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Advances in Brief |
Divisions of Gynecologic Oncology [L. M., L. R., J. F.], Medical Oncology [L. B., V. M., J. W.], and Pathology [J. F.], University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, California 90033; Cancer Immunology Program, Loyola University School of Medicine Chicago, Illinois 60153 [L. A. S., W. M. K.]; Cardinal Bernardin Cancer Center, Maywood, Illinois 60153 [W. M. K., L. A. S.]; and Department of Pathology, City of Hope Medical Center, Duarte, California 91010 [S. W.]
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ABSTRACT |
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Introduction |
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CTLs recognize antigens by the binding of their clonotypic TcRs to the processed endogenous peptides associated with class I molecules. Purified epitope peptides derived from different animal viruses have been shown to induce high-affinity CTLs and protect mice against a lethal challenge with infectious viruses (13, 14, 15, 16, 17, 18) . HLA-restricted HIV epitope peptides emulsified in IFA primed a specific murine CTL anti-HIV response (19 , 20) , and immunization with HPV E6 and E7 peptides with IFA induced protection against lethal challenge with E6- and E7-expressing tumor cells in mice (13) . On the basis of this preclinical rationale, we performed a Phase I clinical trial in which two HPV 16 E7 peptides known to be recognized by CTLs were used in escalating doses with IFA to treat patients with high-grade CIN/VIN. In addition to the toxicity and tolerability of the vaccine, immune, virological and clinical response end points were assessed in this clinical trial.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Patients were excluded if they had an endocervical curettage indicating preinvasive changes or invasive cancer, previous pelvic irradiation, prior in utero diethylstilbestrol exposure, dependence on steroids, known HIV positivity, or active autoimmune disease (systemic lupus, rheumatoid arthritis, and others).
The HPV-16 E7 12–20 peptide (Ref. 21 ; MLDLQPETT) was produced by Peninsula Labs, Inc. (Belmont, CA). The peptide was provided with Montanide ISA 51 under an Investigational New Drug application held by the Cancer Therapy Evaluation Program of the National Cancer Institute. The final vialed product was produced by the Monoclonal Antibody Production Facility/BioWhittaker. The peptide was produced to Good Laboratory Practice standards. Sterile vials of HPV E7 peptide were stored at 2–8°C and protected from light. E7 12–20 peptide was dissolved in water and filtered through a 0.22 µm Millipore filter. Analysis of the lot to be used in the Phase I study of this protocol demonstrated that there was no pyrogenicity in a rabbit assay and <0.01 endotoxin units/100 µl of endotoxin in a Limulus amebocyte assay. General safety testing in BALB/c mice was satisfactory. Vials containing three concentrations of the peptide were available as 207, 617, and 2057 µg/ml. Each vial contained 1 ml of peptide solution. Peptide was provided by the Cancer Therapy Evaluation Program of the National Cancer Institute (Bethesda, MD) as the trifluroacetate salt in DMSO. The vials of peptide contained no preservative. Montanide ISA-5 1 (IFA) was manufactured by Seppic, Inc. and supplied as glass ampuls containing 3 ml of sterile adjuvant solution without preservative.
An appropriate amount of HPV E7 12–20 was diluted with sterile saline and added in a 1:1 volume to Montanide ISA 51 and then mixed in a Vortex mixer (Fisher, Inc., Alameda, CA) for 10 min at room temperature. The resulting emulsion was injected deeply s.c. in the lateral thigh in a volume of 1 or 2 ml using a glass syringe. Alternating thighs were used for a total of four injections, which were done 3 weeks apart.
Good Manufacturing Practice grade lipopeptide consisting of linker peptide (KSS), the helper peptide PADRE-965.10 (AJXVAAWTLKAAA), and the E7 peptide 86–93 [Ref. 22 ; TLGIVZPI, where aminobutyric acid (Z) is substituted for cysteine (C) at position 91 of the HPV epitope] was produced by Cytel Corp. (San Diego, CA). This 24-amino acid oligomer [(PAM)2 (KSSAKXVAAWTLK-AAA-TLGIVZPI)] was provided under a National Cancer Institute, Cancer Therapy Evaluation Program Investigational New Drug application. Vials contained a solution of HPV-16 E7 86–93 lipopeptide at a concentration of 5 mg/ml in DMSO with 0.1% trifluoracetic acid. Each vial contained 2.0 ml of solution for a total of 10 mg of the lipopeptide per vial. Vials of lipopeptide contained no preservative. The Recombinant Protein Production Facility of the Biological Resources Branch, Biological Response Modified program placed the peptide material into vials. The E7 lipopeptide was diluted with sterile saline and injected deeply s.c. in the lateral thigh in a volume of 1 or 2 ml using a glass syringe. Alternating thighs were used for a total of four injections, which were done 3 weeks apart.
Eighteen patients had a leucopheresis with an exchange of 
5 liters
of blood volume performed within 2 weeks before beginning vaccinations
and 3 weeks after the final vaccination to collect PBMCs, which were
frozen for future analysis. Skin tests were performed using 50 µg of
the HPV-16 E7 peptide in aqueous solution injected intradermally in a
volume of 100 µl using a tuberculin syringe and a 27-gauge needle.
Candida extract and mumps provided a positive control, and saline was a
negative control for assessment of delayed-type hypersensitivity. At
least 5 mm of induration or erythema above and beyond that shown by
saline read 48 h after intradermal injection were required to
score a HPV E7 12–20 skin test as positive.
Pheresis samples were processed to purify PBMCs by sedimentation on a Ficoll-Hypaque cushion (Pharmacia, Alameda, CA) and extensive washing in HBSS. Cells were frozen in 40% human AB serum (Gemini Bioproducts, Calabasas, CA), 50% RPMI (Life Technologies, Inc., Grand Island, NY) and 10% DMSO (Sigma Chemical Co., St. Louis, MO) and stored in a liquid nitrogen freezer at -168°C until use.
Cytokine release assays were performed using peptide-stimulated T cells
as effector cells. Peptide-stimulated T cells were produced by
incubating 2 x 106 thawed PBMCs with
10 µg/ml HPV 16 E7 12–20, 86–93, or FLU-MI in wells of a 24-well
plate (Corning, Oneonta, NY). Cells were plated in IMEM with 10% human
AB serum. Two days later, IL-2 (kindly provided by Chiron, Emeryville,
CA) was added at 50 IU/ml. Fresh IL-2 was added every 3–4 days. After
10, days, the T cells were restimulated with thawed autologous PBMCs
pulsed with 10 µg/ml of peptide at 37°C for 2 h and irradiated
with 3000R. IL-2 was again added 48 h later at 50 IU/ml. T cells
were restimulated with peptide-pulsed PBMCs every 7 days and after 3
restimulations were harvested for immune assays. For the cytokine
release assay, 100,000 peptide-stimulated, T cells were harvested at
least 5 days after the last restimulation and incubated with 100,000 T2
cells pulsed with 10 µg/ml HPV E7 12–20, 86–93, or FLU M1 peptide
or Caski cells as targets in a total volume of 1 ml of RPMI 1640
without serum for 18 h in a 5% CO2
incubator at 37°C. Neither the effectors nor the targets were
irradiated. Supernatants were collected, spun briefly at 14,000 x
g to pellet cells and debris and frozen at -80°C until
assays were done. IFN-
was detected in supernatants using an
antihuman IFN- Quantikine ELISA kit (R and D Systems, Minneapolis,
MN).
Chromium release assays were performed using the same effectors and
targets as in the cytokine release assays, but 5000 targets labeled for
2 h with 51Cr were plated in each well of a
96-well, round-bottomed plate (Corning). Effectors (150,000, 50,000,
15,000, and 5,000) were added to a total volume of 200 µl for final
E:T ratios of 30:1, 10:1, 3:1, and 1:1. Twenty-fold excess of K562
cells was added to suppress natural killer activity. E:T mixtures were
spun down at 500 x g for 5 min and then incubated for
4 h in a 5% CO2 incubator at 37°C.
Supernatants were harvested using a Skatron collecting apparatus, and
the liquid-impregnated filters were counted on a Packard gamma counter.
The percentage of specific chromium release was measured as:
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For TcRs assays, testing for signal transduction molecules was done according to the technique described by Nieland et al. (23) . Assays were scored as the percentage of a battery of 10 normal controls.
Specimens for HPV DNA testing of cervical scrapings were collected in transport media (Digene, Silver Spring, MD) and stored at -20 degrees until processing. HPV typing was performed by PCR as described previously (24, 25, 26) . Briefly, DNA extracted from a cervical scraping was amplified with ß-globin primers to confirm the presence of amplifiable DNA. Consensus primers in the HPV L1 gene MY09/MY11, and type-specific primers for HPV 6, 16, and 18 as well as the reaction conditions have been published. Every reaction set up contained appropriate positive and negative controls. The amplified PCR product was electrophoresed in a 2% agarose gel and stained with ethidium bromide for detection of a visual product. Eligibility criteria for the trial included a positive visual product with the consensus as well as the HPV 16 type primers. All HPV 16 isolates were confirmed by Southern blotting and transferred to nylon membranes (MagnaNT; MSI, Inc.). Products were hybridized overnight with 32P randomly labeled probes for HPV 6, 16, and 18. The membranes were washed four times under stringent conditions with 2x SSC with 0.1% SDS at 48°C and 0.1x SSC with 0.1% SDS at 60°C and exposed to X-ray film with an intensifying screen at -80°C for 4 days.
In situ hybridization for HPV 16 RNA was performed as
described previously. After deparaffinization, rehydration, and
proteinase K digestion for 30 min at 1 µg/ml (Boehringer Mannheim,
Indianapolis, IN), the sections were acetylated in 0.25% acetic acid
anhydride and then dehydrated through graded ethanols. HPV riboprobes
prepared from pBluescript plasmids were labeled with
35S-labeled UTP and reduced to 150 bp by
alkaline hydrolysis. Sections were hybridized with both sense and
antisense strand HPV 16 riboprobes at 45°C in hybridization solution
containing 50% formamide, 10% dextran sulfate, 10
mM/l Tris-HCl (pH 7.4), 2x SSC, 1
mM/l EDTA, 500 mg/ml Escherichia coli
tRNA, and 1x Denhardt’s solution. After hybridization, the
slides were washed in 4x SSC for 20 min, incubated with RNase A (10
µg/ml) at 37°C for 30 min, followed by an additional 30-min wash
with 0.1x SSC at 55°C, then dehydrated through graded ethanol
containing 300 mM/l ammonium acetate, and coated
with photographic emulsion (Kodak, New Haven, CT). Duplicate slides
were exposed for 2–4 weeks at 4°C and then developed and lightly
counterstained with hematoxylin. The slides were examined under
dark-field microscopy, and the signal in the epithelium with the most
severe dysplasia were scored 1+ to 3+, with 1+ just above background,
2+ a moderate signal, and 3+ a strong signal with focally very strongly
positive individual cells.
Immunohistochemistry for CD3, CD4, CD8, and S100 was performed on formalin-fixed, paraffin-embedded sections according to standard procedures for heat-induced epitope retrieval, as described previously. CD3, CD8, and S100 antibodies (Dako Corp., Carpinteria, CA) were used at dilutions of 1:100, 1:25, and 1:400, respectively, and CD4 (Novacastra Laboratory, Newcastle-on-Tyne, United Kingdom) at a dilution of 1:40. Positive and negative controls included lymph node (CD3 and CD4) and tonsil tissue (CD8), whereas a multi-tissue block of tumor and neural tissue was the S100 control. Antigen-antibody complexes were detected by avidin-biotin technique (Vector Elite kit; Vector, Burlingame, CA) per the manufacturer’s directions with 3',3'-diaminobenzidine as the chromogen.
To estimate the number of intraepithelial DCs, the number of S100-positive nuclei were counted in 3–5 mm of the cervical epithelium with the highest grade of dysplasia and then averaged per mm. Most of the biopsy specimens had insufficient adjacent uninvolved epithelium to evaluate. In the cone biopsies taken after treatment the number of S100-positive DCs, nuclei were counted in a region of the cone that had histologically normal epithelium and little inflammation.
The number of T cells positive in the superficial stroma that were immunoreactive with CD3, CD4, and CD8 were scored 1+ to 4+ based on the following scale: 1+ showed a few scattered positive cells; 2+, occasional clusters or patchy groups of positive cells; 3+, diffuse infiltrate; and 4+, a heavy infiltrate.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The median age was 29, with 16 CIN
II/III and 2 VIN II/III patients. All patients were HLA-A2 positive,
and all patients had a Virapap showing HPV type 16 by DNA PCR analysis.
All patients but one had a measurable lesion in the cervix or vulva
that was biopsy proven at least 1 month prior to therapy and was
colposcopy positive 1 month after initial biopsy to rule out
spontaneous regression; one patient had a history of carcinoma in
situ of the cervix with positive PAP smears postoperatively but no
lesions positive by biopsy. All patients gave informed consent and had
a pretherapy pheresis within 2 weeks prior to starting vaccinations and
within 4 weeks after the fourth and last vaccine injection.
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below.
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shows the evolution of viral PCR
signals for 18 patients. In the published literature, little
information is available about Virapap detection of HPV by PCR after a
LEEP, but we examined Virapaps over time after LEEP for HPV 16 by DNA
PCR in a sample of 16 patients who were HLA-A2 negative or had an
unsatisfactory colposcopy and could not go on the vaccine study. In 16
of 16 cases, virus was positive prior to LEEP but cleared within 4
months afterward from the mucosae (data not shown). Of note is that for
six patients who had adequate biopsy tissue with viable dysplastic
tissue to study after vaccination, an in situ RNA
hybridization signal was detected in tissue removed from all LEEP or
excision specimens of these patients at the time of their definitive
procedure. Interestingly, 3 of 6 samples (patients 3, 8, and 15) had a
much weaker signal after vaccination (data not shown). The PCR data
from immunized patients in this study suggest that in a significant
proportion of cases (15 of 18), a HPV 16 viral DNA signal derived from
swab material of the cervix or vulva disappears or diminishes after
peptide vaccine therapy and prior to definitive surgery, but that a RNA
signal in the dysplastic tissue itself continues to be detected.
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cytokine release assay or a chromium release assay using
antigen-specific and nonspecific targets to test whether there was an
increase in HPV-specific CTL activity detected in the peripheral blood
after vaccinations compared with before. The data shown below in Fig. 1
for the first 16 patients represent
cross-specificity assays to detect antigen-specific T cells that
release cytokine (IFN-) before and after immunizations. Total PBMCs
were thawed simultaneously and split into two parts. One aliquot before
and after vaccination was repeatedly stimulated with the FLU M1 matrix
58–66, A2-restricted epitope peptide, and the other with HPV E7 12–20
peptide. After three weekly restimulations, the resulting effector
cells were incubated with HLA-A2+ targets expressing the E7 12–20, FLU
58–66, or no peptide. After 18 h, supernatants were collected,
frozen, and later thawed and assayed for IFN- by ELISA. For 10 of 16
patients tested, there is a clear augmentation of HPV E7-specific
IFN- release after vaccination, without a significant change in the
FLU-specific reactivity (FLU data not shown). Positive assays were seen
for patients 1, 4, 5, 7, 8, 10, 11, 13, 15, and 16. FLU-stimulated
cells revealed no E7-specific reactivity relative to background, nor
did E7specific effectors show reactivity against FLU,
demonstrating appropriate cross-specificity and suggesting that there
is a significant augmentation of E7-specific T-cell immunity as a
result of vaccination. There does not appear to be a dose response,
with high levels of cytokine release at all doses of peptide used,
although the numbers are obviously quite small. Patients 11–16
received the 86–93 lipopeptide, and they had separate aliquots of
PBMCs stimulated with the 86–93 peptide, with no responders observed
in cytokine release assays (data not shown). All samples were used for
a repeated cytokine release assay to verify the results of the assay
shown in Fig. 1
, and the results were reproducible (data not shown).
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suggest that there is a correlation
between the cytokine and chromium release assays, because 10 patients
(nos. 1, 4, 5, 7, 8, 10, 11, 13, 15, and 16) exhibited an immune
response with increased cytokine secretion after HPV 16 E7 12–20
stimulation, and 8 of those 10 samples showed augmented E7
12–20-specific cytolysis in Fig. 2
. For the six patients analyzed who
also received the 86–93 lipopeptide, augmented chromium release was
observed in three, shown for patient nos. 11, 13, and 16 in Fig. 3
. The above data suggest that reactive T
cells detected by cytokine release assays after E7 peptide vaccination
are also lytic for E7-expressing targets.
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. In the cone biopsies after
vaccination, the adjacent nondysplastic squamous epithelium had fewer
DCs than did the cervical intraepithelial neoplasia. Fig. 4
shows an example of a CIN III lesion
before vaccination with few infiltrating DCs by immunohistochemical
staining in the left panel and a large increase in the DC infiltrate in
the postvaccination LEEP specimen in the right panel. Decreases in
Langerhans cells, which are antigen-presenting DCs present in
epithelial tissues, are reported in HPV-infected cervical epithelium,
and there is some evidence to suggest that fewer Langerhans cells can
be found in cervical lesions that persist compared with lesions that
regress (27
, 28)
. The current data suggest that in a
modest number of carefully prepared samples at different doses of the
HPV peptide vaccine, significant increases in DC infiltration were seen
after vaccination.
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chain signal
transduction molecules, which have been found to be deficient in cancer
patients in general and women with cervical cancer in particular
(23
, 29) . T cells that have deficient levels of TcR
transduction molecules are unlikely to be fully functional in
vivo or in vitro. Of the first 16 women in the trial,
all except 2 had significant defects in TcR by intracellular flow
cytometry staining (mean TcR, 69.3 ± 4.5% before vaccination and
74.1 ± 11.2% after vaccination). Normal levels were established
using a large number (>10) of normal healthy controls, and the values
for the CIN/VIN patients are expressed as a percentage of normal
controls. No significant changes in TcR were seen after
vaccination. These data emphasize that women without invasive
malignancy have clear evidence of immunosuppression, suggesting that
strategies to boost immune responses and immune competence in
vaccinated women with CIN/VIN are important.
Clinical Results in Vaccinated Patients.
A total of 9 of 17 evaluable patients had partial or complete
regression of their CIN lesions. Three had complete regression, and 6
had >50% shrinkage of their colposcopically measured disease, as
summarized in Table 5
. No clear
correlation was observed between immune responses and clinical
regression or disappearance of CIN. Patients at all doses of vaccine
had regression of disease, indicating that a dose response was
unlikely.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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75% of high-grade cervical intraepithelial lesions
are positive for high- or intermediate-risk HPV types, such as 16 and
18, compared with 40–50% of vaginal and 80–90% of vulvar epithelial
lesions. The spectrum of HPV-associated clinical findings in women
ranges from genital warts to squamous intraepithelial neoplasia and
invasive carcinoma (31)
. HPV types 6 and 11 are associated
with genital warts (condyloma accuminata; Ref. 32
) and
types 16 and 18 with intraepithelial and invasive lesions. HPV 16 is
the most common type found in squamous carcinomas (33)
,
and HPV 18 is most common in adenocarcinomas and small cell
neuroendocrine cervical carcinomas (34
, 35)
. These data
provide a strong justification for devising immunization strategies
against high-risk HPV types to prevent progression from low-grade
CIN/VIN to high-grade disease, recurrence of high-grade dysplasia, and
the occurrence of invasive cervical/vulvar cancer. HPV-specific serological, T helper, and T cytolytic immune responses have been demonstrated in patients with high-grade CIN/VIN and cervical cancer. Seropositivity directed against the E1, E2, E4, and E7 proteins in virus-like particles has been documented in patients positive for HPV 16 by PCR DNA (36, 37, 38) . T-helper responses to the L1, E2, E4, and E7 proteins can be detected by proliferation of peripheral blood mononuclear cells in patients with high grade CIN/VIN (38, 39, 40, 41, 42, 43, 44, 45) . Proliferative T-cell responses correlate with high-grade lesions and HPV 16 DNA positivity by PCR (40 , 44 , 45) . In mice, experiments to detect E7 CTL reactivity have been performed using murine tumor cells transfected with the full-length HPV 16 genome or E7 cDNA. Specific CTL reactivity has been generated in mice immunized with tumor cells that express E7 and the T-cell costimulatory molecule B7 (46) or infected with a vaccinia virus construct containing the full-length E7 cDNA (47) . A murine H-2-restricted E7 epitope peptide has been used to immunize mice in combination with IFA. Immunized mice developed protection against a subsequent challenge with a lethal dose of E7-expressing tumor cells (13) . Specific anti-E7 CTLs were generated using peptide vaccination with IFA or after peptides were pulsed onto autologous murine splenocyte-derived DCs (48 , 49) . The use of a lipid-tailed peptide construct resulted in increased CTL induction in mice compared with the peptide with adjuvant (50) . In mice, a T-cell line that recognizes that epitope eradicated established HPV-16-induced tumors in mice.
Nine- and 10-amino acid peptides from HPV-16 E7 were defined by strong binding to HLA-A2, and the immunogenicity of a number of A2-binding peptides was tested in vivo in HLA-A2 transgenic mice. Three peptides were defined that were immunogenic both in transgenic mice and in CTL induction experiments using PBMCs from HLA-A2 healthy donors derived from the 11–20, 82–90, and 86–93 amino acid sequences (51) .
E7-specific CTL cells have been generated from the peripheral blood and lymph nodal tissue of HPV-16-positive women with cervical dysplasia and cervical cancer by in vitro restimulation with autologous antigen-presenting cells pulsed with HPV-16 E7 peptides (52 , 53) . CTL responses against HPV-16 E7 appeared to be more potent in women who were virus positive but CIN negative than in those who were CIN positive, suggesting a role for specific CTL responses in tumor progression (54) . Autologous peripheral blood cells and DCs pulsed with E7 peptides or protein were potent inducers of anti-HPV immunity in cervical cancer patients. (55, 56, 57) . When normals without HPV infection were tested, no E7-specific CTLs could be detected. Women with stage IV cervical cancer were immunized with a lipopeptide construct identical to that used in this trial, and only six patients mounted a weak immune response against the E7 86–93 peptide sequence, without evidence of clinical benefit (58, 59, 60) . Vaccination with CTL epitope peptide, strong adjuvant, and nonspecific help might prime CTLs against the weakly immunogenic epitope and result in clearance of HPV. It would seem that women with a lower disease burden and preinvasive disease are more logical candidates for an antigen-specific immunotherapy than women with bulky invasive disease, prior chemotherapy, poor performance status, and profound immunosuppression.
The data presented herein suggest that a vaccine consisting of a HPV 16
E7 peptide administered with IFA added to a HPV E7 lipopeptide
stimulates an immune response in a significant proportion of
HLA-A2-positive patients with CIN/VIN II/III evidenced by cytokine or
chromium release assays. The peptide vaccine was well tolerated, with
side effects mostly consisting of local pain and granuloma formation.
No grade III or IV toxicity occurred in this trial of 18 women. All 17
patients with CIN had a definitive excision procedure after their
series of four vaccines was finished, and only 3 of 17 evaluable
patients had complete regression of their lesion pathologically,
although an additional 6 had partial regression of their CIN lesion.
These data must be interpreted with the acknowledgment of a reported
20–30% rate of spontaneous regression noted in patients with
high-grade dysplasia (61)
, and although disappearance of
CIN in 3 patients and partial regression in 6 is encouraging, only the
performance of a randomized trial with a placebo control group will
permit definitive conclusions on the efficacy of this vaccine regimen.
In six pre- and postvaccine specimens examined by microscopy, no change
in CD8+ or CD4+ T-cell infiltrate was seen on immunohistochemical
staining, but significant and consistent increases in infiltrating
S100-positive DCs were observed, suggesting that vaccination resulted
in infiltration of the dysplastic lesion with antigen-presenting DCs.
These results should be interpreted in light of reports that
Langerhan’s cells are decreased in dysplastic cervical epithelium, and
fewer Langerhans cells are found in cervical lesions that persist
compared with lesions that regress (62)
. Because
infiltration of tumors with DCs may correlate with a positive outcome,
augmented infiltration of dysplastic tissue with DCs after vaccination
may represent a beneficial surrogate marker. A possible mechanism to
explain the augmented DC infiltrate might be increased expression of
chemokines that are known to impact on the migration of DCs, such as
MIP-3
or MIP-3ß, by inflammatory cells that infiltrated a
regressing lesion after vaccination (63)
.
In 15 of 18 patients, disappearance or decreased intensity of a HPV DNA PCR signal was detected prior to and at the time of LEEP, indicating that in a sensitive assay, the majority of patients had clearance of HPV 16 from the cervical and/or vulvar tissue after vaccination with peptides. All biopsy specimens of dysplastic tissue tested had evidence of HPV 16 mRNA transcription by in situ hybridization at the time of definitive removal, indicating that virus genetic material was present and had not been cleared from the dysplasia.
The significant findings of this Phase I study were that the majority
of patients had a detectable immune response in peripheral blood cells
after four injections of the peptide E7 12–20 vaccine. Immune
responses in 10 of 16 patients by cytokine release assay was confirmed
by cytolysis assays in 8 of the 10, suggesting that cytokine-secreting
and CTLs were augmented in the peripheral blood after the HPV 16 E7
12–20 peptide vaccine with IFA. All patients except 2 had a positive
FLU-specific cytokine response both before and after vaccination as a
positive control for the assay. One of the patients (no. 2) that did
not respond to FLU M1 or to E7 12–20 was HLA-A2 subtyped by PCR,
revealing the A 0206
subtype,4
which has
been shown to bind A0201-associated peptides such as those used in this
trial at a greatly reduced level (64)
. The augmented
immune responses seen in this trial are similar to those observed in a
peptide trial in cervical cancer (58)
, but these data are
the first that we know of demonstrating that patients with high-grade
cervical/vulvar dysplasia can be vaccinated against HPV 16 and mount a
detectable immune response in the peripheral blood. The data on TcR
transduction molecules described in the text are consistent with
globally depressed immunity similar to that seen in patients with
frank, invasive cancer. The demonstration of decreased levels of class
I molecules on dysplastic and frankly neoplastic cervical cells
(65
, 66)
and the reduction in TcR chain detected in
our patients with preneoplastic dysplasia suggest that strategies to
overcome host immunosuppression will be an important aspect of any
effort to prevent cancer by vaccination of high-risk patients.
Because it is likely that continued expression of E7 is required for proliferation and is necessary but not sufficient for malignant transformation, clearance of virus and/or dysplastic cells expressing E7 might induce regression of CIN/VIN II/III, generate long-lasting immunity against HPV 16, and prevent the premalignant changes associated with HPV 16. Long-lasting protection against cervical/vulvar intraepithelial neoplasia would have a significant impact on the incidence of cervical and vulvar carcinoma. The encouraging initial results from the trial described herein indicate that potent, long-lasting levels of T-cell-mediated immune responses might be beneficial to patients with high-grade dysplasia. In future trials, we will continue efforts to prevent cervical/vulvar cancer by augmenting T-cell immunity to a specific, well-characterized tumor antigen using DNA plasmids and heat shock proteins to deliver the immunogen.
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FOOTNOTES |
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1 Supported by Grants RO1-CA67872, RO1-CA74397,
RO1-CA/AI78399, and 5P30-CA14089 from the National Cancer Institute. 
2 To whom requests for reprints should be
addressed, at University of Southern California/Norris Comprehensive
Cancer Center, Room 6428, 1441 Eastlake Avenue, Los Angeles, CA 90033.
Phone: (323) 865-3919; Fax: (323) 865-0061; E-mail: jweber{at}hsc.usc.edu
3 The abbreviations used are: CIN, cervical
intraepithelial neoplasia; VIN, vulvar intraepithelial neoplasia; HPV,
human papillomavirus; PBMC, peripheral blood mononuclear cell; HLA,
human leukocyte antigen; HIV, human immunodeficiency virus; IFA,
incomplete Freund’s adjuvant; TcR, T-cell receptor; LEEP, loop
electrocautery excision procedure; DC, dendritic cell; IL,
interleukin.
4 F. Marincola, personal communication.
Received 2/29/00; revised 5/30/00; accepted 5/31/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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 M. J.P. Welters, G. G. Kenter, S. J. Piersma, A. P.G. Vloon, M. J.G. Lowik, D. M.A. Berends-van der Meer, J. W. Drijfhout, A. R. P.M. Valentijn, A. R. Wafelman, J. Oostendorp, et al. Induction of Tumor-Specific CD4+ and CD8+ T-Cell Immunity in Cervical Cancer Patients by a Human Papillomavirus Type 16 E6 and E7 Long Peptides Vaccine Clin. Cancer Res., January 1, 2008; 14(1): 178 - 187. [Abstract] [Full Text] [PDF]
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 D.-W. Liu, Y.-C. Yang, H.-F. Lin, M.-F. Lin, Y.-W. Cheng, C.-C. Chu, Y.-P. Tsao, and S.-L. Chen Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients J. Virol., March 15, 2007; 81(6): 2869 - 2879. [Abstract] [Full Text] [PDF]
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M. Nakagawa, K. H. Kim, T. M. Gillam, and A.-B. Moscicki HLA Class I Binding Promiscuity of the CD8 T-Cell Epitopes of Human Papillomavirus Type 16 E6 Protein J. Virol., February 1, 2007; 81(3): 1412 - 1423. [Abstract] [Full Text] [PDF]
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S. J. Piersma, E. S. Jordanova, M. I.E. van Poelgeest, K. M.C. Kwappenberg, J. M. van der Hulst, J. W. Drijfhout, C. J.M. Melief, G. G. Kenter, G. J. Fleuren, R. Offringa, et al. High Number of Intraepithelial CD8+ Tumor-Infiltrating Lymphocytes Is Associated with the Absence of Lymph Node Metastases in Patients with Large Early-Stage Cervical Cancer Cancer Res., January 1, 2007; 67(1): 354 - 361. [Abstract] [Full Text] [PDF]
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S. H. Bae, Y.-J. Park, J.-B. Park, Y. S. Choi, M. S. Kim, and J.-I. Sin Therapeutic Synergy of Human Papillomavirus E7 Subunit Vaccines plus Cisplatin in an Animal Tumor Model: Causal Involvement of Increased Sensitivity of Cisplatin-Treated Tumors to CTL-Mediated Killing in Therapeutic Synergy Clin. Cancer Res., January 1, 2007; 13(1): 341 - 349. [Abstract] [Full Text] [PDF]
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 A. Mahdavi and B. J. Monk Vaccines Against Human Papillomavirus and Cervical Cancer: Promises and Challenges Oncologist, August 1, 2005; 10(7): 528 - 538. [Abstract] [Full Text] [PDF]
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 A. Nencioni and P. Brossart Cellular Immunotherapy with Dendritic Cells in Cancer: Current Status Stem Cells, July 1, 2004; 22(4): 501 - 513. [Abstract] [Full Text] [PDF]
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 A. S. Tsao, E. S. Kim, and W. K. Hong Chemoprevention of Cancer CA Cancer J Clin, May 1, 2004; 54(3): 150 - 180. [Abstract] [Full Text] [PDF]
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P. J. Baldwin, S. H. van der Burg, C. M. Boswell, R. Offringa, J. K. Hickling, J. Dobson, J. St. C. Roberts, J. A. Latimer, R. P. Moseley, N. Coleman, et al. Vaccinia-Expressed Human Papillomavirus 16 and 18 E6 and E7 as a Therapeutic Vaccination for Vulval and Vaginal Intraepithelial Neoplasia Clin. Cancer Res., November 1, 2003; 9(14): 5205 - 5213. [Abstract] [Full Text] [PDF]
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 K. Devaraj, M. L. Gillison, and T.-C. Wu DEVELOPMENT OF HPV VACCINES FOR HPV-ASSOCIATED HEAD AND NECK SQUAMOUS CELL CARCINOMA Critical Reviews in Oral Biology & Medicine, September 1, 2003; 14(5): 345 - 362. [Abstract] [Full Text] [PDF]
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 E. J. Davidson, P. Sehr, R. L. Faulkner, J. L. Parish, K. Gaston, R. A. Moore, M. Pawlita, H. C. Kitchener, and P. L. Stern Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia J. Gen. Virol., August 1, 2003; 84(8): 2089 - 2097. [Abstract] [Full Text] [PDF]
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 G. Parmiani, L. Pilla, C. Castelli, and L. Rivoltini Vaccination of patients with solid tumours Ann. Onc., June 1, 2003; 14(6): 817 - 824. [Abstract] [Full Text] [PDF]
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 M. Stanley Chapter 17: Genital Human Papillomavirus Infections--Current and Prospective Therapies J Natl Cancer Inst Monographs, June 1, 2003; 2003(31): 117 - 124. [Abstract] [Full Text] [PDF]
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 I. D. Davis, M. Jefford, P. Parente, and J. Cebon Rational approaches to human cancer immunotherapy J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29. [Abstract] [Full Text] [PDF]
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G. L. Eiben, M. P. Velders, H. Schreiber, M. C. Cassetti, J. K. Pullen, L. R. Smith, and W. M. Kast Establishment of an HLA-A*0201 Human Papillomavirus Type 16 Tumor Model to Determine the Efficacy of Vaccination Strategies in HLA-A*0201 Transgenic Mice Cancer Res., October 15, 2002; 62(20): 5792 - 5799. [Abstract] [Full Text] [PDF]
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 M. A. Steller Cervical Cancer Vaccines: Progress and Prospects Reproductive Sciences, September 1, 2002; 9(5): 254 - 264. [Abstract] [PDF]
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 S. Dermime, A. Armstrong, R. E Hawkins, and P. L Stern Cancer vaccines and immunotherapy Br. Med. Bull., July 1, 2002; 62(1): 149 - 162. [Abstract] [Full Text] [PDF]
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 S. Zwaveling, S. C. F. Mota, J. Nouta, M. Johnson, G. B. Lipford, R. Offringa, S. H. van der Burg, and C. J. M. Melief Established Human Papillomavirus Type 16-Expressing Tumors Are Effectively Eradicated Following Vaccination with Long Peptides J. Immunol., July 1, 2002; 169(1): 350 - 358. [Abstract] [Full Text] [PDF]
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 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini Cancer Immunotherapy With Peptide-Based Vaccines: What Have We Achieved? Where Are We Going? J Natl Cancer Inst, June 5, 2002; 94(11): 805 - 818. [Abstract] [Full Text] [PDF]
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K. L. Knutson, K. Schiffman, M. A. Cheever, and M. L. Disis Immunization of Cancer Patients with a HER-2/neu, HLA-A2 Peptide, p369-377, Results in Short-lived Peptide-specific Immunity Clin. Cancer Res., May 1, 2002; 8(5): 1014 - 1018. [Abstract] [Full Text] [PDF]
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B. Klencke, M. Matijevic, R. G. Urban, J. L. Lathey, M. L. Hedley, M. Berry, J. Thatcher, V. Weinberg, J. Wilson, T. Darragh, et al. Encapsulated Plasmid DNA Treatment for Human Papillomavirus 16-associated Anal Dysplasia: A Phase I Study of ZYC101 Clin. Cancer Res., May 1, 2002; 8(5): 1028 - 1037. [Abstract] [Full Text] [PDF]
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M. P. Velders, S. McElhiney, M. C. Cassetti, G. L. Eiben, T. Higgins, G. R. Kovacs, A. G. Elmishad, W. M. Kast, and L. R. Smith Eradication of Established Tumors by Vaccination with Venezuelan Equine Encephalitis Virus Replicon Particles Delivering Human Papillomavirus 16 E7 RNA Cancer Res., November 1, 2001; 61(21): 7861 - 7867. [Abstract] [Full Text] [PDF]
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S. Weijzen, S. C. Meredith, M. P. Velders, A. G. Elmishad, H. Schreiber, and W. M. Kast Pharmacokinetic Differences Between a T Cell-Tolerizing and a T Cell-Activating Peptide J. Immunol., June 15, 2001; 166(12): 7151 - 7157. [Abstract] [Full Text] [PDF]
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M. P. Velders, S. Weijzen, G. L. Eiben, A. G. Elmishad, P.-M. Kloetzel, T. Higgins, R. B. Ciccarelli, M. Evans, S. Man, L. Smith, et al. Defined Flanking Spacers and Enhanced Proteolysis Is Essential for Eradication of Established Tumors by an Epitope String DNA Vaccine J. Immunol., May 1, 2001; 166(9): 5366 - 5373. [Abstract] [Full Text] [PDF]
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