype Phase II
lymphoma Immunological adjuvant: n=26
KLH and GM-CSF
FAV-ID-01 Low-grade non-Hodgkin All stages Idiotype Phase II
lymphoma Immunological adjuvant: n=25
KLH and GM-CSF
NCI-00-C-0201 Multiple myeloma Stage II–III after allogeneic stem-cell Idiotype Phase I–II
transplantation Immunological adjuvant: n=22
KLH and GM-CSF
DM99-412 Chronic lymphocytic Binet stage A DNA plasmid encoding Phase I–II
leukaemia idiotype
NCI-01-C-0069 B-cell non-Hodgkin Stages III and IV de novo disease or recurrent Autologous tumour Phase II
lymphoma (follicular) disease Immunological adjuvant: n=20
interleukin 2
JHOC-JO115, Multiple myeloma De novo disease Autologous tumour Phase I–II
K0007 Immunological adjuvant: n=15
GM-CSF (secreted by
bystander cell line)
JHOC-JO115, Acute myeloid leukaemia De novo disease Autologous tumour Phase I–II
K0009 Immunological adjuvant: n=20
GM-CSF (secreted by
bystander cell line)
H11541 Chronic lymphocytic Untreated disease or complete remission Autologous tumour Phase I dose
leukaemia engineered with escalation
interleukin 2 and CD40
ligand
MDA-DM-97325 Acute myeloid leukaemia, Accelerated phase Peptide PR-1 Phase I–II
chronic myeloid leukaemia HLA-A2 restriction Immunological adjuvant: n=60
Not eligible for conventional therapy incomplete Freund’s adjuvant
MSKCC-99012 Chronic myeloid leukaemia Chronic phase BCR-ABL1 peptide Phase II
t(9;22) and b3a3 breakpoint-positive disease Immunological adjuvant: n=24
QS-21
KLH, keyhole limpet haemocyanin; GM-CSF, granulocyte-monocyte colony-stimulating factor.

tumour-associated antigen, the patient will ultimately relapse outcome.

with a tumour that no longer expresses the tumour-
associated antigen against which they were vaccinated.
Furthermore, with few exceptions, it is still unclear whether
tumour-associated antigens identified so far are
immunodominant proteins to which effective immune
responses can be generated. Because polyvalent vaccines are
made of a pool of several tumour-associated antigens, they
are thought to overcome the limitations of
immunotherapeutic strategies based on single tumour-
associated antigens.
However, most tumour-associated antigens of polyvalent
vaccines are molecularly unknown and immunological
monitoring of patients given polyvalent vaccination is either
non-specific (eg, measurement of delayed-type
hypersensitivity) or is limited to the tumour-associated
antigen known to be present in the vaccine formulation,
making it difficult to correlate immune response with clinical
Whole-cell polyvalent vaccines
A vaccine formulation that contains the tumour cell has a
broad range of tumour-associated antigens that could serve as
targets for the immune system. Because of advances over the
past decade in gene-transfer techniques various tumour cells
have been genetically modified to either secrete cytokines (eg,
interleukin 2 and GM-CSF), or to express components of the
cell membrane such as adhesion molecules or costimulatory
molecules.54 This approach increases the immunogenicity of
malignant cells either by enhanced presentation of tumour
antigens, or by enhanced costimulatory signals to the T-cell
arm of the immune system.
However, development of this vaccination strategy is
hindered by the time needed for labour-intensive
preparation of the vaccine and by the variability in the
cytokine production of each patient’s vaccine formulation.

Oncology Vol 5 December 2004 http://oncology.thelancet.com 733

 Review
To overcome such drawbacks, investigators have developed
an allogeneic bystander cell line (called K562) that secretes
large and stable amounts of GM-CSF.55 This cell line can be
grown easily in suspension and has no detectable expression
of HLA class I or class II molecules, and thus minimises the
likelihood of anti-bystander allogeneic responses with
multiple vaccinations. This strategy of universal bystander
vaccine obviates the need for gene modification for each
individual tumour source and ensures uniform cytokine
production, thereby eliminating intra-patient and inter-
patient variability with each vaccination. This means of
active specific immunisation is presently being tested in two
trials (table 3).
Chronic lymphocytic leukaemia
Wierda and co-workers56 transduced the tumour cells of
11 patients with CLL by use of an adenoviral expression
vector encoding the mouse gene Tnfsf5 (tumour necrosis
factor ligand superfamily member 5, previously called CD40
ligand). This study was based on the preclinical observation
that CLL cells transduced with Tnfsf5 became highly
proficient at antigen presentation and induced autologous
T-cell responses that generated specific cytotoxic
T-lymphocytes. Each patient received a single infusion of
the genetically modified CLL cells in a phase I dose-
escalation trial. 4 weeks after treatment, an in vitro assay
showed an increase in T cells that secreted interferon ␥ in
response to CLL cells activated by TNFRSF5 (tumour
necrosis factor receptor superfamily member 5). 6 months
after therapy, T cells maintained vigorous proliferation and
higher interferon-␥ release than baseline values. When a
sizeable number (ie, more than 39%) of the infused cells
expressed the Tnfsf5 transgene, there was a decrease in
circulating tumour-cell load as well as regressions of lymph-
node enlargements. However, the design of the study meant
it was not possible to define whether the immunological
and clinical effects of treatment were attributable to
expression of Tnfsf5 or to the adenovirus vector backbone.
Multiple myeloma
A phase I assessment of vaccination with interleukin 2 and
virally transduced autologous plasma cells by Trudel and
colleagues34 found that vaccine was effective in seven of
eight patients. 2 months after high-dose therapy, six
patients received between one and five injections of the
vaccine. Moreover, injection with tumour cells induced a
local inflammatory response consisting mainly of CD8-
positive T cells and TIA1-positive T cells, a finding also
reported in patients with melanoma metastases who
responded to peptide-based active specific immunisation.57
The multiple myeloma-specific immune response was not
enhanced after vaccination, but only one patient was
assessed. Similarly, there was no clinical response in the one
patient who had measurable disease at time of vaccination.
These results support the feasibility that generation of
plasma-cell vaccines modified by an adenovirus vector is
technically feasible and can be administered safely after
transplantation. Further studies of immunological and
clinical effectiveness of plasma-cell vaccines are needed.
734
Vaccines for haematological tumours
Heat-shock proteins
These intracellular molecules act as chaperones for
peptides,58 and complexes of heat-shock proteins and
peptides can be isolated—a fairly easy way to obtain a
molecularly undefined cocktail of epitopes derived from
tumour-associated antigens. When extracted from tumour
samples, heat-shock proteins can be administered as
vaccines to humans; generation of a cellular immune
response will need cross-presentation of the peptides
associated with these proteins. Dendritic cells have a specific
receptor for heat-shock proteins called low-density
lipoprotein receptor-related protein 1, engagement of
which leads to maturation of dendritic cells.59 Thus, heat-
shock proteins function both as multiple-peptide carriers
and as natural immunological adjuvants.
For solid cancers, some trials of anticancer vaccination
based on heat-shock proteins have been published and
some randomised studies are under way;60 however, only
one trial enrolling patients with haematological malignant
disorders has been published. In this study, Younes35 treated
ten patients who had indolent non-Hodgkin lymphoma
with autologous heat-shock proteins and recorded partial
tumour regression in one patient (table 2).
Dendritic-cell-based vaccines
A pivotal factor for an effective antitumour immune
response is the optimum processing and presentation of
tumour-associated-antigen epitopes to T cells in the
appropriate HLA context. Dendritic cells are the most
potent antigen-presenting cells that initiate an effective
T-cell response. The enhanced priming of T cells by
dendritic cells, as well as new techniques for obtaining large
numbers of human dendritic cells, has made possible the
use of these cells for therapeutic vaccination.61,62 However,
vaccine preparation remains time consuming and labour
intensive, which is probably why no randomised trials have
yet been done for any type of cancer.63 Two general
strategies have been used to obtain human dendritic cells
for clinical studies: first, purification of immature
dendritic-cell precursors from peripheral blood; and
second, differentiation in vitro of dendritic cells from
peripheral-blood CD14-positive monocytes or from CD34-
positive haemopoietic stem cells.
Although blood dendritic cells might not need extensive
culture, they must be activated through molecularly defined
triggers such as toll-like receptors and TNFSF5 before
reinfusion, especially since non-activated (or improperly
activated) dendritic cells can cause tolerance instead of
immunisation.64 Culturing of dendritic cells from CD14-
positive monocytes or CD-34 positive haemopoietic stem-
cell cultures occurs in two steps. First, immature dendritic
cells are generated initially in the presence of GM-CSF and
interleukin 4 for 5–7 days. These cells have high expression
of MHC, adhesion, and costimulatory molecules, and can
process and present antigens to T cells. Second, further
culture with tumour-necrosis factor ␣, TNFSF5, or a
monocyte-conditioned medium results in stable maturation
of the dendritic cells.
Oncology Vol 5 December 2004 http://oncology.thelancet.com
 Vaccines for haematological tumours
Multiple myeloma
In a pilot study by Lim and co-workers,38 six patients with
IgG multiple myeloma were vaccinated with intravenous
infusions of dendritic cells derived from peripheral-blood
mononuclear cells pulsed with autologous idiotype.38
Although both a B-cell and a T-cell immune response were
found in most cases tumour responses were only minor,
which consisted of a modest (25%) but consistent decrease
in serum concentration of idiotype in one patient. In two
other clinical trials on dendritic-cell-based active specific
immunisation, investigators combined idiotype-pulsed
infusions of dendritic cells with idiotype-booster
immunisations. Reichardt and colleagues39 reported on
12 patients who had undergone autologous peripheral
stem-cell transplantation and who were then given a series
of monthly immunisations of two intravenous infusions of
idiotype-pulsed autologous dendritic cells followed by
boost immunisations with subcutaneous idiotype-KLH.39
This strategy was tolerated well: patients had only minor
side-effects. Furthermore, two of 12 patients developed
idiotype-specific cellular proliferative immune response,
and one of three patients developed an idiotype-specific
cytotoxic-T-cell responses despite recent high-dose therapy.
Specificity was proven in this assay by artificial expression
of idiotype genes in autologous fibroblasts by use of
adenoviral gene transfer. The researchers concluded that
dendritic-cell-based idiotype vaccination is feasible after
peripheral-blood stem-cell transplantation and can elicit
idiotype-specific T-cell responses in patients with multiple
myeloma. The lower rate of induction of immune responses
in this group of patients compared with patients with non-
secreting lymphomas who were vaccinated on a similar
schedule might be a result of the inhibitory effect of
circulating paraprotein, or caused by immunosuppressive
effects of the previous high-dose chemotherapy. Although
no firm evidence for clinical benefit was found, two patients
who developed a cellular idiotype-specific immune
response remained in complete remission.
Finally, Titzer and colleagues40 showed the feasibility of
vaccination with CD34-positive stem-cell-derived dendritic
cells incubated ex vivo with autologous idiotype. Infusions
were followed by vaccination with idiotype-GM-CSF or
idiotype-pulsed dendritic cells. Treatment evoked
increasing anti-idiotype titres in three of ten vaccinated
patients, and in four patients an increase in the frequency of
idiotype-specific T cells was measured by ELISPOT assay for
interferon ␥. One patient showed a significant decrease in
plasma-cell infiltration of the bone marrow.
Maier and co-workers43 have used dendritic-cell-based
vaccination for treatment of patients with cutaneous T-cell
lymphoma. Patients were given weekly intranodal injections
of mature monocyte-derived dendritic cells pulsed with
autologous tumour lysate admixed with KLH. Tumour-
specific immune response was recorded in three of eight
patients (delayed-type hypersensitivity) and in three of five
patients (proliferation and cytokine production). Tumour
regression was seen in five of ten patients, with one
complete response and four partial responses. As in the
study by Titzer and colleagues,40 these researchers showed
Oncology Vol 5 December 2004 http://oncology.thelancet.com
Review
that continuation of vaccination with new tumour lysate
reinduced regression of progressive disease in two patients.
Chronic myeloid leukaemia
Fusion of tumour cells with dendritic cells (ie, a
tumour–dendritic cell hybrid) has been proposed for the
preparation of vaccines for solid cancers.65 Leukaemic-cell
progenitors have the unique feature of acting as professional
antigen-presenting cells that express the entire repertoire of
tumour-associated antigens, and are thus an enticing tool
for development of autologous vaccine formulation in the
presence of the necessary immunological costimulation.66
Several groups have reported the generation of leukaemic-
derived dendritic cells obtained by the culturing of
leukaemic blasts in the presence of various combinations of
GM-CSF, interleukin 4, tumour necrosis factor ␣, and
TNFSF5.14–17
Ossenkoppele and colleagues44 reported on the clinical
implementation of such an immunisation strategy. Accrual
was prematurely interrupted because of the availability of
imatinib for chronic myeloid leukaemia resistant to
treatment with interferon alfa.67 Although no haemato-
logical or cytogenetic responses were achieved, two of three
patients developed strong delayed-type hypersensitivity
against leukaemic dendritic cells and uncultured chronic
myeloid leukaemic cells. Because many patients with
chronic myeloid leukaemia who have complete cytogenetic
response after imatinib are still BCR–ABL1 positive at PCR
analysis and because resistance to imatinib might be
acquired, the researchers conclude that the potential of
active specific immunisation should be investigated further
in the adjuvant setting.
Conclusion
Conventional approaches to most haematological
malignant disorders can achieve clinically evident tumour
responses in a substantial number of patients. However,
these treatments lack tumour specificity and have been
associated with high systemic toxic effects. Although early
clinical studies of cancer vaccines showed that specificity
could be achieved without high toxic effects, so far they
have shown a limited effect in terms of clinical benefits.
Reasons for this are multifactorial and still largely
unknown. A central issue is the difficulty to overcome the
intrinsically poor immunogenicity of tumour-associated
antigens so far identified and used in the clinical setting.
Several efforts have been made to increase the
immunogenicity of tumour-associated antigens, but clinical
implementation of recent technologies and immunological
insights (eg, vaccines based on dendritic cells and
genetically engineered vaccines) is still in its infancy.
However, after the sequencing of the human genome, the
rapid diffusion of high-throughput technologies such as
DNA array, proteomics should accelerate the discovery of
new and more immunogenic tumour-associated antigens
suitable for anticancer vaccination.68,69 The immune
responses seen in several studies together with low rates of
tumour regression suggest that current methods of
immunological follow-up might be inadequate to show an
735
 Review
Search strategy and selection criteria
Data for this review were identified by PubMed using the
searches terms “cancer vaccines”, “haematological
malignancies”, “leukaemia”, “lymphoma”, “multiple myeloma”,
“clinical trials” and the types of cancer vaccination (eg,
“idiotype vaccine”). Continuing clinical trials were also
searched at National Cancer Institute (NCI) website (http://
www.cancer.gov/clinicaltrials). Only papers published in
English were reviewed.
effective immunisation status.70 Alternatively, excessive
tumour burdens might overwhelm the immune system,
even in the presence of tumour-specific lymphocytes.
Although complete haematological or cytogenetic
remissions are often achieved in several haematological
tumours after conventional treatments, persistence of
minimal residual disease is likely to underlie the lack of
survival benefit often reported in these patients. Therefore,
the adjuvant setting seems to be the most suitable therapeutic
scenario for active specific immunisation, where the
immunosuppressive effects of bulky disease are almost
absent and the effector-target ratio is favourable. These
theoretical advantages might be counterbalanced by the
global immune suppression that accompanies the early
period after transplantation. However, studies in animals
have shown the presence of an autologous graft-versus-host
effect early after transplantation, which is normally transient
but might be extended after vaccination.71,72
Overall, the exceptional complexity of the immune
network and of the interactions between the tumour and the
immune system make the task of identifying the optimum
vaccine formulation (eg, tumour-associated antigen,
immunological adjuvant, and vector) and regimen (eg, dose,
route, and schedule) highly challenging, so too will be the
task of identifying the clinical setting needed
to achieve the greatest clinical benefit.73 Available data in
humans do not allow any definitive conclusion to be drawn
on the role of active specific immunisation as part of
standard treatment for any haematological malignant
disease; however, they do support the hypothesis that
appropriate therapeutic interventions might redirect
adaptive immunity against malignant cells. In the near
future, completion of randomised phase III trials of cancer
vaccines, as well as clinical implementation of the most
recent insights into tumour immunology that aim to break
immune tolerance towards malignant cells, should allow
investigators to define the actual role of active specific
immunisation in the management of haematological cancers.
Conflict of interest
We declare no conflicts of interest.
Acknowledgemnts
This work was supported in part by grants from the Italian Association
for Cancer Research and from the Italian Ministry of Health 2002.
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