ARTICLE
Auteur(s) : Victor
I Seledtsov, Alexey A Shishkov, Mariya A Surovtseva, Denis M
Samarin, Galina V Seledtsova, Natalya A Niza, Dmitriy V
Seledtsov
Department of Cell Biotechnologies, Institute of Clinical
Immunology, Russian Academy of Medical Sciences, 14 Yadrintsevskaya
str., 630099 Novosibirsk, Russia
accepté le 1 Juillet 2006
Malignant melanoma is a cancer with one of the most rapidly
increasing incidence rates [1]. Surgical resection of the
early-stage localized disease is the only curative treatment.
Metastatic melanoma is usually resistant to the standard cytotoxic
therapy, including highly toxic combinations. Hence, immunotherapy
has become the mainstay of treatment in advanced melanoma. An
active specific immunotherapy (vaccination) is a strategy using
tumor-associated antigens (TAAs) for inducing an antitumor immune
process. However, only few of the TAAs capable of evoking immune
responses have been identified [2]. Moreover, there is an antigenic
diversity, even in the same histological tumor of cancer patients
[3]. Since whole tumor cells elicit a broad spectrum of immune
responses to different TAAs, they should be more applicable to
constructing cancer vaccines, compared to a single,
tumor-associated, antigenic peptide. Although both autologous and
allogeneic cell vaccines are well studied, we favor a xenogenic
vaccine. Evidence has been accumulated to demonstrate that the
xenogenic TAAs can be much more effective in eliciting antitumor
immune responses than their homological analogs [4-8]. It is
reasonable to suggest that xenoantigens may potentially represent
an “altered self”, with sufficient differences from self-antigens
to render them immunogenic, but with sufficient similarities to
allow reactive T cells to maintain recognition of self. The vaccine
under study is composed of disrupted murine B16 melanoma and LLC
carcinoma cells. This xenogeneic polyantigenic vaccine (XPV) is
intended to stimulate responses against multiple targets expressed
on cancer cells.In this paper, we report for the first time, the
toxic, immunological and clinical effects of administrating XPV in
advanced melanoma patients.
Patients and methods
Patients
This study was performed in exact accordance with the protocol
approved by the Scientific Council and Ethics Committee at the
Institute of Clinical Immunology. Informed consent was obtained
from every subject who was enrolled in this study.
Eligibility criteria included histologically proven, measurable
disease, no prior immunosuppressive therapy for a minimum of four
weeks, a good performance status (Karnofsky scale, 70% or more) and
adequate marrow, renal and hepatic functions.
Vaccine preparation
B16 melanoma and LLC carcinoma cells, both of C57 BL/6 (B6;
H-2b) origin, were grown in RPMI 1640 medium
supplemented with 10% fetal calf serum, 2 mM L-glutamine, and
antibiotics (all reagents were from Sigma). After being harvested
and washed, the cells were stored at – 20 °C
until use. One vaccinal dose contained 50 × 106 B16 and
25 × 106 LLC dead cells.
Preparation of the cell lysates for immunoreactivity
assays
B16 and LLC cells were harvested, washed extensively and further
stored at – 20 °C until use. A suspension of
B6 spleen cells (SCs) was prepared with the aid of a glass
homogenizer using gentle pressing of organ fragments in the cold
(4 °C) RPMI medium. After being washed the cells
were stored at – 20 °C until use. The human
tumor cells were prepared from histologically proven metastases,
which had been surgically isolated from soft tissues of 3 melanoma
patients. The cells were washed, pooled in equivalent quantities
and further stored at – 20 °C until use.
Peripheral blood mononuclear cells (PBMCs) were isolated from the
same patients by centrifugation over Ficoll-Verografin in the
standard way. Like melanoma cells, the PBMCs were washed, pooled
and further stored at – 20 °C.
Preparation of soluble antigens (Ags) for immunoreactivity
assays
B16 and LLC cells, as well as B6 SCs, were lysed by freeze-thaw
procedures. Next, the solutions were separated from cell detritus
by centrifugation, filter sterilized using a 0.22 μm filter
unit (Sartorius), adjusted to a final protein concentration of 2.0
mg/mL, and stored at – 20 °C until use.
Treatment plan
An inducing vaccinal course consisted of 10 subcutaneous
immunizations (five at weekly and five at fortnight intervals) and
took about three months. Twenty-four hours following each of the
first five vaccinations, each patient was given subcutaneously a
low dose of a non-oxidated recombinant interleukin-2 (Ronkoleukin,
250000 U; Biotech, St. Petersburg, Russia) to potentiate
vaccine-induced immune responses. Throughout the subsequent
consolidating treatment each trial patient was vaccinated monthly.
Vaccine toxicity grading system
For systemic toxicities, the National Cancer Institute Common
Toxicity scale was used. For local vaccine toxicities, the
following scale was used: grade I, erythema and induration <
20 mm; grade II, erythema and induration 20 mm without
ulceration; grade III, ulceration or painful adenopathy; and grade
IV, permanent dysfunction related to local toxicity.
Delayed-type hypersensitivity (DTH) skin testing
Patients were given intradermal inoculations of antigenic solutions
(2.0 mg/mL) in 0.1 – mL volumes of physiological solution.
Erythemas were measured 24 hours later in two perpendicular
diameters.
Proliferation assay
PBMSs were cultured at 2 × 105 per well with antigenic
stimuli in RPMI 1640 medium supplemented with 5 mM HEPES, 2 mM
L-glutamine, 5 × 10–5 M mercaptoethanol, antibiotics
(all reagents from Sigma) and 10% autological plasma, in a 96-well
round-bottom plate (BDSL, Ayrshire, UK) for five days. The lysates
of either B16, LLC or spleen cells (each 5 × 104/ well)
were added in cultures as murine Ags, whereas the lysates of human
melanoma cells or PBMCs (each 12 × 103/well) were used
as human Ags. As a control the responding PBMSs were cultured in
the medium alone. The cell proliferation was measured by a
3H-thymidine assay in the standard way. The results are
expressed as the mean of triplicates. A stimulation index was
calculated for each triplicate by dividing the mean radioactivity
(cpm) of stimulated cells by that of unstimulated cells.
Antibody (Ab) assay
The solutions with soluble antigens (2 mg/mL) prepared from cell
lysates using the above procedure were placed in a 96-well ELISA
plate (Costar) in a volume of 50 μL per well and left overnight.
The next morning the plate was washed extensively before adding 4%
casein solution (Vector-Best; Kolzovo, Russia) into all wells to
block non-specific absorption sites. Plasma samples diluted 1:100
were incubated in the wells for 1 hour and Ig G bound to the
absorbed antigens was detected with peroxidase-conjugated rabbit
anti-human Ig G monoclonal Ab (Vector-Best), using
tetramethylbenzidine as an enzyme substrate. The reaction was
quantified at 450 nm in an ELISA reader. The assay was performed in
duplicate for each serum sample. The values were expressed in titer
units, which were calculated according a 5-point curve of measuring
Ab titer in the pooled positive sera obtained from 3 vaccinated
patients.
Serum cytokine assay
Interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were determined
in the patient’s sera, using commercially available ELISA-kits,
according to the manufacture’s instructions (Vector-Best; Kolzovo,
Russia).
Statistics
The paired Student’s test was used to determine the significance of
the observed differences. The Kaplan-Meier method was employed to
estimate overall survival.
Results
Toxicity
A total of 40 patients (14 with III and 26 with IV disease stage;
26 females and 14 males) aged from 18 to 71 years (mean age 50
years) completed an inducing course of vaccinotherapy consisting of
10 immunizations and had adequate follow-up to monitor toxicity and
immune responses. No III-IV grade systemic toxicity associated with
the vaccine administration was noted. During 24-to-48 h post
vaccination 19 patients (47%) exhibited an influenza-like syndrome
in the form of a body temperature rise up to 38 °C
and musculoskeletal discomfort, which was usually self-limiting.
The local I-II grade toxicity in the form of developing irritation
at the injection site was noted in 29 (72%) of evaluable patients.
Such local manifestations usually disappeared within
24-to-48 h post-vaccination. There were no treatment-related
hospitalizations or mortalities.
Cell blood parameters, as well as renal and hepatic functions,
remained within the initial ranges throughout the observation
period. There were no significant changes in subpopulation
composition of PMBCs tested for expression of CD3, CD4, CD8, CD20,
and CD16 surface markers. Serum Ig A, G, M also remained in the
initial ranges (data not shown).
Serum concentrations of a rheumatoid factor, but also of Abs
specific to DNA, cardiolipin, thyreoglobulin, and microsomal
fraction of thyreocytes were measured in sera before and after the
inducing the vaccinal course. No statistically significant changes
in these parameters were noted (data not shown). Consistent with
these data, the XPV-treated patients exhibited no clinical evidence
for developing any systemic autoimmune disorders.
Immunoreactivity
As a consequence of inducing vaccinations, a remarkable increase in
skin immunoreactivity (by 5 mm or more) to B16 Ags was found
in 28 (70%) of 40 evaluable patients ( (figure 1) ). The median
quantitative increase in anti-melanoma reactivity of the patients
was statistically significant (P < 0.01).
The majority (32 of 40, 80%) of assessable patients initially
had a high (10 mm or more) skin reactivity to LLC Ags. As can
be seen from ( figure
1 ), this reactivity did not undergo any significant
XPV-related changes.
Before the treatment, skin reactivity to non-tumor SC antigens
in most evaluable patients was 5 mm or less and its increase
owing to vaccinations was moderate ( (figure 1) ).
To assess the reactivity of PBMCs to vaccinal Ags, a
proliferative assay was used. As can be seen in ( figure 2 ), a statistically
significant increase in reactivity to both tumor-associated and
non-tumor xenogenic Ags occurred in the XPV-treated patients (P
< 0.02). It should be noted, however, that with the vaccinations
the B16 and LLC Ags became more effective stimulators of PBMC
proliferation compared to the SC Ags.
An important aim of our study was to determine whether or not
murine TAAs would be capable of contributing to the generation of
immune responses specific to human melanoma-associated Ags. As
shown in ( figure
3 ), inducing vaccinations gave rise to a marked
augmentation of the proliferative responses of patients’ PBMCs to
the human melanoma-associated Ags, while not affecting their
reactivity to the control alloantigens.
As shown in ( figure
4 ), the vaccinotherapy resulted in a noticeable elevation
of the serum level of Ab binding to vaccinal B16 or LLC Ags (p <
0.02). It should, however, be noted that a similar elevation was
also attributable to Abs binding SC Ag.
Two types of T cells are categorized as T helper 1 and T helper
2 on the basis of their cytokine production. T helper 1 lymphocytes
mainly produce interferon-gamma (IFN-γ) and mediate cellular immune
response, whereas T helper 2 lymphocytes mainly produce IL-4 and
mediate humoral responses. The data presented in table 1( Table 1 ) indicate that the inducing
vaccinations led to a detectable increase in serum concentrations
of both IFN-γ and IL-4 (P < 0.05).
Table 1 Serum cytokine concentrations (pg/ml, M ± m) in
the patients before and after 10 vaccinations
|
Cytokine
|
Before vaccinotherapy
|
Three months after onset of vaccinotherapy
|
|
INF-γ
|
799 ± 210
|
1371 ± 196
|
|
IL-4
|
40 ± 15
|
55 ± 15
|
Overall survival
Although the primary end points of this trial were toxicity and
immune-mediated responses to XPV, we also evaluated the overall
3-year survival in 32 XPV-treated patients with stage IV disease.
Their characteristics are presented in table 2( Table 2 ). All patients had measurable or evaluable
disease. The control group was composed retrospectively of the
patients who received conventional therapy. Each control patient
was randomly selected to be a clinically comparable counterpart of
a trial patient, so that control and trial groups were evenly
balanced by both prognostic and clinical parameters. Throughout the
follow-up period the trial patients received no other systemic
therapy other than immunotherapy. If it was reasonable and
possible, both trial and control patients underwent cytoreductive
palliative surgery.
As shown in ( figure
5 ), the median survival of the XPV-treated patients was
significantly longer (P < 0.05) than that of the control
patients (13.8 vs. 5.8 months). The longer survival of the XPV in
our study appears to have been associated with a high DTH skin
reactivity to the vaccinal B16 Ags. The median survival of the six
patients, whose reactions after inducing vaccinations were <
10 mm, was 10 months, whereas that of the 26 remaining
patients with high reactions (≥ 10 mm) was 20 months.
The overall 3-year survival rate in XPV-treated and control
patients was 25% and 2%, respectively. A clinical effect of various
grades with a duration not shorter than 6 months was observed in 21
(66%) of the 32 trial patients: complete response, partial response
and disease stabilization was achieved in 5 (16%), 2 (6%) and 14
(44%) patients, respectively. Thus, the results suggest that
xenovaccinotherapy may significantly prolong the lifetime in a
significant proportion of advanced melanoma patients.
Xenovaccinotherapy is potentially able to provide regression of
visceral metastases. The magnetic resonance imaging (MRI) scans
shown in ( figure
6 ) indicate the disappearance of liver metastatic lesions
in a 25 year-old female patient 6 months post immunotherapy
initiation. This patient exhibited a long-term complete remission
(the follow-up time was 3 years) and has had no evidence of further
disease to date. Also of interest is that the development of
connective tissue cysts in the sites of former visceral metastases
was noted in several patients (data not shown).
Table 2 Characteristics of the patients assessable for
survival
|
Characteristic
|
Trial
|
Control
|
|
Number of patients
|
32
|
32
|
|
Males/females
|
10/22
|
10/22
|
|
Age, years (median, range)
|
48.8 (18–69)
|
48.2 (24–77)
|
|
Site of metastases:
|
|
|
|
Lymph node, skin/soft tissue
|
23 (70%)
|
26 (81%)
|
|
Lung
|
10 (31%)
|
6 (19%)
|
|
Liver
|
7 (22%)
|
7 (22%)
|
|
Other organs
|
7 (21%)
|
8 (25%)
|
|
Prior treatment:
|
|
|
|
surgery
|
17 (53%)
|
16 (50%)
|
|
surgery + chemotherapy
|
9 (28%)
|
10 (31%)
|
|
surgery + immunotherapy (IFN)
|
0 (0%)
|
1 (3%)
|
|
surgery + chemotherapy + immunotherapy (IFN)
|
2 (6%)
|
2 (6%)
|
|
surgery + chemotherapy + physiotherapy
|
2 (6%)
|
0 (0%)
|
|
surgery + chemotherapy + radiotherapy
|
1 (3%)
|
0 (0%)
|
|
no treatment
|
1 (3%)
|
3 (9%)
|
Discussion
Immunizations with unmodified homologous (autological or
allogeneic) tumor cells have demonstrated only limited therapeutic
success in cancer patients. There are two major reasons for the low
immunogenicity of homologous tumor vaccines. Firstly, most of the
homological TAA represent self-Ags, which are not inherently
immunogenic. T cells that recognize self-Ags with high avidity are
believed to undergo a negative selection through clonal deletion in
the thymus or anergy in the periphery [9]. Secondly, homologous
tumor cells are not recognizable by antigen-presenting cells (APC)
as cells that should be internalized and their antigens processed
[10-12]. Presentation of tumor-associated peptides on the
professional APC in association with classes I and II MHC molecules
is a prerequisite for the activation of tumor-specific cytotoxic
and helper T cells, respectively [10-12].
Because of their structural distinctions from homologous
analogs, xenoantigens appear to be capable of effectively
overcoming immune tolerance to self-Ags, including tumor-associated
ones [4-8]. On the other hand, all humans possess natural
(preexisting) Abs, which provide an acute rejection of any
non-primate cells and function as a major barrier for the
transplantation of animal organs to humans [13]. A significant part
of these Abs represents the Ig G specific to the α-gal epitope that
is expressed abundantly on glycoproteins and glycolipids of
non-primate mammals and New Word monkeys [13, 14]. By the
opsonization of xenogenic tumor cells, the natural Abs could
promote internalization of tumor material in APC via a
Fcγ-receptor-mediated mechanism, and thereby enhance greatly the
immunogenic presentation of tumor-associated Ags to tumor-specific
T lymphocytes. This proposition is consistent with the data
indicating a critical role of the FcγR-receptors in generating an
effective antimelanoma immunity [15], as well as with the published
results showing that the rejection of alphaGal positive melanoma
cells can efficiently boost the immune response to other
tumor-associated antigens present in alphaGal negative melanoma
cells [16].
The majority of tumor-associated Ags are well known to be
common. In other words, they can express on different types of
tumors. To broaden the spectrum of immunizing targets, carcinoma
(LLC)-associated Ags were included in XPV in addition to the B16
melanoma-associated Ags. Before vaccinations, the majority of
melanoma patients demonstrated a relatively high (10 mm or
more) and early (for the first several hours) skin response to the
LLC Ags. In our view, by stimulating a local inflammation at the
vaccination site, the LLC products might act as an adjuvant in
developing the overall XPV-specific immune process.
Presumably, the positive role in generating antitumor responses
by a xenovaccine might belong not only to the xenogenic Ags
associated with tumor cell phenotype, but also to the xenoantigens
that are not related with cell malignization. Consistent with this
proposition are published data indicating that in a model of active
immunization, mouse gp75 expressed in insect cells may be
significantly more effective in protecting mice from lung
metastases, compared to gp75 expressed in homologous cells [4].
The results presented herein point out that the XPV-based
therapy is safe for clinical use and has much less toxicity than
current standard therapy for melanoma. Noteworthy is that the
vaccine-treated patients exhibited no evidence of systemic
autoimmune disorders, of which development could not be excluded
initially because of the broad range of different Ags present in
XPV.
It appears that the xenogenic Ags, both tumor-associated and
inherent to normal cells, can be involved in XPV-induced immune
responses. As evidenced by both DTH and PBMC proliferation, the
immunoreactivity to B16 Ags observed in the XPV-treated patients
was detectably greater than the immunoreactivity to SC Ags. This
may imply that the cell-mediated, immune sensitization of the
XPV-treated melanoma patients was due to melanoma-associated Ags
more than to other xenogenic Ags present in normal murine cells.
This, however, did not appear to be the case for Ab-mediated
responses. No significant differences between the anti-B16 Ab
titers and the anti-SC Ab-titers were found in the XPV-treated
patients. These findings suggest that those antigenic determinants,
which are present in not only malignant, but also non-malignant
cells, may be preferentially responsible for XPV-generated Ab
responses.
Of the utmost importance is that the XPV administration
stimulated cell-mediated reactivity not only to murine, but also to
human melanoma-associated Ags, thereby supporting the theoretical
basis of applying xenovaccinotherapy in cancer patient management.
One more matter of principle is whether or not the XPV is able to
induce immune responses directed against the patient’s own tumor.
With vaccinations, an increase in T-cell mediated responses to self
melanoma-associated Ags was observed by us in 2 out of 4 assessable
patients. This increase was detectable in both proliferative and
macrophage inhibitory factor production assays (data not shown).
Unfortunately, we were unable to evaluate immune responses to self
melanoma-associated Ags in other patients because autological tumor
material was not available.
As shown in this paper, the XPV administration resulted in serum
level elevations of not only IFN-γ, but also of IL-4, suggesting
intensification of both T helper 1- and T helper 2-mediated immune
responses in XPV-treated patients. Similar results were obtained in
colorectal cancer patients (data not shown). These findings are of
great importance, in the light of previously reported data that
indicate a critical role for cooperating cell- and Ab-mediated
mechanisms in generating anti-melanoma cytotoxicity in vivo
[15].
The results of the present study suggest that the
xenovaccinotherapy is capable of significantly prolonging survival
of advanced melanoma patients. The three-year survival rate of the
XPV-treated patients was, however, inferior to the survival rate
that had been previously reported for stage IV melanoma patients
treated with allogeneic cell vaccines. One possible reason for this
may be that a significant portion (66%) of the patients entered in
our own study had initially unresectable metastases, whereas in the
aforementioned trials all patients were vaccine-treated as early as
possible following complete metastasis ablation [17].
According to our own experience, xenovaccinotherapy can result
in eradicating visceral metastatic lesions in some melanoma
patients. Nevertheless, stabilization of the disease appears to be
the most common outcome of effective immunotherapy in advanced
cancer patients. The XPV-based therapy is not an exception in this
regard. Unlike the cytotoxic chemotherapy, tumor vaccine-based
approaches may permit the host to reach a state of balance with the
tumor, in which the net result of tumor growth and destruction is
zero. That might lead to more significant survival benefits than a
rapid destruction and rapid regrowth of the tumor following
cytotoxic therapy.
In conclusion, this study represents the first demonstration of
the safety, immunogenicity, and feasibility of administering a
xenogeneic composite cell vaccine in humans, and outlines its
relevance to overall anticancer therapy. Although the results are
encouraging, they must be interpreted with caution because they are
based on a small number of patients with very advanced disease.
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