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Vaccination with p53 peptide-pulsed dendritic cells is associated with disease stabilization in patients with p53 expressing advanced breast cancer; monitoring ...
Cancer Immunol Immunother DOI 10.1007/s00262-007-0293-4

O RI G I NAL ART I C LE

Vaccination with p53 peptide-pulsed dendritic cells is associated with disease stabilization in patients with p53 expressing advanced breast cancer; monitoring of serum YKL-40 and IL-6 as response biomarkers Inge Marie Svane · Anders E. Pedersen · Julia S. Johansen · Hans E. Johnsen · Dorte Nielsen · Claus Kamby · Svend Ottesen · Eva Balslev · Eva Gaarsdal · Kirsten Nikolajsen · Mogens H. Claesson Received: 13 November 2006 / Accepted: 13 January 2007 © Springer-Verlag 2007

Abstract p53 mutations are found in up to 30% of breast cancers and peptides derived from overexpressed p53 protein are presented by class I HLA molecules and may act as tumor-associated epitopes in cancer vaccines. A dendritic cell (DC) based p53 targeting vaccine was analyzed in HLA-A2+ patients with progressive advanced breast cancer. DCs were loaded with 3 wild-type and 3 P2 anchor modiWed HLA-A2 binding p53 peptides. Patients received up to 10 sc vaccinations with 5 £ 106 p53-peptide loaded DC with 1– 2 weeks interval. Concomitantly, 6 MIU/m2 interleukine-2 was administered sc. Results from a phase II

I. M. Svane (&) · D. Nielsen · C. Kamby Department of Oncology, Copenhagen University Hospital, Herlev, Denmark e-mail: [email protected] I. M. Svane · H. E. Johnsen · E. Gaarsdal · K. Nikolajsen Center for Cancer Immune Therapy, Department of Hematology, Copenhagen University Hospital, Herlev, Denmark J. S. Johansen Department of Rheumatology, Copenhagen University Hospital, Herlev, Denmark E. Balslev Department of Pathology, Copenhagen University Hospital, Herlev, Denmark A. E. Pedersen · M. H. Claesson Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Copenhagen, Denmark S. Ottesen Department of Oncology, Roskilde Hospital, Roskilde, Denmark

trial including 26 patients with veriWed progressive breast cancer are presented. Seven patients discontinued treatment after only 2–3 vaccination weeks due to rapid disease progression or death. Nineteen patients were available for Wrst evaluation after 6 vaccinations; 8/19 evaluable patients attained stable disease (SD) or minor regression while 11/19 patients had progressive disease (PD), indicating an eVect of p53-speciWc immune therapy. This was supported by: (1) a positive correlation between p53 expression of tumor and observed SD, (2) therapy induced p53 speciWc T cells in 4/7 patients with SD but only in 2/9 patients with PD, and (3) signiWcant response associated changes in serum YKL-40 and IL-6 levels identifying these biomarkers as possible candidates for monitoring of response in connection with DC based cancer immunotherapy. In conclusion, a signiWcant fraction of breast cancer patients obtained SD during p53-targeting DC therapy. Data encourage initiation of a randomized trial in p53 positive patients evaluating the impact on progression free survival. Keywords Dendritic cells · Breast cancer · p53 peptides · Immunotherapy · Biomarkers

Introduction Dendritic cells (DCs) are utilized as natural adjuvant in cancer vaccines as they are extremely eYcient antigenpresenting cells and potent stimulators of memory-B and -T cells as well as resting or naïve T cells [1–4]. Thus, several vaccines have been constructed comprising peptide-pulsed DCs based on prior identiWcation of cytotoxic T-cell (CTL)-deWned synthetic peptide

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Cancer Immunol Immunother

epitopes and the presenting major histocompatibility complex (MHC) molecules. DCs for antigen loading can be generated in clinical scale from blood leukapheresis products followed by in vitro manipulation and growth factor stimulation [5, 6], and thereafter loaded with the synthetic MHC binding peptides known to stimulate CTLs. p53 is an obvious target for cancer vaccination therapy [7]. Mutations that inactivate the p53 protein or members of the p53 pathways are the most common genetic alterations found in human cancers, and mutations in the p53 gene are found in approximately 50% of human tumors including about 30% of breast carcinomas associated with a poor prognosis following conventional therapy [8–10]. More than 85% of p53 mutations result in single amino-acid substitutions that lead to the synthesis of stable, inactive p53 protein, which accumulate in the nucleus and cytosol of the tumor cells. Tumor cells expressing high cytosolic levels of p53 are potential targets for recognition, and lysis by p53 speciWc CTLs as peptides derived from accumulated p53 protein are presented by class I MHC molecules on the cell surface, thereby, acting as tumor-associated epitopes. In accordance with this, CTLs with HLA class I restricted speciWcity for wild-type p53 peptides have been observed in peripheral blood of cancer patients suggesting immune surveillance of tumors expressing these peptides [11–14]. Due to the diversity of p53 mutations, peptides representing wild-type sequences are preferable as basis for a broad-spectrum p53-targeting cancer vaccine. We have established a p53-targeting vaccine using autologous DCs pulsed with six HLA-A2 binding p53 peptides for treatment of metastatic breast cancer. Three of the peptides represent wild-type sequences and were chosen to avoid the need for individualized vaccine targets [15, 16]. The other three p53 peptides included a single P2 anchor amino acid modiWcation to increase HLA-A2 binding capacity and induction of p53-speciWc CTLs [17]. Preclinical studies have shown that these wild-type p53 derived HLA-A2 binding peptides are able to activate human T cells and generate eVector T cells, which are cytotoxic to human HLAA2+, p53+ tumor cells [18, 19]. In a recently published phase I trial, the safety of the p53-DC vaccine was analyzed in six patients with progressive breast cancer [20]. Induction of p53 speciWc T cells and indications of clinical eVect were observed in some of the patients without any toxicity of signiWcance. Application of response biomarkers are important for identiWcation of patients responding to treatment. The cytokine interleukine-6 (IL-6) and the protein YKL-40 could be potential candidates. They are nor-

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mally produced by immune cells; T-and B-cells (IL-6) [21], macrophages, and granulocytes (YKL-40) [22]. However, they are also found to be constitutively expressed by a variety of tumor cells among these breast carcinoma cells [22, 23]. IL-6 can inhibit DC diVerentiation [24], and both IL-6 and YKL-40 are able to promote tumor growth by up-regulating antiapoptotic proteins in the tumor cells and remodeling of the extracellular matrix during cell proliferation and invasiveness [25, 26]. Studies in several types of cancer patients including breast cancer have shown a negative impact on prognosis of elevated serum levels of YKL-40 and IL-6 [22, 27, 28]. However, knowledge of the value of serum IL-6 as predictive and response biomarker is very sparse and the applicability of monitoring serum YKL-40 during anticancer treatment for evaluation of response is previously not described. In this phase II trial, autologous DCs loaded with a cocktail of 3 wild-type and 3 modiWed p53-peptides are analyzed in 26 HLA-A2+ patients with progressive metastatic breast cancer. Patients are evaluated for toxicity, clinical response, and induction of p53 speciWc T-cell immunity. Furthermore, measurement of selected serum biomarkers during treatment is carried out to evaluate their feasibility as indicators of clinical response.

Methods Patients and eligibility criteria Between June 2002 and June 2004, 26 patients with progressive metastatic breast cancer were enrolled in this phase II trial, in agreement with the inclusion criteria: (1) histologically proven metastatic or locally advanced carcinoma of the breast, (2) progressive disease and no standard systemic treatment indicated, (3) at least one measurable lesion or osteolytic bone metastasis, (4) expression of the HLA-A2 allele, (5) WHO performance status 0–2, and (6) life expectancy more than 3 months. Main exclusion criteria included: (1) evidence of brain metastasis, (2) use of immunosuppressive drugs such as glucocorticoids, (3) radiation therapy or chemotherapy within the prior four weeks, (4) signiWcantly increased blood liver-enzyme level (>2.5£ upper normal limit), (5) other malignancies, or (6) pregnancy. The study protocol was approved by the Institutional Ethical Committee, Copenhagen County and the Danish Medicines Agency. Written informed consent was obtained from all patients.

Cancer Immunol Immunother

Vaccine preparation Generation of DCs All procedures were performed according to Good Laboratory Practice standards as approved by the Danish Medicines Agency. Patients underwent unmobilized leukapheresis using a continuous Xow blood cell separator for isolation of large scale (>2 £ 109) mononuclear cells. The remaining red blood cells were lysed with Orthomune lysing solution (provided by hospital pharmacy). Peripheral blood mononuclear cells (PBMC) were washed, resuspended in culture medium (CM) (X-VIVO15, 2% L-glutamin 200 mM, 1% autologous heat inactivated plasma) at 7 £ 106 cells/ml, and separated by 1 h adherence to plastic Nunclon dishes (Nunc, Biotech Line, Slangerup, Denmark). Nonadherent cells were removed and adherent cells were subsequently cultured for 7 days in CM supplemented with 250 U/ml rh-IL-4 (CellGenix, Freiburg, Germany) and 1000 U/ml GM-CSF (Leucomax, Schering Plough, Farum, Denmark). Cells were harvested at day 7 using a cell-scraper and cells with typical DC morphology were counted by light microscopy. Aliquots of a minimum of 5 £ 106 DCs were frozen in 85% autologous serum, 10% DMSO (BDH limited pool, UK, GMP), and 5% Glucosteril 40% (Fresenius, Albertslund, Denmark) using automated cryopreservation (Planer Freezing Unit, Planer, UK). Sterility controls of DCs were negative at all times. Aliquots of the cultured cells were subjected to phenotypic analysis at time of cryopreservation. The expressions of the cell surface antigens, HLA-A2, HLA-DR, CD1a, CD11c, CD33, CD40, CD54, and CD86 were analysed [29]. Peptide loading of in vitro generated DCs On the day of vaccination, one vial of DCs was thawed and washed twice; DC viability as determined by tryphan blue staining was 85–95%. DCs were resuspended in 500 l RPMI and pulsed for 2 h at 37°C with a mixture of the six HLA-A*0201 binding p53-derived peptides and the pan-MHC class II binding peptide, PADRE [30]; each peptide in a concentration of 40 g/ ml. After incubation, cells were washed twice resuspended in 500 l RPMI and transferred to a 0.5 ml insulin syringe for injection. Treatment Eligible patients were to receive a total of 10 immunizations, with at least 5 £ 106 peptides pulsed autologous DCs. The Wrst four vaccinations were given

weekly; thereafter biweekly. The vaccine was administered sc near the inguinal region on the same thigh each time. Concomitantly with each vaccination, 6 MIU/m2 interleukin-2 (Proleukine, Swedish Orphan, Denmark) was administered sc. Synthetic epitope peptides DC were loaded with 6 diVerent HLA-A*0201 binding p53 derived peptides (Table 1); three wild type peptides with high HLA-A*0201 binding aYnities [18, 19, 31–33] and three position 2 anchor modiWed HLA-A*0201 binding peptides [17]. An 11-amino acid pan-MHC class II binding peptide, PADRE (aKXVAAWTLKAAa), was added for induction of in vivo T-helper activation [30]. Purity of all peptides for clinical uses was >95%; these peptides were suspended at 1 mg/ml in RPMI, Wltered through a 0.22 micron Wlter and tested for sterility. ELISPOT control peptides IMP58–66 GILGFVFTL from the inXuenza matrix protein, BMFL1280–288 GLCTLVAML from the immediate-early lytic protein of Epstein Barr virus and RTase309–317 ILKEPVHGV from the reverse transcriptase of HIV-1 virus. Wildtype HLA-A*0201 binding p53 derived peptides: p53149–157 STPPPGTRV (S9V), p53139–147 KTCPVQLWV (K9V) and p53103–111 YQGSYGFRL (Y9L). All peptides were purchased from Schafer N, Copenhagen, Denmark. Clinical monitoring Patients receiving one vaccination or more were evaluable for toxicity, and patients receiving four vaccinations or more were evaluable for tumor response. With the Table 1 HLA-A2 binding p53 peptides used for DC loading prior to vaccination Peptide number

Peptide name

Wild type p53 peptides 1 R9V 2

L9V

3

G11V

ModiWed p53 peptides 4 S9V 5

K9V

6

Y9L

Peptide sequence

p53 65–73 (RMPEAAPPV) p53 264–272 (LLGRNSFEV) p53 187–197 (GLAPPQHLIRV) p53 149–157 (SLPPPGTRV) p53 139–147 (KLCPVQLWV) p53 103–111 (YLGSYGFRL)

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exception of patients with osteolytic bone metastasis, all patients underwent response evaluation according to the RECIST criteria [34, 35] at baseline, one week after the sixth (9 weeks after Wrst treatment) and one week after the tenth (16 weeks after Wrst treatment) DC vaccination. If treatment was continued, evaluation was performed every three months. Toxicity was graded according to the NCI Common Toxicity Criteria. Blood samples Heparinized peripheral blood samples were collected pre-immunization and post-immunization after the fourth, sixth, and tenth vaccinations. PBMC were separated by centrifugation on a Lymphoprep (NYCOMED, Norway) density gradient using standard procedures. Aliquots of PBMC were frozen in RPMI with 10% AB-serum and 10% DMSO. In vitro stimulation of PBMC Triplicates of PBMC (105/well) were cultured in X-VIVO15 medium (Biowhittaker, Wokingham, England) containing 2% inactivated human AB and 10 g/ml relevant peptide in 96 well microtiter plates (Nunc, Denmark). RhIL-2 (Proleukine from Chiron, The Netherlands) 300 IU/mL was added on day 2. On day 10, cells were tested for peptide speciWc IFN- production in the ELISPOT assay. ELISPOT assay The IFN- ELISPOT assay was used to quantify p53 peptide speciWc CTLs during the vaccination trial. It was performed as previously described [20]. Each well of PBMC expanded for 10 days with the relevant peptide as described above were harvested separately and transferred directly to 96 well ELISPOT plates (Multiscreen, MAHAS4510 from Millipore, Molsheim, France) precoated with 7.5 g/ml anti-human IFN- (M-700A from Endongen, USA). After 24 h culture at 37°C with relevant peptide, the ELISPOT plates were developed with 0.75 g/ml biotinylated anti-human IFN- (M-701B from Endogen, USA) and HRP-streptavidin (DAKOCytomation, Denmark) and substrate (41CN from Sigma). The number of spot-forming cells was determined with computer-assisted image analysis software (KS ELISPOT, Zeiss, Munic, Germany). DTH Delayed-type hypersensitivity (DTH) reaction tests against: (1) RPMI (control), (2) mixed p53 peptides

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(0.5 g/l), and (3) mixed p53 peptides + PADRE peptide were applied intradermally at the same time points as the blood samples were drawn. DTH skin test reaction was measured as the diameter of induration and Xushing at the injection site after 48 h. ELISA for serum IL-6 and YKL-40 Serum samples were stored at ¡80°C until analysis for YKL-40 and IL-6. Serum concentrations of YKL-40 and IL-6 were determined using commercially available Elisa kits (YKL-40: #8020, Quidel, San Diego, CA [36]; IL-6: #HS600B, R&D Systems, Minneapolis, Minnesota). Sensitivity of the ELISA was 10 g/l for YKL40 and 0.11 ng/L for IL-6. The intra- and interassay coeYcients of variation were less than 5.0 and 10.2% for YKL-40, and 10.5 and 17.7% for IL-6. All samples from each patient were analyzed on the same ELISA plate. The serum YKL-40 concentration in healthy subjects (n = 245; median age 49, range 18–79) is 43 g/ l, and the plasma IL-6 concentration in healthy subjects (n = 320; median age 48, range 18–64) is 1.4 ng/l. Immunohistochemical staining for p53 Sections of formalin-Wxed, paraYn-embedded tissue from the primary breast carcinoma were cut and stained with antibody against p53 DO7, (DAKOcytomation), 1:50. In brief, the slides were immersed in citrate buVer solution for antigen retrieval and boiled in microwave for 10 min and washed in buVer solution (TBS). They were incubated with primary antibody for 1 h at room temperature and then washed in TBS. After 1 h of incubation in the secondary antibody, the sections were incubated with streptavidin-biotin-complex (DAKOcytomation). A multi-block including several diVerent tissues was used for positive and negative controls. For estimation of positive reaction, only the strongly stained nuclei were counted as a percentage of all tumor nuclei. Less than 5% were estimated as negative [37]. Statistics Survival was measured from the Wrst vaccination until death or date of last follow-up. Survival distribution between the stable disease (SD) and progressive disease (PD) group was estimated using the Kaplan– Meier method. T-tests were performed to test for signiWcant diVerences in mean values for DC yield and phenotype between the SD and PD group. Fisher’s exact test was used to test the association between clinical outcome and tumor p53 expression and vaccination induced p53 speciWc immune response.

Cancer Immunol Immunother

Serum marker values were logarithmically transformed prior to analysis by Wilcoxon signed rank test applied to test for signiWcant diVerences between preand post treatment values within the SD and PD groups. For inter-group comparisons of treatment associated changes in serum marker values, post values were divided with pre values in the respective groups and the computed ratios were subjected to Mann– Whitney test. All tests were two-sided and P-values less than 0.05 were considered signiWcant.

had signiWcant lower monocyte (P = 0.02) and DC yield (P = 0.04). The Wnal DC cell population was phenotyped with a panel of antibodies and analyzed by four color Xow cytometry. The phenotype was characteristic for intermediate mature DCs with some inter-patient variations [29], but no signiWcant correlations between the DC parameters and clinical outcome or induced p53 speciWc immune responses were observed (data not presented). Safety and toxicity

Results Patient characteristics A total of 26 patients with progressive metastatic breast cancer were included. Characteristics of the treated patients are summarized in Table 2. All patients were HLA-A2 positive, and they had a mean age of 53 years. In 11 patients, metastatic disease was only present in one region, while the remainder of the patients had more widespread disease involving two to six regions. The majority (18/26) of the patients had previously received up to Wve diVerent chemotherapy regimens, and 22/26 patients had received up to three diVerent endocrine treatment regimes. All but one patient had at least one measurable tumor lesion; the remaining patient exclusively had osteolytic bone metastases. In 11/26 patients, tissue samples from the primary breast tumor stained positive (>5% positive cells) for p53 by immunohistochemistry. Dendritic cell preparations The median number of PBMCs collected by leukapheresis was 4.2 £ 109 (range 0.7–10.3 £ 109) cells. Monocytes constituted median 14% (range 6.7–21.1) of the harvested PBMC and median 64% (range 17.4–100) of the monocytes developed into DC during culturing. According to the protocol, patients were to receive a minimum of 5 £ 106 viable DCs pr vaccination; each patient was treated with a constant number of DCs pr vaccination but the DC content varied between patients with a median of 16 £ 106 (range 8–43 £ 106) DCs/vaccination as a consequence of DC preparation outcome. None of these parameters were found to vary signiWcantly between patients with PD or SD as clinical outcome; however, a tendency towards higher monocyte concentration (P = 0.15), DC yield (P = 0.15), and vaccine DC content (P = 0.16) were found in the SD group. Patients with early termination of treatment

The vaccine was well tolerated. None of the patients developed skin toxicity at the site of vaccine injection. No signs of autoimmunity were observed. The most common side eVect was mild to moderate local reaction at the site of proleukine injection. Furthermore, as expected, patients experienced CTC grade 1–2 Xu-like symptoms 12–24 h after proleukine injection. All patients received IL-2 as described except for a single patient (pt. no. 19), where IL-2 administration was stopped after the seventh vaccination due to noticeable joint and muscle pain. Clinical response Nineteen patients were available for Wrst evaluation after six vaccinations; none of the patients achieved objective response according to the RECIST criteria; eight patients obtained SD or minor regression (n = 2) and 11 patients had PD. Seven patients did not reach the of evaluation as treatment was discontinued after only 3–4 immunizations (2–3 weeks) due to clinical signs of rapid disease progression or death (one patient). This group of patients was designated ‘early termination’ and categorized as PD. Thus, a total of 18 patients had PD during treatment. One patient obtained minor regression (no. 37) as CT showed regression of an axillary lymph node (LN) from 4.5 to 3.0 cm. Patient no. 21 had end stage disease including weekly need for drainage of bilateral pleural eVusions and ascites at inclusion. At time of evaluation, ascites production had ceased and the pleural eVusions signiWcantly decreased. Furthermore, minor regression of axillary LNs from 1.2/1.6 cm to 0.9/1.0 cm was noted. Due to appearance of a new lesion in the breast, she was excluded after ten vaccinations but survived for another year. Only Wve patients completed all ten planned immunizations. Two patients (no. 19 and 29) received additional monthly immunizations in continuation of the study for 16 and 9 months, respectively, due to prolonged SD.

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According to clinical outcome, patients were divided into two groups: SD (n = 8) and PD (n = 18). In Table 3, disease extent and previous treatment intensity are listed for the two groups; it appears that in the PD group, a higher fraction of patients had more wide spread disease at time of inclusion. The previous exposure to endocrine therapy did not diVer considerably; however, even though the SD group also included patients previously treated with several diVerent chemotherapy regimes, 5/8 patients in the SD group were chemo-naïve compared to only 3/18 in the PD group (Table 2). The mean duration of survival diVered

signiWcantly (P = 0.04) with a median survival of 13.8 months in the SD group compared to 4 months for patients with PD at Wrst evaluation (Fig. 1). Positive p53 expression of the primary tumor as measured by immunohistochemistry was not used as inclusion criteria. However, p53 expression was found more frequently in tumors from patients achieving SD during treatment (Table 2); thus, 5/6 patients tested in the SD group expressed p53 whereas only 6/18 in the PD group (P = 0.06), indicating a possibly correlation between p53 expression and clinical outcome.

Table 2 Patient and treatment characteristics Patient Number

Age

Metastatic regions

Number Previous therapy of regions (number of regimes)

p53 expr (%)

Total number of vacc

Clinical outcome after six vacc

Induced or increased p53 immune response

Survival after one vacc (months)

Chemo Endocrine Response at one evaluation 14 58 liver 15 47 nodal 17 46 nodal 19 64 nodal 21 44 pleura, bone, nodal, ascites 29 58 lung 35 74 lung, liver 37 66 nodal, skin PD at one evaluation 13 72 nodal, skin 16 54 pleura, liver, bone 20 59 skin, lung , bone, nodal 22 33 lung, pleura, liver, bone, nodal 23 46 nodal 26 51 lung 27 64 liver, bone 28 64 lung, pleura, liver, bone, nodal, ascites 31 52 bone 36 64 skin 38 62 pleura, liver, bone, nodal Early termination 10 51 lung, bone, nodal 11 61 nodal 12 56 lung, liver, nodal 18 36 liver, bone 30 44 skin, lung, pleura, liver 32 58 skin 33 56 liver,bone, nodal

1 1 1 1 4

0 0 0 0 0

2 0 2 2 1

0 80 40 30 30

10 9 9 >10 10

SD SD SD SD PR minor

+ + NA ¡ ¡

14.5 9 26.5 21 12.5

1 2 2

3 2 5

0 2 1

90 nd nd

>10 10 8

SD SD PR minor

+ + ¡

13 5.5 >21

2 3

1 2

1 2

0 0

7 5

PD PD

(+) ¡

4 2

4

3

1

100

6

PD

NA

7.5

5

2

1

0

5

PD

NA

2

1 1 2 6

1 3 2 2

0 1 2 1

90 0 0 50

7 6 6 5

PD PD PD PD

¡ ¡ ¡ ¡

13 10 2 2

1 1 4

0 1 1

2 2 2

10 0 0

7 6 6

PD PD PD

¡ ¡ (+)

3 >21 9

3

0

2

0

3

PD

¡

1

5

3

6

12

Number of previous therapy regimes

0–1

>1

0–1

>1

Chemotherapy Endocrine

5 4

3 4

9 9

9 9

Table 4 Vaccination related immune response to p53 peptide 1–6 as evaluated by Elispot

1.0 SD, n = 8 PD, n = 11

Proportion surviving

0.8

Patient number

Pre-existing

p = 0.04 0.6

0.4

0.2

0.0 0

6

12 18 Survival after first vaccination

p53 peptide speciWc T cell reactivity (peptide number according to Table 1)

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Fig. 1 Kaplein–Meier plot of survival after Wrst vaccination for patients with SD at Wrst evaluation contra patients with PD at Wrst evaluation

p53 speciWc CTL response IFN- ELISPOT assay was applied to analyze the induction of speciWc CTLs for each of the six included p53 peptides. Among 26 patients, 25 were available for evaluation of immune response; however, in three patients, the background level of IFN- production was too high for data interpretation; thus, 22 patients were evaluable for immune reactivity. Eight of these 22 patients had induced or increased numbers of CTLs against one or more p53 derived peptides after immunization (Table 4), while 14 patients had no measurable induction of or increase in p53 speciWc CTLs. Response against wild-type peptides (1–3, Table 1) and P2 anchor modiWed peptides (4–6, Table 1) seems to be induced equally well (Table 4). As seen from Table 2, four out of seven patients with SD at Wrst evaluation had induction or increase in p53 speciWc CTLs, compared to only 2 out of 11 patients with PD at Wrst evaluation (P = 0.14). Furthermore, these two

Induced

Response at one evaluation 14 1,2,3,4 5,6 15 4,5,6 29 1,2,3,4,5,6 35 1,2,4 PD at 1.evaluation 13 1 27 3,4 36 1,2,3,4,6 38 1 Early termination 12 3,4,6 1,5 18 2,3,4,5,6 1

Increased

Reduced

2,4

3,4 1,2,3,4,6

6 2,5,6

Elispot responses were deWned as positive when the number of spots was >20 and more than 2 background (vehicle). Induced or increased responses was deWned as responses >2 pre-vaccine levels, and reduced responses was deWned as responses 2< prevaccine levels at a given time point. Data represents means of triplicates with background subtracted

patients with PD only generated p53 speciWc reactivity against a single peptide while reactivity against multiple peptide epitopes were seen in patients obtaining SD (Table 4). It also appeared that pre-existing p53 speciWc CTL reactivity was prevailing in patients exhibiting PD, and reduction of pre-existing reactivity was only observed in patients with PD (Table 4). In most cases, an increase in the number of p53 peptide speciWc CTLs during vaccination was measured (Fig. 2a). However, there are some Xuctuations with low CTL levels at certain time points and a tendency towards a more marked decline at late time points after vaccination; for most peptides, this reduction did not reach pre-vaccination levels. The modiWed peptides in the vaccine are supposed to induce CTLs in vivo with preserved speciWcity for the corresponding native unmodiWed peptides that is processed by the tumour cells. We therefore tested

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Cancer Immunol Immunother

a

p53(149-157)m p53(139-147)m p53(103-111)m

p53(65-73) p53(264-272) p53(187-197) Cmp53 014

Cmp53 014 >200

SFC/100.000 PBMC

SFC/100.000 PBMC

>200 150 100 50 0 pre vac

4. vac 6. vac

150 100 50 0 pre vac

3 month

10. vac

SFC/100.000 PBMC

SFC/100.000 PBMC

40 30 20 10 4. vac 6. vac

40 30 20 10 10. vac

100

SFC/100.000 PBMC

SFC/100.000 PBMC

4. vac 6. vac

Cmp53 029

Cmp53 029

50

0 pre vac

4. vac 6. vac

50

0 pre vac

10. vac

SFC/100.000 PBMC

50 40 30 20 10 0 pre vac

4. vac 6. vac

4. vac 6. vac

10. vac

Cmp53 035

Cmp53 035 SFC/100.000 PBMC

3 month

50

0 pre vac

10. vac

100

b

10. vac

Cmp53 015

Cmp53 015 50

0 pre vac

4. vac 6. vac

50 40 30 20 10 0 pre vac

10. vac

4. vac 6. vac

10. vac

Cmp53 014

SFC/100.000 PBMC 100 75 50 25 0 1

2

3

4

5

Peptides

whether CTL reactivity against unmodiWed versions of the modiWed vaccine p53 peptides were also induced during immunization. As an example, Fig. 2b illustrates

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Post 10. vac pre vac 6

7

an increased CTL reactivity against corresponding modiWed and unmodiWed p53 peptides induced during vaccination.

Cancer Immunol Immunother

䉳 Fig. 2 a CTL reactivity against modiWed and unmodiWed p53 peptides in breast cancer patients with SD during vaccination. PBMC were analyzed for p53 speciWc CTLs in the ELISPOT assay. Data is presented as mean of triplicates with background subtracted. b CTL cross-reactivity against modiWed and unmodiWed p53 peptide variants in a breast cancer patient before and after

None of the patients developed positive DTH skin reaction against naked p53 peptides during immunization. Serum biomarkers Several serum disease markers were measured including lactate dehydrogenase (LDH), alkaline phosphatase (ALP), YKL-40, and IL-6. Figure 3a and b depicts patient speciWc changes and mean serum levels during treatment, illustrating the diVerent patterns for patients with PD and SD. Two types of comparisons were performed testing the association with clinical outcome; one evaluating changes in serum levels for the diVerent markers pre- and post-treatment within the SD and PD groups, respectively, and another comparing the magnitude of treatment associated changes between the SD and PD groups. LDH and ALP are well known serum disease markers. Comparable mean serum levels were found in the PD and SD groups prior to therapy (Fig. 3b); however, as it appears from Fig. 3a, a few patients in the PD group had deviating high pre-values. Both serum LDH and ALP increased signiWcantly (P < 0.05) in the PD group after four vaccinations, in contrast to the SD group where LDH decreased (P = 0.06), while ALP remained stable. The treatment associated changes in LDH and ALP were found to diVer signiWcantly (P < 0.05) between SD and PD patients. High serum IL-6 and YKL-40 levels have been veriWed to have independent prognostic value in several types of cancer. Both factors were measured in 18 of the patients during treatment to assess their feasibility as response markers in connection with DC based cancer immunotherapy. As for LDH and ALP, noticeable diVerences were found among SD and PD patients. In general, YKL-40 increased in PD patients (P = 0.06) during treatment but remained unchanged in SD patient (Fig. 3b). This diVerence in treatment associated changes in serum YKL-40 between SD and PD patients was signiWcant (P = 0.03). As for LDH and ALP, some patients in the PD group had relatively high serum YKL-40 and IL-6 pre-values. The pattern of serum IL-6 changes diVered from the other serum markers as a signiWcant decline was observed in both SD (P = 0.02) and PD patients (P = 0.04) after fourth vaccination. However, the

vaccination. PBMCs from patient no. 14 were analyzed for p53 speciWc CTLs in the ELISPOT assay as described in [20]. Peptides 1 p53149–157modiWed, 2 p53139–147modiWed, 3 p53103–111modiWed, 4 p53149–157wild type, 5 p53139–147wild type, 6 p53103–111wild type, 7 vehicle background

relative decline in serum IL-6 was signiWcantly higher in the SD group (P = 0.01). The accentuated curve in Fig. 3a represents patient no. 21, who suVered from extensive disease dissemination and had clinical beneWt from the treatment. Combined phase I and II data This phase II study was carried out in direct continuation of the previously published phase I study [20] using exactly the same inclusion criteria and vaccination regime according to a common phase I/II protocol. Six patients were treated in the phase I part; thus, a total of 32 patients were treated in all and clinical outcome included 11 SD (of these, three patients had minor regression), 20 PD, and 1 mixed response (MR). Statistical analysis of the correlation between clinical outcome and p53 expression were applied to the combined data. In total, 30 patients were evaluable for p53 expression; 7/9 patients with SD were p53 positive compared to 7/21 patients with PD (P = 0.046). Thus, 7/ 14 (50%) p53 positive patients obtained SD while only 2/16 (12.5%) of p53 negative patients, reXecting a signiWcant diVerence with an increased chance of treatment beneWt for p53 positive patients. Combined phase I and II data was also evaluated for correlation between clinical outcome and induced immune response; there was a trend towards a correlation between SD and induced p53 immune reactivity (P = 0.10) but no signiWcance was reached.

Discussion This phase II study was carried out in continuation of a recently published phase I study [20] in which six patients with progressive advanced breast cancer were treated. Immunization with p53-peptide pulsed autologous DC was found to be safe and not associated with signiWcant toxicity. Therapy associated expansion of p53 speciWc peripheral blood T-cells was demonstrated and, importantly, indications of a possible anti-tumor activity of the treatment were found. Now, additional 26 patients have been treated in the phase II part of the study. Unfortunately, a signiWcant fraction of patients did not reach the evaluation time point due to disease deterioration within the Wrst few weeks of treatment, which emphasizes the

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Cancer Immunol Immunother

a

Serum alkaline phosphatase

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2000 1500 1000 500 0

pre vacc 4. vacc

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Fig. 3 a Individual changes in serum alkaline phosphatase (U/l), LDH (U/l), YKL-40 (g/L), and Il-6 (ng/l) during treatment for patients with PD and SD. The red curve illustrates the course of serum IL-6 in patient 21, who suVered from extensive disease dissemination and had clinical beneWt from the treatment. b Mean

serum level §SD of alkaline phosphatase (U/l), LDH (U/l), YKL-40 (g/l), and IL-6 (ng/l) pre-vaccination and after fourth vaccination for patients with PD and SD. The treatment associated changes in these serum markers all diVered signiWcantly (P < 0.05) between SD and PD patients (detailed in the text)

diYculties in conducting therapeutic vaccination trials in end stage cancer patients. Theoretically, when using tumor antigen immunization strategies such as ours, a time span of at least 6–8 weeks is necessary to establish a CTL response with a potential clinical capacity; furthermore, immunocompetent patients are a prerequisite.

No objective responses according to the RECIST criteria were achieved; however, nearly 1/3 of the patients, all having veriWed progressive disease at time of treatment initiation, attained disease stabilization during therapy. Two patients had minor regression of single lesions; and in two patients, one with nodal and

13

Cancer Immunol Immunother

the other with lung metastasis, prolonged stabilization was observed. Patients within the SD group survived signiWcantly longer; however, this might be due to less wide spread disease at time of inclusion. Also, more patients in the SD group were chemo-naïve, which sustain the assumption that immunization therapy is more likely to succeed in less pretreated patients. Hence, in the present study a signiWcant fraction of the breast cancer patients experienced clinical beneWt in association with the DC-p53 vaccination treatment, suggesting that vaccine-induced immunity may have therapeutic eVect in some patients with deWned tumor characteristics. To support this observation, it was important to document a p53 dependency of the observed clinical responses. Wild-type p53 is only present in low levels in normal cells whereas mutant p53 has a prolonged half-life causing an accumulation of p53 protein. This diVerential level of p53 expression potentially allows the immune system to discriminate between normal and malignant cells and provides the basis for p53 targeting immunotherapy. Tumor p53 over-expression was however not used as inclusion criteria, but p53 expression of the primary tumor was measured by immunohistochemistry allowing us, retrospectively, to evaluate the inXuence of tumor p53 status on clinical outcome. Phases I and II data were combined to obtain a larger patient material, and we were thereby able to demonstrate that signiWcantly more SD patients than PD patients had p53 over-expression in their primary tumor samples; a correlation which crucially sustains the p53 dependency of this p53 targeted immunotherapy. So, in spite of the fact that p53 over-expression is in general a predictor of poor prognosis and therapeutic response [38], these results indicate that p53 expression might be advantageous when therapy is targeting p53 speciWcally. Noticeably, if only p53+ patients had been included in this trial, it would have resulted in SD rates of up till 50%. Tumor cells might, however, lose antigens and therefore display a reduced susceptibility to vaccineinduced immunity in the course of vaccinations. The use of multi-epitope vaccine strategies are a potential way to avoid this phenomenon; therefore, in combination with p53, we have introduced survivin [39, 40] and telomerase [41] as additional targets. In an ongoing trial, breast cancer patients are immunized with autologous DC loaded with up to 26 diVerent HLA epitopes, including p53, survivin, and telomerase. Despite the fact that p53 is an obvious candidate for immunotherapeutic strategies, p53 speciWc immunization has only been tested in very few clinical trials [42– 45] and, to our knowledge, this is the Wrst time that

clinical and immunologic eYcacy of p53 peptide loaded DCs have been tested in cancer patients. In one previous p53 vaccination study, the safety and eYcacy of administration of a canarypox virus encoding the human wild-type p53 gene (ALVACp53) was evaluated in colorectal cancer patients [44, 45]. In another recent study, DC infected with an adenoviral construct containing the full length wild-type p53 was tested for treatment of patients with extensive small lung cancer [42]. Here, p53-speciWc T cell responses against ALVAC-p53 were observed in about half of all patients using IFN- ELISPOT, and against the L9V HLA-A*0201 binding p53 peptide in more than half of the HLA-A2+ patients. We were able to detect measurable induction of p53 peptide speciWc immunity by IFN- ELISPOT in one-third of the immunized patients; possibly, this lower frequency can be explained by diVerences in immunization concept. Utilizing DCs pulsed with predeWned p53 peptides oVers the possibility of more easily following the induced immunity and evaluate the immunological and clinical eYcacy of these speciWed epitopes. Furthermore, the peptide immunization strategy circumvents the use of viral vectors, which holds the intrinsic problem of potential interference with adjuvant components due to antiviral immune reactivity. However, both antigen presentation spectrum and probably also presentation time are more limited in DC-peptide based vaccines. The identiWcation of class I binding peptides does not ensure that the epitopes are also processed by e.g. tumour cells. For the peptides used in this study, such processing and sensitivity of target cells to CTL killing were tested in preclinical studies [17–19, 32, 33]. Similarly, induced immune responses against the three modiWed p53 peptides, with improved HLA class I binding, are directed against non self peptides per se, and it is therefore of importance to test cross-reactivity against wild-type peptides. Additional experiments conWrmed that a parallel induction of CTL reactivity against modiWed and corresponding unmodiWed peptides could be attained. These results have important implications for the generation of anti-tumor reactivity against self epitopes as they indicate that the use of anchor-modiWed p53 peptides with increased aYnity for HLA class I in cancer vaccines is capable of expanding CTL responses against the corresponding wild-type peptide epitopes. A higher fraction of SD than PD patients had measurable induction of immune reactivity and also against a higher number of p53 peptides; furthermore, decrease of p53 reactivity was only observed in PD patients. Even though statistic signiWcance was not

13

Cancer Immunol Immunother

reached, these observation are central as they point toward a possible connection between immune response and clinical outcome. Interestingly, in several patients with SD, we observed Xuctuations in the number of p53 peptide speciWc CTLs during vaccination with low CTL levels at certain time points and decline at late time points. Since repeated IL-2 treatments have been shown to shift tumor speciWc T-cells from the blood to the tumor site [46], our data might suggest that p53 peptide speciWc T-cells leave the blood stream. To the contrary, induction of tolerance due to repeated injections of the antigen can not be ruled out [47], but does not seem likely as a reduction to background level is only seen in a few patients; further studies investigating the vaccine associated induction of p53 speciWc regulatory T cells are ongoing in order to clarify this. IdentiWcation of response biomarkers are important to facilitate the identiWcation of patient subgroups responding to treatment. In immunotherapy response, biomarkers is a particular important issue as objective responses frequently are lacking and clinical beneWt often appears in the form of induced or prolonged stable disease with variable correlation to a measurable immune response. The protein YKL-40 (also named CHI3L1) is a 40 kDa glycoprotein and a member of “mammalian chitinase-like proteins”. It is expressed by non-malignant cells such as macrophages and neutrophils and by several diVerent types of malignant cells including breast cancer cells. The biological function of YKL-40 is partially unknown, but it is believed to be involved in inXammation and remodeling of the extracellular matrix through growth factor activity. Furthermore, several studies have indicated a role for YKL-40 in cancer cell proliferation and invasiveness [22]. A number of studies have suggested that one single measurement of serum YKL-40 in cancer patients at time of diagnosis, or at time of relapse, is a potential independent biomarker of short survival [22]. Three studies have suggested that serum YKL-40 may be a useful biomarker to monitor in cancer patients [48–50]. The cytokine IL-6 is produced by both normal cells (e.g. macrophages, lymphocytes, and endothelia cells) and cancer cells. It plays a major role as immune modulator but also acts as a paracrine and autocrine growth factor for cancer cells and inhibits radio- and chemotherapy induced apoptosis of cancer cells [51]. The serum IL-6 concentration is increased in breast cancer patients and in patients with other kinds of solid tumors and is related to disease stage. In breast cancer patients, a high serum level of IL-6 is associated with shorter survival [27, 52]; only a few studies with IL-6 as response marker exist [53, 54].

13

In addition to LDH and ALP, we decided to measure serum IL-6 and YKL-40 during treatment to test their feasibility as markers of response to DC therapy. We found signiWcant response related changes in the serum disease markers LDH; ALP as SD was associated with cessation of serum LDH and ALP rise. Serum YKL-40 and IL-6 changes were also signiWcantly related to clinical outcome, as the YKL-40 level remained stable in SD patients, who also displayed the highest relative decline in IL-6 levels. These results imply that the role of serum YKL-40 and IL-6 as biomarkers of response in connection with DC based cancer immunotherapy should be further explored. The phenotype and functionality of DCs employed in vaccination trials are believed to be decisive for the immunological and thereby the clinical outcome of the treatment [55]. To facilitate the ongoing optimization of clinical DC preparation procedures, it is therefore mandatory to collect relevant quality assessment data. The procedures for generation of DCs from leukapheresis separated PBMC used in the present work resemble those used by other authors [56, 57]. Large granular cells comprised around 60% of the cells in the Wnal therapeutic DC preparations and the cells displayed a phenotype characteristic for intermediate-to-mature DCs, even though no maturation cytokine cocktail was employed. In a recently published study, we tested more thoroughly the function of DCs from breast cancer patients included in vaccination trials [29]. Here, we found an unimpaired capacity of the patientderived DCs for cross-presentation of naïve (KLH) as well as recall (CMV and Tetanus) antigens. Only a few human cancer vaccine studies have been made that directly compare immature and mature DCs. However, these studies clearly demonstrate that in vitro DC maturation is optimal for induction of a potent T cell response, also in immunotherapeutic settings [56, 58]. Phase I of our clinical trial was initiated in 2001 when no consensus regarding maturation of DCs for clinical applications existed; therefore, the protocol was planned without in vitro DC maturation. To maintain a homogenous treatment of all included patients, this strategy was not changed during phase II. However, immature DCs are also able to induce peptide speciWc CD8+ T cells [59] and the DCs used in our study induced signiWcant levels of CTL activity despite the lack of in vitro maturation. Protocols that apply immature DC might not induce tolerance as feared, simply because the in vitro generation and manipulation of the DCs induce some degree of maturation. In addition, immature DCs pulsed with MHC class II binding epitopes, like PADRE employed in this protocol [30], may induce conditioning of the DC in vivo.

Cancer Immunol Immunother

In conclusion, a signiWcant fraction of the breast cancer patients obtained disease stabilization during p53-DC vaccination and correlation to tumor p53 expression, induction of p53 speciWc immunity, and observed changes in biomarkers supported the clinical results. All patients in this trial had metastatic breast cancer often with a high tumor burden, and the patients were frequently heavily pre-treated. As a consequence, transformation of a p53 speciWc activation of the immune system into signiWcant tumor regression might not be obtainable in these patients. More relevant could be the ability of the treatment to induce prolonged survival, a question this study was not scaled to answer. However, the clinical results obtained here encourage further clinical studies at an earlier stage of the disease with progression free survival as endpoint. Acknowledgment This work was supported by grants from Dansk Kræftforsknings Fond, The Danish Cancer Society, Direktør Leo Nielsen og Hustru Karen Margrethe Nielsens Legat for Lægevidenskabelig Grundforskning, Michaelsen Fonden, and Aase og Ejnar Danielsens Fond.

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