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Keywords:

  • immunotherapy;
  • ovarian cancer;
  • p53;
  • vaccine

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53-SLP) vaccine, twenty patients with recurrent elevation of CA-125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53-SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no ≥ grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN-γ producing p53-specific T-cell responses were induced in all patients who received all 4 immunizations as measured by IFN-γ ELISPOT. An IFN-γ secretion assay showed that vaccine-induced p53-specific T-cells were CD4+, produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53-specific response. P53-specific T-cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53-specific T-cells. As best clinical response, stable disease evaluated by CA-125 levels and CT-scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine-induced immunity. This study shows that the p53-SLP vaccine is safe, well tolerated and induces p53-specific T-cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper-1 polarization and clinical efficacy. © 2009 UICC

Ovarian cancer is the most frequent cause of death from gynecological malignancies. As a result of the absence of specific symptoms, the majority of patients present with advanced stage disease. Despite standard treatment almost all patients will relapse, with a median progression free survival of only 18 months. Notwithstanding advances in chemotherapeutic strategies in the last decades, 5-year survival remains low at ∼40%.1, 2 Immunotherapy is considered to be a promising potential novel therapeutic strategy to treat ovarian cancer as a far more favorable prognosis is observed for those of whom the tumor is strongly infiltrated by T cells.3, 4

One of the possible targets for intra-tumoral T-lymphocytes identified in ovarian cancer is the tumor-suppressor protein p53. This protein is activated upon DNA damage and arrests the cell cycle to allow for DNA repair or apoptotic cell death. Overexpression of the p53 protein is observed in 50–60% of ovarian cancers5, 6 and is associated with mutations of the p53 gene in 50% of these cases.7 Because of altered processing and expression of p53 in tumor cells when compared with normal cells, p53 could serve as a target tumor antigen for the immune system.8

A large body of evidence shows that p53 can function as a target for both the humoral and cellular arm of the immune system in cancer patients. P53-specific serum auto-antibodies (p53-Aab) can be detected in patients with many types of cancer9 and in ∼18–25% of ovarian cancer patients, indicating the presence of p53-specific T-helper (Th) cells.10–14 Indeed, circulating and tumor-infiltrating p53-specific memory T-cells could be detected and isolated from patients with ovarian cancer but not benign ovarian tumors.15 In addition, p53-specific memory T cells have been observed in patients with colorectal cancer15–17 and breast cancer,18 indicating that tumor-derived p53 can effectively be presented to the immune system and activate circulating p53-specific T-lymphocytes. Although accumulating evidence suggests that the p53-specific CD8+ T-cell repertoire is restricted by self tolerance,19, 20 the CD4+ T-cell repertoire is not affected.21 P53-specific CD4+ T cells by themselves are expected to play an important role in combating cancer because IFN-γ secreting CD4+ Th1-cells are key in orchestrating and sustaining the local immune attack by CD8+ cytotoxic T-lymphocytes (CTL) and innate immune effector cells, even in the case of MHC class II-negative cancers.22–24 Indeed, infusion of p53-specific CD4+ Th-cells supported the immune response against p53 over-expressing tumors in a murine tumor model.21, 25 Moreover, Th1-cells can activate dendritic cells (DC) that have ingested tumor material, allowing DC to launch an effective CTL response against the unique tumor antigens that are present in tumor cells.26 In view of the fact that p53 is over expressed in many types of tumors, p53-specific CD4+ T cells may act as “universal” T-helper cells for cancer and act analogous to the observed clinical remission and immunological responses to antigens other than NY-ESO-1 after the infusion of NY-ESO-1-specific T-helper cells in a patient with metastatic melanoma.27 Our analyses of the p53-specific CD4+ Th-cell repertoire in patients with cancer showed that in general these responses were weak15, 16 and failed to produce any of the Th1 or Th2 key cytokines17 or were polarized towards a non-effective Th2 response,15 suggesting that the induction of a strong p53-specific Th1-response through immunization may enhance the efficacy of the anti-tumor response.

In our current study, we used overlapping synthetic long peptides (SLP), constituting the most immunogenic part of the p53 protein,15–17 to enhance the p53-specific immune response in patients with cancer. Because of their length, SLP are predominantly taken up by professional antigen presenting cells (APC) where they are processed for presentation by both MHC class I and II molecules.28 Mixes of SLP are likely to contain multiple HLA class I and II T-cell epitopes which allows the use of this type of peptide vaccines in all patients irrespective of the type of HLA of each patient.29 The efficacy of SLP vaccines to induce effective CD4+ and CD8+ T-cell responses was demonstrated in rodents30 and patients with cervical cancer.31, 32 In parallel, injection of p53-SLP resulted in a strong p53-specific CD4+ T-cell response to several different epitopes in mice.21

To evaluate the safety and immunogenicity of the p53-SLP vaccine in patients with ovarian cancer, we performed a phase II clinical trial in patients with recurrent disease after primary standard treatment. Evaluation of clinical responses to the p53-SLP vaccine was a secondary objective of this study.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Participants

Adult patients with epithelial ovarian cancer and rising CA-125 level after prior systemic treatment (terminated ≥4 weeks) who were not (yet) eligible for renewed chemotherapy were included. Additional inclusion criteria were WHO performance status 0–2, life expectancy >3 months, and adequate bone marrow function.Exclusion criteria were high-dose immunosuppressive medication, symptoms consistent with CNS metastases, secondary malignancies and severe cardiac, neurological or psychiatric comorbidity.

Vaccine and treatment scheme

The p53-SLP vaccine consisted of 10 synthetic 25–30 amino acids long overlapping peptides, spanning amino acids 70–248 of the wt-p53 protein (Table I; patent number WO2008147186). Peptides were prepared at the GMP facility of the Department of Clinical Pharmacy and Toxicology of the LUMC.31, 32 At the day of immunization, peptides (0.3 mg/peptide) were dissolved in dimethyl sulfoxide (DMSO, final concentration 20%) admixed with 20 mM phosphate buffer (pH 7.5) and emulsified with an equal volume of Montanide ISA-51. The vaccine was administered subcutaneously 4 times with a 3-week interval. Dose and route of immunizations were chosen based on results from HPV16 E6/E7 synthetic long peptides trials.31, 32 To ascertain immunizations did not result in severe toxicity, the first 5 patients were extensively monitored up to 3 hr after each immunization. In the absence of ≥ CTC grade 3 toxicity after a minimum of 2 immunizations in these 5 patients, inclusions would continue. Study treatment was stopped in case of progressive disease necessitating other forms of anti-tumor therapy or persisting severe toxicity. Immunizations were administered between July 2006 and August 2007.

Table I. Position and Sequence of Amino Acids Used for p53 Synthetic Long Peptides
Vaccine peptidePositionAmino acid sequence
170–99APPVAPAPAAPTPAAPAPAPSWPLSSSVPS
286–115APAPSWPLSSSVPSQKTYQGSYGFRLGFLH
3102–131TYQGSYGFRLGFLHSGTAKSVTCTYSPALN
4126–155YSPALNKMFCQLAKTCPVQLWVDSTPPPGT
5142–171PVQLWVDSTPPPGTRVRAMAIYKQSQHMTE
6157–186VRAMAIYKQSQHMTEVVRRCPHHERCSDSD
7174–203RRCPHHERCSDSDGLAPPQHLIRVEGNLRV
8190–219PPQHLIRVEGNLRVEYLDDRNTFRHSVVVP
9206–235LDDRNTFRHSVVVPYEPPEVGSDCTTIHYN
10224–248EVGSDCTTIHYNYMCNSSCMGGMNR

Clinical monitoring

Toxicity was evaluated in patients who received ≥1 immunization and graded according to Common Terminology Criteria for Adverse Events v3.0 (CTC). Relationship to treatment was evaluated for all adverse events. At each visit, patients were assessed by physical examination, vital signs, complete blood count with differential and serum biochemistry. Anti-nuclear antibodies (ANA) were evaluated before the first and after the last immunization. Patients were asked to keep a diary to record temperature, adverse events and concomitant medication. Tumor response to treatment, a secondary endpoint, was evaluated according to GCIG criteria33 by combining serum CA-125 levels obtained at every visit with computerized tomography performed 6–9 weeks after the last immunization and evaluated according to RECIST criteria.34

Immunomonitoring

Lymphocytes and sera

Blood for immunological assays was obtained at each visit. Immunogenicity was evaluated in patients with ≥ 1 postimmunization sample. Sera were isolated from clotted blood and cryopreserved. PBMC isolated from heparinized blood by Ficoll-Paque density centrifugation were frozen until use in liquid nitrogen. A 6 mm skin biopsy was obtained from the last immunization site 3 weeks after the last immunization to determine presence of p53-specific T-cells. Biopsy tissue was manually cut into small pieces and the infiltrating lymphocytes were expanded by homeostatic proliferation as reported previously15, 35 and according to standard operation procedure. Briefly, to stimulate homeostatic proliferation, tissue was suspended in 4 ml of medium mix containing 10% autologous serum, 5% TCGF, 4 μl IL-15 (5 ng/ml), 8 μl Gentamicine (20 μg/ml) and 4 μl IL-7 (5 ng/ml) and distributed into 4 wells of a 48-wells plate. Depending on cell growth, samples were split into more wells and medium mix was added until enough cells were available (2–3 weeks of culture) for the detection of p53-specific T-cells by proliferation. In the absence of sufficient visible growth within the first 4 days, feeder cells (irradiated autologous PMBC) were added on day 4. Twice weekly culture medium was refreshed with new medium mix (without IL-7).

Antigens used in immunological assays

The different GMP-grade p53-SLP peptides were divided into 4 pools: p1–2 (aa 70–115), p3–4 (aa 102–155), p5–7 (aa 142–203) and p8–10 (aa 190–248). Memory recall mix (MRM), a mixture of tetanus toxoid (150 limus flocculentius/ml; RIVM, Bilthoven, the Netherlands), M. tuberculosis sonicate (2.5 μg/ml; generously donated by Dr P Klatser, KIT, Netherlands) and C. albicans (0,005% HAL Allergenen Lab, Haarlem, the Netherlands) was used to control the capacity of PBMC to proliferate in response to typical recall antigens.36

IFN-γ elispot assay

The IFN-γ ELISPOT assay optimized to measure p53-specific T-cell responses was performed according to standard operation procedures as described previously.15, 32, 36 Briefly, 1–2 million PBMC were stimulated with the indicated vaccine peptide pools (10 μg/peptide/ml) or MRM (1:50) and cultured in Iscove's medium (Gibco) containing 10% human AB serum (sigma), penicillin, streptomycin and β-mercapto-ethanol for 4 days. Then cells were counted and seeded in quadruplicates in an IFN-γ ELISPOT plate at 100,000 cells per well. After 16 hr, the plates were developed and analyzed by ELI.SCAN ELISPOT scanner (AELVIS GmbH, Hannover, Germany). A response was considered p53-specific if {(mean number of spots in experimental wells) − (mean number of spots in medium + 2 × SD)} ≥ 10 spots/105 PBMC. A vaccine-induced response was defined as a p53-specific response which exceeded the pre-existing immune response at least three-fold.32

Proliferation assays

Freshly isolated PBMC were stimulated with vaccine and nonvaccine peptide pools (10 μg/peptide/ml) or MRM (1:156) in a 6-day lymphocyte stimulation test (LST) according to standard operation procedures as previously described.32 Skin-biopsy derived lymphocytes were analyzed for the presence of p53-specific T cells by stimulation with autologous monocytes pulsed overnight with the indicated vaccine peptides in a standard 3 day proliferation assay.35 Supernatants, isolated at day 5 (PBMC) or day 2 (skin biopsy derived lymphocytes) of the proliferation assay were cryopreserved. A p53-specific response was defined as 3H-thymidine incorporation >1000 counts/min and a stimulation index ≥ 3 (SI = mean of p53-induced proliferation/mean of medium control).15 A vaccine-induced response was defined as a p53-specific response with proliferation after 4 immunizations/proliferation before immunization ≥ 2.

IFN-γ secretion assay

To analyze if p53-specific responses were CD4 or CD8 mediated, PBMC were stimulated for 4 days with the vaccine peptides or medium as described above for the ELISPOT assay and analyzed by IFN-γ secretion assay (Miltenyi Biotec, Utrecht, The Netherlands) according to manufacturer's instructions. Briefly, stimulated PBMC were harvested, washed with ice-cold buffer (PBS/BSA 0.5%/EDTA 2 mM) and incubated in 90 μl cold medium (per 106 cells) containing anti-IFN-γ antibody conjugated to cell surface specific monoclonal antibodies (10 μl/106 cells) for 5 min on ice. Next, PBMC were 10× diluted in warm medium and incubated for 45 min at 37°C under slow continuous rotation. After incubation, PBMC were washed and incubated in 90 μl ice-cold buffer containing 10 μl anti-IFN-γ (per 106 cells), anti-CD4 APC (IQproducts, Groningen, the Netherlands) and anti-CD8 PECy5 (IQ Products, Groningen, the Netherlands). After 10 min incubation on ice, PBMC were washed, suspended in 300 μl ice-cold buffer and analyzed by flow cytometry for the presence of CD4+IFN-γ+ or CD8+IFN-γ+ T-cells.

IFN-γ production by CD4+ or CD8+ T-cells was considered to be p53-specific if after stimulation with vaccine peptides the percentage of IFN-γ producing CD4+ or CD8+ T-cells within the CD4+ or CD8+ T-cell population was at least twice as high as the percentage of nonstimulated (medium only) IFN-γ producing T-cells. A vaccine-induced response was defined as a p53-specific response after immunization that was ≥ 3 fold higher than the percentageof IFN-γ+ T-cells in the preimmunization sample.

Cytokine bead array

Production of IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α was evaluated in supernatants of proliferation assays by cytokine bead array (LINCOplex kit, Linco Research, St. Charles, MO) as described earlier.15 P53-specific cytokine production was defined as concentration of cytokine ≥ 2 medium control and if concentration ≥ 100 pg/ml (IFN-γ), or ≥ 10 pg/ml (other cytokines). A vaccine-induced response was defined as a p53-specific postimmunization concentration ≥ 2 preimmunization concentration (PBMC only).

Immunohistochemistry

Primary tumors were evaluated for p53 expression (DO-7, 1:1000 in PBS 1% BSA, DAKO).15 Tumors with > 50% moderate or strong immunostaining were considered to have p53-overexpression.6, 37

Flow cytometry

PBMC were evaluated for CD3, CD4, CD8, CD19, CD56 (IQ Products, Groningen, the Netherlands) and FoxP3 (eBioscience, San Diego, CA) by flow cytometry (FACSCalibur from BD Biosciences, Erembodegem, Belgium) according to manufacturer's instructions. Skin biopsy derived lymphocytes were analyzed for CD3, CD4, CD8, and if possible Foxp3.

Ethics statement

The study protocol was approved by the Medical Ethical Committee of the University Medical Center Groningen and conducted in adherence with the principles of the Declaration of Helsinki. All patients gave written informed consent. An independent agency (Trial Coordination Center, Groningen, The Netherlands) was contracted to monitor the study and adherence to GCP principles.

Statistical analysis

It was calculated that to ascertain a 95% probability of an immunologic response rate of at least 15%, minimally 19 patients would have to be immunized. Differences between pre- and postimmunization were evaluated using a t-test for paired comparisons or a Wilcoxon's signed-ranks test. Differences in response at different time points for normally distributed quantitative variables were evaluated by repeated measures analysis with linear mixed modeling, or Mantel-Haenzel (MH) statistics. For qualitative variables, overall differences were evaluated using McNemar's test or MH-strategy. MH-statistics were used to determine if average responses differed at the different time points (QSMH). Statistical significance was defined as p < 0.05. Statistical Analysis System version 9.1 (SAS Institute, Cary, NC) was used for all analyses.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

P53-SLP vaccine is safe and well tolerated

Informed consent was obtained from 23 patients but 3 patients failed screening (P07, P10 and P16). Because of the rapidly progressive disease, 2 patients received 2 immunizations only (P04 and P12). All other patients (N = 18) received 4 immunizations. P53-overexpression in the primary tumor was demonstrated by immunohistochemical analysis in 50% (10/20) of patients (Table II). As no vaccine-related CTC grade 3 or 4 adverse events were observed after a minimum of 2 immunizations in the first 5 patients, it was considered safe to continue inclusions. Similarly, no severe vaccine-related adverse events occurred in the subsequently included and immunized patients (Table III). No major vaccine-induced alterations were observed in serum biochemistry and complete blood count. Mild to moderate inflammatory symptoms at the injection site occurred in nearly all patients; redness and swelling lasted 7–8 days on average, and pain and itch 3–4 days on average. Activation of local inflammatory symptoms at prior injection sites was seen in 60% of patients after subsequent immunizations. We did not find evidence of vaccine-induced auto-immunity, assessed by clinical symptoms and levels of serum ANA levels (data not shown). P53-autoantibodies, present in 40% (8/20) of the patients before immunization, were associated with p53-overexpression in the primary tumor (p = 0.023; Fischer's exact test). After 1 or more immunizations, p53-autoantibodies were present in 45% (9/20) of patients. Flow-cytometric analysis of the major lymphocyte subpopulations (T-cells, B-cells, NK-cells) revealed that these remained relatively stable during immunization albeit that the percentage of CD3+ T-cells was somewhat lower after 2 (mean ± SEM: 63.2 ± 3.4) and 3 immunizations (65.9 ± 2.4) when compared with before immunization (70.4 ± 1.7). Within the CD3+ T-cell population, the percentages of CD4+, CD8+ T-cells and CD4+FoxP3+ T-cells remained constant at 40%, 25% and 7%, respectively (data not shown).

Table II. Patient Characteristics
PatientAgeFIGO stageHistologyGradeResidual disease >2cm1Prior chemotherapyCT scan at inclusion2p53-overexpression in primary tumor3HLA-DR genotype
  • FIGO = International Federation of Gynecology and Obstetrics.

  • NED = no evidence of disease.

  • 1

    residual disease after primary surgery.

  • 2

    ED = evidence of disease.

  • 3

    P53 expression in the primary tumor analyzed by immunohistochemistry using the p53-specific antibody DO-7.

P0143IIIcMucinous3NoSecond lineED+DRB1*01
P0250IIIcClear cell3YesSecond lineED+DRB1*13,*15;DRB3; DRB5
P0346IIIcSerous3YesFirst lineEDDRB1*01,*03;DRB3
P0450IIIcEndometrioid3NoSecond lineEDDRB1*03,*07;DRB3;DRB4
P0547IVSerous3YesFirst lineNED+DRB1*04,*15;DRB4;DRB5
P0668IIcEndometrioid3UnknownFirst lineED+DRB1*13,*14;DRB3
P0851IIIcSerous2NoSecond lineNEDDRB1*03,*11;DRB3
P0966IIIcClear cell3YesFirst lineED+DRB1*01,*13;DRB3
P1147IIIcSerous1NoFirst lineEDDRB1*10,*15;DRB5
P1244IcClear cell2NoSecond lineEDnot evaluated
P1369IIISerous3YesSecond lineEDDRB1*08,*13;DRB3
P1467IIIbSerous3NoFourth lineEDDRB1*01,*04;DRB4
P1556IIIbSerous2NoFirst lineNED+DRB1*04,*15;DRB4; DRB5
P1743IIIcEndometrioid3YesFirst lineNEDDRB1*04,*07;DRB4
P1850IIIcSerous2UnknownFirst lineED+DRB1*04,*11; DRB3; DRB4
P1955IIIcSerous3UnknownFirst lineNED+DRB1*11,*13;DRB3
P2051IVSerous3NoSecond lineED+DRB1*03,*04; DRB3; DRB4
P2158IIIcSerous1NoFirst lineEDDRB1*01,*11;DRB3
P2248IIIcSerous2NoFirst lineEDDRB1*03,*13; DRB3
P2352IIIcSerous3NoFirst lineEDDRB1*11,*11; DRB3
Table III. Number of Adverse Events and Number of Patients With Adverse Events
CTC grade1Number of events (number of patients)
12345
  • 1

    Adverse events are graded according to CTC criteria v3.0.

  • 2

    Related to disease progression.

ALAT2 (2)
ASAT3 (3)
LDH4 (4)
Anemia5 (5)
Leucocytes8 (8)1 (1)
Platelets5 (5)
Hematoma (not injection site)2 (2)1 (1)
Cardiovascular8 (5)1 (1)
Thrombosis/Embolism1(1)2
Peripheral Edema2 (2)1 (1)
Pulmonary1 (1)1 (1)
Dyspnea4 (3)
Constitutional symptoms12 (8)4 (3)
Fatigue2 (2)3 (1)
Diarrhea1 (1)
Nausea3 (3)-
Vomiting1 (1)
Musculoskeletal pain8 (6)9 (5)
Abdominal pain9 (7)6 (3)1 (1)2
Dermatology8 (6)
Hernia Cicatricialis2 (2)
Neurology4 (3)1 (1)
Headache18 (6)1 (1)
Anxiety2 (1)
Conjunctivitis1 (1)1 (1)

IFN-γ producing T-cells are induced by the p53-SLP vaccine in all patients

We analyzed p53-specific T-cell responses after every consecutive immunization by IFN-γ ELISPOT. Before immunization, responses against peptides included in the vaccine were present in patients P11 (all vaccine peptide pools), P12 (vaccine peptides 3–4) and P19 (vaccine peptides 1–2, 3–4 and 5–7). In all patients who completed the immunization scheme (N = 18), vaccine-induced IFN-γ producing p53-specific T-cells could be detected at 2 or more time points (Table IV). A number, but not all, of the pre-existing p53-specific responses were boosted (Table IV). The strength of the vaccine-induced p53-specific T-cell response was at its peak in the circulation after 1 immunization (median number of cumulative spots against all vaccine peptide pools 276, interquartile range (IQR) 91-400) after which the number of circulating IFN-γ producing p53-specific T-cells decreased after each subsequent immunization (median (IQR): 214 (63–266), 169.0 (65–188), 93(28–122) respectively after 2, 3 and 4 immunizations). The responsiveness to the mix of recall antigens (MRM) remained stable over time (p = 0.625; Wilcoxon's signed rank test), indicating that the change in p53-reactivity was the result of p53-specific immunization.

Table IV. Vaccine-Induced P53-Specific Immune Responses in PBMC of Ovarian Cancer Patients Immunized With the P53-SLP Vaccine as Analyzed by IFN-γ Elispot
Patient1After one vaccination (I)After two vaccinations (II)After three vaccinations (III)After four vaccinations (IV)
vac p1–p22vac p3–p4vac p5–p7vac p8–p10vac p1–p2vac p3–p4vac p5–p7vac p8–p10vac p1–p2vac p3–p4vac p5–p7vac p8–p10vac p1–p2vac p3–p4vac p5–p7vac p8–p10
  • 1

    Patients analyzed for p53-specific responses before and after every immunization (time points I-IV) by IFN-γ ELISPOT.

  • 2

    The pool of p53 vaccine peptides used to stimulate patient-derived PBMC in vitro for 4 days.

  • 3

    Only vaccine-induced p53-specific responses are shown (see definition in Material and methods). Responses are depicted as number of specific spots per 105 PBMC (mean of experimental wells – (mean + 2xSD) of medium control). Responses to medium stimulated wells were low (median 3.75, IQR 1.5–8.75 spots per 105 PBMC). Pre-existing responses were present in P11 against p1–2, p3–4, p5–7, p8–10 (19, 12, 11 and 30 spots respectively); P12 against p3–4 (25 spots); and P19 against p1–2, p3–4, p5–7 (19, 19 and 11 spots respectively). – = no vaccine-induced p53-specific response. na = PBMC were not available.

P0162331710614332056
P0228216627
P0352371662915814358132152057
P04nananananananana
P051919112918
P0612472221
P081101312412207613020030342113442195932156
P093138551018201412
P1171120138783528
P12266773nananananananananananana
P13656581378313314416125934921
P1460221248
P1518222014
P176346215220
P1888106120416145604113
P1918394756023
P203411281053
P21221191001788881323338321216
P22148025736721572012957721734564
P2350106213520552213

The p53-SLP vaccine induces proliferating p53-specific T-cells

In addition, the capacity of p53-specific T-cells to proliferate upon antigenic stimulation was analyzed before and after 4 immunizations. Pre-existing p53-SLP vaccine-specific proliferative responses were detected in patients P18 (vaccine peptides 5–7, SI 4.4) and P19 (vaccine peptides 1–2 (SI 3.5), 5–7 (SI 5.9) and 8–10 (SI 3.7)). After 4 immunizations, vaccine-induced p53-specific responses against the vaccine peptides were observed in 82.4% (14/17) of patients (Fig. 1b). The pre-existing p53-specific proliferative responses present in P18 and P19 were not significantly boosted by immunization. Similar to the results of the IFN-γ ELISPOT the p53-specific proliferative responses against all, but vaccine peptides 1–2, were higher after 4 immunizations than before immunization (p < 0.02; Wilcoxon's signed rank test). Furthermore, all patients except for 2 (P2 and P17) responded to at least 2 different peptide pools. No differences were detected in the proliferative response to MRM (p = 0.927; Wilcoxon's signed rank test).

thumbnail image

Figure 1. P53-specific responses in ovarian cancer patients immunized with the p53-SLP vaccine. (a) Boxplots comparing responses to vaccine peptide pools as analyzed by IFN-γ ELISPOT using PBMC after I-IV immunizations. The number of p53-specific IFN-γ producing cells (per 105 PBMC) was calculated by subtracting the mean number of spots+2xSD of the medium from the mean number of spots of the experimental wells (vertical axis). (b/c) P-53 specific responses to vaccine peptides as measured by proliferation assay using PBMC obtained before immunization and after 4 immunizations (b) or skin biopsy derived lymphocytes from the last injection site (c). The vertical axis represents the stimulation index calculated as the mean 3H-thymidine incorporation of the experimental wells divided by the mean of the medium control.

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Differences in immunogenicity and magnitude of responses to p53-SLP

To make a distinction in the immunogenicity of the injected p53-peptides, we compared the strength of the p53-specific T-cell response to each of the 4 different peptide pools (vaccine peptides 1–2, 3–4, 5–7 and 8–10) as measured by IFN-γ ELISPOT (Fig. 1a). The strength of the responses against p53 vaccine peptides 3–4, 5–7 and 8–10 were at all time points higher than responses against vaccine peptides 1–2 (p < 0.01; Wilcoxon's signed rank test), suggesting that the immunogenicity of the first pool of peptidesis low within this patient population. After 1, 2 and 3 immunizations, responses against p53 vaccine peptides 8–10 were also higher than responses against vaccine peptides 3–4 (p < 0.02; Wilcoxon's signed rank test). Similar differences were observed in the strength of proliferation induced by the 4 different pools of peptides (Fig. 1b).

Vaccine-induced p53-specific T-cell immunity is mediated predominantly by CD4+ type 2 T-cells

To analyze if the vaccine-induced p53-specific T-cell responses were CD4- or CD8-mediated PBMC of 5 patients (P01, P08, P21, P22, P23), selected on basis of their strong reactivity in the IFN-γ ELISPOT assay, were stimulated for 4 days with the pools of vaccine peptides that induced high IFN-γ responses in the ELISPOT and then analyzed by an IFN-γ secretion assay using flow cytometry. Similar to the results obtained by IFN-γ ELISPOT, we did not observe an IFN-γ associated p53-specific response in the preimmunizationsamples of these patients, whereas vaccine-induced IFN-γ producing T-cell responses were detected in all patients after immunization. All the p53-specific IFN-γ responses were mediated by CD4+ T-cells (Fig. 2a; Table V). No vaccine-induced CD8+ T-cell responses could be detected.

thumbnail image

Figure 2. Phenotyping of p53-specific responses induced by the P53-SLP vaccine. (a) An illustrative example (P23) of induction of IFN-γ secreting CD4+ T-cells, but not CD8+ T-cells after immunization as analyzed by IFN-γ secretion assay. (b) Th1/Th2 cytokine production in PBMC and skin biopsy derived lymphocytes after 4 immunizations with the p53-SLP vaccine. Cytokines were measured in supernatants of cultures with a vaccine-induced (PBMC) or p53-specific response (skin biopsies) as measured by proliferation assay. Vaccine-induced p53-specific responses (PBMC) or p53-specific responses (skin biopsies) in proliferation assays are represented by a box. Vaccine-induced increase in production of a cytokine (PBMC) or positive production of a cytokine (skin biopsy) is indicated by a filling (white: <2× cut off value; yellow: 2–5× cut-off; orange: 5–15× cut-off; red: 15–50× cut-off; and dark red: >50× higher than cut-off). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Table V. P53-Specific IFN-γ Production by CD4+ and CD8+ T-Cells as Analyzed by IFN-γ Secretion Assay
PatientVac. pepIFN-γ producing CD4+ T-cells (%)IFN-γ producing CD8+ T-cells (%)
Pre-immun.Post-immun.Pre-immun.Post-immun.
  1. The percentage of IFN-γ producing T-cells before immunization (pre-immun.) and after immunization (post-immun.) upon stimulation with the p53 vaccine peptide pools is shown. Responses are depicted as the percentage of IFN-γ production by the peptide-stimulated cells – the medium control (median 0.115, IQR 0.100–0.205). Vaccine-induced p53-specific T-cell responses are shown in bold (see definition in Material and methods).

P01vac p8–p10–0.030.360.050.10
P08vac p5–p7–0.080.03–0.07–0.17
 vac p8–p100.050.50–0.22–0.11
P21vac p3–p4–0.010.380.150.06
 vac p8–p10–0.010.380.000.05
P22vac p5–p7–0.010.44–0.090.02
 vac p8–p100.000.66–0.080.06
P23vac p5–p7–0.020.290.040.03

To determine the polarization of the vaccine-induced T-cell response, the p53-specific production of both Th1 (IL-2, IFN-γ and TNF-α) and Th2 cytokines (IL-4, IL-5 and IL-10) was measured in the supernatants isolated from the proliferation assays of a subset of 8 patients of whom both PBMC and lymphocytes cultured from skin biopsies (see below) displayed a p53-specific proliferative response (Fig. 2b). Analyses of the culture supernatants of these proliferation assays revealed that p53-specific proliferation coincided with the production of both Th1 and Th2 cytokines. In PBMC, vaccine-induced production of both Th1 cytokines (median [range] IL-2: 19.7 [12.1–27.3] pg/ml; IFN-γ 193 [123–486] pg/ml; TNF-α 109 [41.8–1255] pg/ml) and Th2 cytokines was detected (IL-5 68.6 [14.4–224] pg/ml; IL-10 145 [45.8–347] pg/ml) as determined by cytokine bead array. IL-5 and IL-10 were more frequently produced than IL-2 and IFN-γ. In lymphocytes cultured from skin biopsies, also vaccine-induced production of both Th1 cytokines (IL-2: 275 [28.3–708] pg/ml; IFN-γ 262 [111–461] pg/ml; TNF-α 426 [88.0–676] pg/ml) and Th2 cytokines (IL-4: 653 [45.4–22924] pg/ml; IL-5 1606 [35.7–10242] pg/ml; IL-10 1167 [62.0–3555] pg/ml) was found. Based on predefined cut-offs, all analyzed patients showed a predominance in the p53-specific Th2 response, as the frequency of Th2 responses and the amounts of Th2 cytokines produced are higher than Th1 cytokines (Fig. 2b).

Vaccine-induced p53-specific T-cells migrate into the immunization sites

To analyze the capacity of p53-specific T-cells to migrate to sites where p53 antigen is presented, we cultured lymphocytes from skin biopsies taken at the fourth injection site (n = 17) using a successful protocol to obtain antigen-specific T-cells, which is based on the induction of homeostatic proliferation and as such does not strongly affect the phenotype of the infiltrating lymphocytes, as published previously.15, 35, 38 Only in 2 cases (P15 and P20) we were not able to obtain sufficient numbers of lymphocytes from the skin biopsy to analyze the p53-specific T-cell response by proliferation assay. The median yield of cells was 1.25 × 106 (range 0.06 × 106–11.1 × 106) after 2–3 weeks of culture. Flow cytometric-assisted phenotyping of the infiltrating lymphocytes showed a preponderance of CD3+ T-cells in skin biopsy derived lymphocytes (mean ± SEM: 72.5% ± 7.8), of which 58.4% ± 7.2 were CD4+, 12.6% ± 2.4 were CD8+ and the percentage of CD4+Foxp3+ cells was 2.7% ± 0.7. Vaccine-site infiltrating p53-specific T-cells were detected in 52.9% (9/17) of the tested patients (Fig. 1c). The p53-specific responses inskin-derived lymphocytes and PBMC were not always directed against the same epitopes (Fig. 2b).

Tumor responses

Two patients received only 2 immunizations due to rapidly progressive disease. All other patients were evaluated for tumor response after the fourth immunization. Two patients (10%) had stable disease as evaluated by CA-125 and computerized tomography (P17, P23). In both patients, vaccine-induced p53-specific responses were present. The remainder of patients (18/20; 90%) had clinical, biochemical and/or radiographic evidence of progressive disease.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This phase II clinical trial shows that a synthetic p53 long peptide (p53-SLP) vaccine, constituting the middle part of the p53 protein, emulsified with Montanide ISA-51 is safe, well-tolerated and capable of inducing p53-specific CD4+ T-cell responses in 90% of ovarian cancer patients as detected on at least 2 different time points. The number of circulating IFN-γ-producing p53-specific T-cells peaked after the first stimulation and then subsided, but was always higher than before immunization (Fig. 1). A similar decrease in vaccine-induced p53-specific T-cell reactivity after multiple immunizations has also been observed in patients with breast cancer who were vaccinated with p53-peptide pulsed dendritic cells.39 Formally, it cannot be excluded that the constant release of p53 peptides from the immunization sites may have resulted in the partial induction of hypo-responsiveness or apoptosis of p53-specific T-cells, but the isolation of poly-functional p53-specific T-cells from the immunization sites argues against this. In addition, an increase in regulatory T-cell activity due to repeated immunizations cannot be ruled out. This would imply that immunization resulted in the induction of p53-specific regulatory T-cells. Despite the fact that immunization with HPV16 peptides induced HPV16-specific regulatory T-cells in patients with cervical cancer,32 p53-specific regulatory T-cells were not detected following p53-specific immunization of patients with colorectal cancer.40 Moreover, the number of CD4+Foxp3+ (regulatory) T-cells remained constant before and after immunization (not shown). It is more likely that the decrease in the number of circulating p53-specific T-cells is due to the emigration of vaccine-activated p53-specific T-cells from the blood into the extra-lymphoid organs41 as illustrated by the detection of p53-specific T cells in the immunization sites (Figs. 1 and 2).

The p53-SLP-vaccine induced p53-specific T-cells against multiple epitopes in the majority of patients (Table IV, Figs. 1 and 2), but responses to the first part of the vaccine (p1–p2) were infrequently observed. This is reminiscent of our prior studies examining spontaneous response to p53. In these studies, 3 large pools of peptides were used and the amino acid sequence covered by p1–p2 was present in the first pool of peptides. This first pool of peptides was only sporadically recognized, whereas the second pool of p53 peptides (covering p3–p10) was predominantly recognized,15, 16 suggesting that peptides 1 and 2 of the p53-SLP do not encode HLA class II epitopes for the particular types of MHC class II molecules within this group of Dutch patients. Notably, scrutiny of vaccine-induced immunity vs. HLA class II (Table II) did not reveal any correlation between responsiveness and a particular HLA class II type indicating that the responses are likely to be restricted by multiple MHC class II molecules.

Although p53-SLP immunization resulted in the expansion of p53-specific Th1 and Th2 CD4+ T-cell responses, the production of Th2 cytokines dominated both in frequency and amount (Fig. 2). Notably, Th2 cytokines were also the main product of the spontaneous immune response against p53 in patients with ovarian cancer.15 Likewise, colorectal cancer patients with tumor-induced17 or p53-SLP vaccine-induced p53-specific T-cells40 produced only low amounts of IFN-γ or none of the key signature cytokines of Th1 or Th2 cells at all. Together these 2 clinical studies suggest that the current p53-SLP vaccine is capable of activating or reinforcing the same type of T-cell response as the one that spontaneously occurs in patients, albeit the vaccine-induced response is stronger and more pronounced. Based on our study in colorectal cancer, we reasoned that a prolonged immunization scheme (i.e. multiple instead of 2 injections) may result in a stronger polarized Th1 response.40 Our current trial, however, in which patients were vaccinated 4 times, reveals that this is not the case and indicates that the vaccine needs to be supplemented with strong Th1 polarizing agents. Ligands of Toll-like receptors (TLR) have been shown to act as strong Th1/CTL immunity polarizing adjuvants for vaccines (reviewed in28, 29). A combination of such an adjuvant with the p53-SLP vaccine may result in the induction of an effective CD4+ Th1 response and p53-specific CTL as well. Here, we did not detect CD8+ T-cell reactivity and this was not unexpected as the p53-specific CD8+ T cell, but not the CD4+ T cell repertoire is severely restricted by self tolerance and might only consist of lower affinity p53-specific CD8+ T cells.19, 21 However, p53-specific CD8+ T cells have been identified in cancer patients,42, 43 and we can thus not rule out the possibility that with the help of an effective p53-specific Th1 response also p53-specific CD8+ T-cells are activated. In favor of this are our observations that only those cervical cancer patients who were able to mount an E7-specific Th1 response upon HPV16-SLP immunization were also able to mount E7-specific CD8+ T-cell immunity.32 A trial to improve Th1 polarization has been started in patients with colorectal cancer.

We observed stable disease in 2 patients with vaccine induced p53-specific T-cell responses, however, disease stabilization could not be attributed to vaccine-induced immunity and is more likely the natural course of disease in these patients. This raises the issue at which point during the course of disease immunotherapy is most effective. In ovarian cancer, clinical response rates up to 80% are obtained with first-line treatment1 and at this point during disease pre-existing p53-specific T-cell responses are present in about half of the patients.15 However, as in the majority of patients disease will recur,44 this poses an excellent niche for immunotherapy in the adjuvant setting. Perhaps the clinical response rate would have been better when we administered the vaccine in a truly adjuvant setting, instead of in patients with recurrent, yet limited disease, who had a strikingly lower detection rate of pre-existing p53-specific T-cell immunity than expected (20%; this study). Alternatively, one could combine immunotherapy with chemotherapy. Lung cancer patients vaccinated with an adenoviral vector expressing p53 displayed better clinical responses to chemotherapy administered after immunotherapy when they had also mounted a vaccine-induced p53-specific T-cell response, hinting at a possible benefit of combining chemotherapy and immunotherapy.45 As the clinical activity of the p53-SLP vaccine can also be impeded by the incapacity of T-cells to infiltrate tumor tissue as is found in a substantial number of patients3, 46 p53-SLP immunization in combination with an endothelin B receptor (ETbR) antagonist may augment T-cell homing to tumors and reduce tumor growth similar to that found in previously ineffective immunotherapy models.46 Another problem which may inhibit anti-tumor responses and obstruct clinical efficacy are ovarian cancer infiltrating regulatory T-cells.47 Although the number of circulating CD4+FoxP3+ (regulatory) T-cells in our patient group (7.0%) as well as in another study of ovarian cancer patients48 is relatively low and comparable to what is found in healthy subjects,48 their presence and recruitment to the tumor fosters tolerance to the tumor.47 In cancer patients, low doses of cyclophosphamidewere shown to selectively deplete regulatory T cells49 and enhance the induction of antigen-specific T-cells as well as increase survival when combined with immunotherapy.50 We have now started a new clinical trial in which p53-SLP immunization is combined with cyclophosphamide to test whether this increases immunity and clinical activity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank all patients who participated in this study, Klaske ten Hoor for assistance with immunohistochemistry, and the Trial Coordination Center of the UMCG for study monitoring and statistical analyses.

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  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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