close

Вход

Забыли?

вход по аккаунту

?

381

код для вставкиСкачать
Int. J. Cancer: 65,450-453 (1996)
0 1996 Wiley-Liss, Inc.
EX VIVO ras PEPTIDE VACCINATION IN PATIENTS WITH ADVANCED
PANCREATIC CANCER: RESULTS OF A PHASE 1/11 STUDY
Marianne K . GJERTSEN',Arne BAKKA~,
Jarle BREIVIK~,
Ingvil SAETERDAL~,
Tobias GEDDE-DAHL
IIIl, Kjell T. STOKKE~,
Bjarte G. sOLHEIM4,Tor S. EGGE5, Odd S ~ R E I D Erik
E ~ , THORSBY~
and Gustav GAUDERNACK',~
Institute of Transplantation Immunology, 2Departmentof Surgery B and SDepartmentof Radiology, The National Hospital,
University of Oslo; 3Pronova a.s.; 4Red Cross and National Hospital Blood Centre, Oslo, Noway.
In a pilot phase 1/11 study we have tested synthetic ras
peptides used as a cancer vaccine in 5 patients with advanced
pancreatic carcinoma. The treatment principle used was based
on loading professional antigen-presenting cells (APCs) from
peripheral blood with a synthetic ras peptide corresponding to
the ras mutation found in tumour tissue from the patient.
Peptide loading was performed ex vivo and the next day APCs
were re-injected into the patients after washing to remove
unbound peptide. Patients were vaccinated in the first and
second week and thereafter every 4-6 weeks. In 2 of the 5
patients treated, an immune response against the immunising
ras peptide could be induced. None of the patients showed
evidence of a T-cell response against any of the ras peptides
before vaccination. The treatment was well tolerated and could
be repeated multiple times in the same patient. Side effects
were not observed even if an immunological response against
the ras peptide was evident. We conclude that ras peptide
vaccination according to the present protocol is safe and may
result in a potentially beneficial immune response even in
patientswith advanced malignant disease.
o 1996 Wiley-Liss,Inc.
Pancreatic adenocarcinoma carries a poor prognosis. Most
patients are biologically or technically inoperable when diagnosed. For these patients there is no effective radiation or
chemotherapy option available, and median survival time is
only 3 4 months (Warshaw and Fernandez-del Castillo, 1992).
New treatment approaches of advanced pancreatic cancer are
urgently needed. Ras genes carrying mutations at specific
positions have been identified in up to 90% of pancreatic
adenocarcinomas (Alrnoguera et al., 1988). The spectrum of
mutations is limited and confined to position 12 of the m s
oncogene in pancreatic adenocarcinoma (Capella et al., 1991).
The human immune system has the potential to recognise cells
expressing such rus mutations (Fossum et al., 1995).
We have previously shown that different human lymphocyte
antigen (HLA) molecules can bind ras peptides and present
them to T cells, thereby initiating an immune response
(Fossum et al., 1993; Gedde-Dahl et al., 1994b). T cells from
both healthy individuals (Jung and Schluesener, 1991) and
cancer patients (Gedde-Dahl et al., 199%; Fossum et al., 1994)
specifically recognise mutant p21 ras and corresponding peptides. The effector cells involved in this recognition are T
helper (CD4+) and cytotoxic (CD8+) T cells. Both of these
T-cell subsets are able to specifically kill or inhibit growth of
colon cancer cell lines presenting the corresponding ras mutation (Gedde-Dahl et al., 1994~;Fossum et al., 1995). Together
these findings demonstrate that the T-cell repertoire in both
healthy individuals and cancer patients contain T cells capable
of recognising the same s e h of common mutant ras peptides.
The purpose of vaccination is therefore to selectively expand
these T cells in cancer patients.
These observations formed the basis for the present pilot
clinical study, where peptide loaded professional antigenpresenting cells (AF'Cs) were administered to cancer patients.
Our primary objectives were to determine the safety and
toxicity of ras peptide vaccination in patients with adenocarcinoma of the pancreas and to evaluate whether vaccination with
mutant ras peptides induces an immunological response against
the immunising peptide.
PATIENTS AND METHODS
Eligibilig criteria
All patients had histologically confirmed adenocarcinoma of
the pancreas. Inoperability was based on computerised tomography (CT), visceral angiography and/or laparotomy. The ras
mutation was identified in tumour tissue by enriched PCR and
sequencing of K-rus genes (Kahn et al., 1991). Immunocompetence in the patients was verified using purified protein
derivative of M. tuberculosis (PPD; The Veterinary Institute,
Oslo, Norway), phytohemagglutinin (PHA, Wellcome, Dartford, UK) and superantigen (SEC-3; Toxin Technology, Sarasota, FL). The protocol required a Karnofsky performance
status of 60 or greater and an estimated life expectancy of
more than 8 weeks. Patients treated with chemotherapy or
radiation within 3 months prior to vaccination were excluded.
A written, formal consent to participate in this study was
required from all patients. This vaccination protocol was
approved by the Regional Ethics Committee and was performed according to the Helsinki declaration (1989 revision).
Medication to relieve symptoms was allowed.
Vaccination protocol included 1 to 4 x lo9 peripheral blood
mononuclear cells (PBMC) collection of by leucapheresis.
PBMC were isolated by density centrifugation over FicollHypaque (Lymphoprep; Nycomed, Oslo, Norway). Cells were
diluted in X-VIVO 10 (BIO-Whittaker, Walkersville, MD)
supplemented with indomethacin (Confortid, Dumex, final
concentration 5 pg/ml) to prevent monocyte activation. The
synthetic ras peptides (Pronova, Oslo, Norway), encompassing
residues 5-21 of p21 ras (Table I), were synthesised and
purified as described earlier (Gedde-Dahl et al., 1992a). The
ras peptide representing the mutation found in the tumour
tissue of each patient (Table 11) was dissolved in sterile water
before filter sterilisation and added to the cells at a final
concentration of 25 pM. After overnight incubation at 37°C in
5% COz, cells were washed to remove unbound peptide and
diluted in isotonic saline supplemented with heparin (Novo
Nordisk, Bagsvaerd, Denmark; final concentration 20 IE/ml)
and human serum albumin (Octapharma, Vienna, Austria;
final concentration 2.5%). The cell suspension was re-infused
into a peripheral vein during 30-60 min. All patients were
vaccinated at day 0, and booster vaccinations were performed
similarly on days 7-10 and at 1 month. Further booster
vaccinations were performed at 4-6-week intervals or as long
as the patient's immunological response and overall performance status permitted over a period of 2-10 months.
6To whom correspondence and reprint requests should be sent, at
Section for Immunotherapy, Department of Immunology, Institute of
Cancer Research, N-0310 Oslo, Norway.
Received: October 2,1995.
PILOT STUDY OF RAS PEPTIDE VACCIINATION
TABLE 1 - AMINO-ACID SEQUENCES OF 6 SYNTHETICPEPTIDES
ENCOMPASSING POSITIONS 5-21 OF PZ1 ras, CARRYING DIFFERENT
MUTATIONS IN POSITION 12 OR 13
12 13
S
21
'The 5-21, 12Gly, 13Gly peptide represents the native, nonmutated ras peptide.
TABLE I1 -PATIENT CHARACTERISTICS
Sex
(F/M)
Extent of
disease
Regional
Regional
Metastatic
(liver)
Regional
Metastatic
2
3
Val(12)
Arg(l2)
Asp(l2)
M
M
M
4
5
Asp 12
Asp{l2]
F
1
pearance of tumour-related pain was called "pain relief' in
those patients who no longer needed any kind of analgesic.
Amino-acidseauence
Position
Peptide
Patient m z i o n
451
F
Age Performance Number of
(yrs)
(KPS)
vaccinations
49
63
69
100
60
39
70
80
80
80
7
2
3
4
3
Evaluation of safety
Patients were followed closely for signs of toxicity during
and after vaccination. They were questioned about side effects
by the responsible physician before each booster vaccination.
Physical examinations were recorded at every follow-up at the
hospital. Standard haematological and biochemical analyses
were performed prior to and after each vaccination. Treatmentrelated toxicity was documented using the grading of the
World Health Organisation (WHO; Miller et al., 1981).
Evaluation of immunological response
ACD-blood (10 ml) was drawn weekly, separated on Lymphoprep (Nycomed) to obtain fresh PBMC and tested in vitro for
proliferative T-cell responses. Cells were seeded lo5per well in
round-bottomed 96-well plates (Costar, Cambridge, MA) in
100 pl X-VIVO 10 medium supplemented with the immunising peptide at 25 pM with or without recombinant interleukin-2 (rIL-2), 1 Uiml (Amersham, Aylesbury, UK). As controls we seeded PBMC without peptide, with 5 homologous
peptides differing only in one amino acid in position 12 or 13 of
p21 ras (Table I) and with 1 kg/ml PHA, 2 pg/ml PPD and 1
pg/ml SEC-3. Proliferation was measured at day 7 (day 4 with
PHA) after overnight incubation with 3H-thymidine 3.7 X 104
Bq/well (Amersham). Values are given as mean counts per
minute (cpm) from triplicates. An antigen-specific response
was considered positive when the stimulatory index (i.e.,
response with antigen divided by response without antigen)
was above 3.
Evaluation of clinical state
Tumour mass was evaluated by CT and magnetic resonance
(MR) scans every 4 weeks provided that at least one diameter
was 2 3 cm. Tumour response (complete or partial) was
evaluated according to accepted criteria (WHO). No changes
in tumour mass for more than 2 months was defined as no
change (NC), while progressive disease (PD) denoted a 25%
or more increase in tumour mass. Occurrence of ascites was
also defined as disease progression. Ambulatory performance
status was evaluated and registered according to the scale of
Karnofsky et al. (1948). Patients suffering from tumour-related
pain received morphine-based analgesics at inclusion. Disap-
RESULTS
Safety /toxicity
Five patients entered the study. All patients received at least
2 vaccinations and were evaluable for safety analysis and
immunological response. All patients had a primary tumour
and 2 had additional metastases. Details of patient and tumour
characteristics are given in Table 11. All immunising treatment
was performed under special safety conditions, and patients
were hospitalised for at least 2 days. No sign of toxicity and no
adverse events following peptide vaccination were observed.
No abnormalities in haematology and blood chemistry, other
than those related to the nature of the disease, were found. No
patients had to be withdrawn due to treatment-related side
effects or toxicological reactions.
Immunological response
All patients were tested weekly for proliferative T-cell
responses against the immunising peptide. In 2 of the 5
patients vaccinated, a proliferative T-cell response in PBMC
around day 40 after onset of vaccination was found (Table 111).
In the first patient, the T-cell response was strictly peptidespecific, and in the other patient the T cells showed crossreactivity against the other homologous peptides. Neither of
these 2 patients showed any sign of T-cell responsiveness
against the rus mutation before vaccination. The T-cell response was transient in both patients and disappeared during
the following weeks. Repeated vaccination did not result in the
re-appearance of responding T cells in peripheral blood. The
first patient experienced a slight increase in body temperature
(38.lo-38.6"C) approximately 7 days after booster vaccinations. This was not the case for the other responding patient.
Tumour evaluation
No complete or partial response to therapy was found.
Three patients had stable disease, while 2 patients experienced
disease progression. Three patients with NC in tumour mass,
with tumour-related pain, noticed pain relief. The 2 patients
with immune response against the immunising peptide experienced enhanced quality of life. The median duration of
survival was 10.5 months for the 2 responding patients,
compared to 4.5 months for the non-responding patients
(Table IV).
TABLE I11 -IMMUNOLOGICAL RESPONSES ON DAY 40 AFTER ONSET OF
mas PEPTIDE VACCINATION'
~~
Patient
PHAZ
ppD3
Z
1
3
11.879
31:222
24,140
4
32-15
_ _> - - -
37.771
-31021
3,662
13.986
4,032
5
NT7
- - I
~
~ ~ c - 3 4 Immunising
peptide5
28.725
91440
10,355
89.809
731529
10.789
'136
504
31.976
914
Control
peptide
5-21
12G&
1.195
152
518
22.987
1;451
'Values are given as mean counts per minute (cpm) from
triplicates.-2PHA, phytohemaggl~tinin.-~PPD,purified protein
derivative of M. tuber~ulosis.-~SEC-3,
staphylococcalenterotoxin.SThe peptide that corresponds to the ras mutation found in the
tumour tissue from the patient.-6The peptide representing the
normal sequence of amino acid residue 5-21 in p21 ~ ~ s . - ~ Nnot
T,
tested in this assay. Figures in bold type show positive responses to
peptide stimulation; background response in the absence of
peptide was below 500 cpm.
452
GJERTSEN ETAL.
TABLE N - RESPONSE TO THERAPY
Patient
Immunological
response
Sulvival time
(months)
Tumour
evaluation
Yes
no
no
Yes
no
12
3.5
NC’
PD2
NC
NC
PD
5
9
4.5
‘NC, no change.-ZPD, progressive disease.
DISCUSSION
This pilot study involving 5 patients with inoperable pancreatic adenocarcinoma was designed to assess whether vaccination with synthetic ras peptides is a safe and tolerable
procedure. Unlike cytostatic drugs and biological responsemodifying agents, peptide vaccines are not expected to have
any general effects on cellular metabolism. The synthetic ras
peptides used will have no direct effect on the cancer cells but
are designed to elicit an immune response specific for a single
amino acid residue discriminating a mutant p2lras molecule
from its normal counterpart. This treatment modality is safe,
and in the 5 patients we have treated, no toxic reactions or
other side effects were observed. Nineteen treatment sessions
with 3 different synthetic ras peptides have been carried out,
and the vaccinations were well tolerated when given repeatedly. One might have anticipated that in patients showing an
immune response directed against the immunising peptide, a
subsequent vaccination could give rise to a strong immune
reaction with possible side effects, eg., “flue-like” symptoms,
normally observed during an infection. This was not found to
be the case in the 2 patients who demonstrated an immune
response against the ras peptides, though in one of the
patients, a slight increase in body temperature was noted after
booster vaccination. The lack of a strong response is probably
related to the fact that we are using a single peptide representing a limited number of epitopes and that the number of
corresponding clonal T cells that have the capacity to recognise
the peptide in any patient is low. This is in accordance with a
very low frequency of precursor cells specific for a single rus
mutation found in normal individuals (Gedde-Dahl et ul.,
1992~).
A second primary end-point of this study was to determine
whether vaccination with synthetic ras peptides results in a
T-cell response against the peptide. Our data clearly demonstrate that immunisation with synthetic ras peptides in vivo can
give rise to such an immune response directed against the
peptides used for vaccination. Thus, peptide vaccination of
patients according to this protocol is feasible and can induce T
cells specific for a mutation found only in the cancer cells of the
patient and not in the normal cells. No patient showed
evidence of a T-cell response against mutated rus before the
onset of treatment. The success rate of peptide stimulation in
vivo (2 of 5 ) is similar to what we have experienced in
experiments with healthy human volunteers using in vitro
stimulation to generate ras peptide-specific T-cell responses
(data not shown).
The similarity between the mutant ras peptides and the
peptide representing normal p21 ras can give rise to T-cell
clones that may show varying degrees of cross-reactivity
between the homologous ras peptides, including the normal
peptide. This opens the possibility that some T-cell clones may
be autoreactive and therefore potentially harmful. In one of
our patients, a cross-reactive T-cell response was observed
after initiation of vaccination, and the responsive cells continued to be present in circulation for 3 weeks, after which they
disappeared. During this period a scheduled booster vaccination was postponed, and the patient was carefully observed for
possible side effects caused by these potentially autoreactive T
cells. No side effects were, however, observed, and treatment
was continued after disappearance of the responding T cells
from peripheral blood. The lack of an apparent autoreactivity
may be due to a too low expression of normal p21 ras in
non-malignant cells, compared to aberrant forms of p21 ras
which are known to be over-expressed in tumour cells (Slamon
eta/., 1984).
In all patients, the tumour eventually progressed, leading to
a fatal outcome. The speed of the progress varied between the
different patients, but it is our impression that tumour progression was slower in those patients who mounted an immune
response against the peptide vaccine. Whether this simply
reflects that patients with a good performance status live
longer or is a result of the induction of a partly protective
anti-cancer immune response remains to be determined.
We conclude that ras peptide vaccination according to the
present protocol is safe and may result in a potentially
beneficial immune response even in patients with advanced
cancer. The design of the study and the small number of
patients treated do not allow any conclusions regarding the
tumour response to ras peptide vaccination.
ACKNOWLEDGEMENTS
We thank the patients and their families for their confidence, Miss I. Knutsen for sequencing of the rus mutations and
Miss L. Killingbergtr@for helping with clinical management.
This workwas supported by the Norwegian Cancer Society, the
Norwegian Research Council for Science and the Humanities
and Pronova a.s.
REFERENCES
ALMOGUERA,
C., SHIBATA,
D., FORRESTER,
K., MARTIN,J., ARNHEIM,-DP, and -DQ restricted T cells. Europ. J. Immunol., 23, 2687-2691
N. and PERUCHO,
M., Most human carcinomas of the exocrine (1993).
pancreas contain mutant c-K-rusgenes. Cell, 53,549-554 (1988).
FOSSUM,
B., OLSEN,
A.C., THORSBY,
E. and GAUDERNACK,
G., CD8+T
from a patient with colon carcinoma, specific for a mutant p21 ras
CAPELLA,
G., CRONAUER-MITRA,
S., PEINADO,
M.A., and PERUCHO,cells
derived eptide (13Gly + Asp), are cytotoxic towards a carcinoma cell
M., Frequency and spectrum of mutations at codons 12 and 13 of the line
harfouring the same mutation. Cancer. Immunol. Immunother.,
c-K-rusgene in human tumors. Environ. Health Perspect., 93, 125-131 40,165-172
(1995).
(1991).
111, T., ERIKSEN,
J.A., THORSBY,
E. and GAUDERNACK,
GEDDE-DAHL
B., GEDDE-DAHL
111 T., BPSIVIK,J., ERIKSEN,
J.A., SPURKLAND,G., T-cell responses against products of oncogenes: generation and
FOSSUM,
A., THORSBY,
E. and GAUDERNACK,
G., p-21-ras-peptide-specific T-cell characterization of human T-cell clones specific for p21 ras-derived
responses in a patient with colorectal cancer. CD4+ and CD8+T cells synthetic peptides. Hum. Immunol., 33,266-274 (1992~).
recognize a peptide corresponding to a common mutation GEDDE-DAHL
111, T., NILSEN,
E., THORSBY,
E. and GAUDERNACK,
G.,
(13Gly + Asp). Int. J. Cancer, 56,4045 (1994).
Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T
B., GEDDE-DAHL
111, T., HANSEN,
T., ERIKSEN,
J.A.,THORSBY,cells s ecific for mutant p21 ras. Cancer. Immunol. Immunother., 38,
FOSSUM,
E. and GAUDERNACK,
G., Overlapping epitopes encom assing a point 12’7-1f4 (1994~).
mutation (12Gly+Arg) in p21 ras can be recognizefby HLA-DR, GEDDE-DAHL
HI,T., SPURKLAND,
A,, ERIKSEN,
J.A., THORSBY,
E. and
PILOT STUDY OF RAS PEPTIDE VACCIINATION
GAUDERNACK,
G., Memory T cells of a patient with follicular thyroid
carcinoma recognize peptides derived from mutated p21 ras
(Gln + Leu61). fnt. Immunol., 4,1331-1337 (199%).
GEDDE-DAHL
111, T., SPURKLAND,
A., FOSSUM,
B., WITTINGHOFER,
A,,
E. and GAUDERNACK,
G., T cell epitopes encompassing the
THORSBY,
mutational hot spot position 61 of 21 ras. Promiscuity in ras peptide
binding to HLA. Europ. J. Immuno& 24,410-414 (1994b).
JUNG,S. and SCHLUESENER,
H.J., Human T lymphocytes recognize a
peptide of single point-mutated, oncogenic ras proteins. J. exp. Med.,
73,273-276 (1991).
KAHN,S.M., JIANG,W., CULBERTSON,
T.A., WEINSTEIN,
B., WILLIAMS,
G.M., TOMITA,N. and RONAI,Z., Rapid an sensitive non-radioactive
453
detection of mutant K-rus genes via “enriched” PCR amplification.
Oncogene, 6,1079-1083 (1991).
.
.
URNOFSKY,
D.A., ABELMANN,
W.H., CRAVER,L.F. and BURCHENAL,
J.H., The use of the nitrogen mustards in the palliative treatment of
carcinoma. Cancer, 1,634-656 (1948).
MILLER,A.B., HOOGSTRATEN,
B., STAQUET,M. and WINKLER,A.,
Reporting results of cancer treatment. Cancer, 47,207-214 (1981).
SLAMON,D.J., DE KERNION,J.B., VERMA,I.M. and CLINE, M.J.,
Expression of cellular oncogenes in human malignancies. Science, 224,
256-262 (1984).
WARSHAW,
A. and FERNANDEZ
DEL CASTILLO,
C., Pancreatic cancer.
New Engl. J. Med., 326,455-465 (1992).
Документ
Категория
Без категории
Просмотров
3
Размер файла
434 Кб
Теги
381
1/--страниц
Пожаловаться на содержимое документа