Immunostimulatory cancer chemotherapy using local ingenol-3-angelate and synergy with immunotherapies

Vaccine 27 (2009) 3053–3062

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Immunostimulatory cancer chemotherapy using local ingenol-3-angelate and synergy with immunotherapies Thuy T.T. Le a , Joy Gardner a , Diem Hoang-Le a , Chris W. Schmidt a , Kelli P. MacDonald a , Eleanore Lambley a , Wayne A. Schroder a , Steven M. Ogbourne b , Andreas Suhrbier a,∗ a b

Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia Peplin Inc., Brisbane, Queensland 4006, Australia

a r t i c l e

i n f o

Article history: Received 23 September 2008 Received in revised form 11 March 2009 Accepted 15 March 2009 Available online 3 April 2009 Keywords: Chemotherapy Cancer vaccines Metastases

a b s t r a c t Ingenol-3-angelate is a new local chemotherapeutic agent in clinical trails that induces primary necrosis of tumour cells and transient local inflammation. Here we show that cure of subcutaneous tumours with ingenol-3-angelate (PEP005) resulted in the generation of anti-cancer CD8 T cells that could regress metastases. Furthermore, PEP005-mediated cure synergized with several CD8 T cell-based immunotherapies to regress further distant metastases. PEP005 was shown to have adjuvant properties, being able to upregulate CD80 and CD86 expression on dendritic cells in vivo, and to promote CD8 T cell induction when co-delivered with a protein antigen. PEP005 thus emerges as a unique local chemotherapeutic immunostimulatory debulking agent that could be used in conjunction with immunotherapies to promote regression of metastases. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Ingenol-3-angelate (PEP005) is a new topical chemotherapeutic agent currently in phase III clinical trials for the treatment of actinic keratosis and phase II for non-melanoma skin cancer [1]. Three daily topical treatments with 10–20 ␮g of PEP005 cured a range of mouse and human tumours grown subcutaneously in C57BL/6 and Foxn1nu mice [2]. The mechanism of action of PEP005 is unique for a chemotherapeutic agent as it causes primary necrosis of tumour cells, and causes a local moderate acute inflammatory response, which resolves over 5–10 days leaving a favourable cosmetic effect [2]. The PEP005-induced inflammation is associated with a pronounced infiltrate of neutrophils, which prevent relapse of the treated tumour by mediating antibody-dependent cellular cytotoxicity against residual tumour cells [3]. PEP005 also activates protein kinase C (PKC) at 4–1000 ng/ml [4,5], and this activity is likely to be important for stimulating the inflammatory response [3]. PKC activation does not appear to be required for inducing primary necrosis, which requires 80–100 ␮g/ml of PEP005 in vitro [2]. Here we show that a single intratumoural (i.t.) injection of PEP005 was able to cure subcutaneous (s.c.) tumours, which here-

Abbreviations: i.t., intratumoural; s.c., subcutaneous; i.v., intravenous; PKC, protein kinase C; PEP005, ingenol-3-angelate; OVA, ovalbumin; DC, dendritic cell; CFA, Complete Freund’s Adjuvant. ∗ Corresponding author. Tel.: +61 7 33620415; fax: +61 7 33620107. E-mail address: [email protected] (A. Suhrbier). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.03.025

after are nominally referred to as “primary tumours”. Importantly, we describe for the first time the unique immunostimulatory properties of PEP005 treatment wherein PEP005-mediated cure of primary tumours resulted in the regression of smaller distant untreated tumours, nominally referred to hereafter as “secondary tumours”. This activity was apparent in prophylactic settings, and more importantly was retained in therapeutic settings, where secondary tumours were established several days prior to PEP005 treatment of primary tumours. PEP005 emerges as a novel immunostimulatory chemotherapeutic agent that not only ablates the treated tumour, but in-so-doing also generates anti-cancer CD8 T cells that can synergize with CD8 T cell-based immunotherapies to regress distant secondary tumours. 2. Materials and methods 2.1. Growth of tumours B16 and B16 cells expressing ovalbumin (OVA) (provided by Dr K. Rock, Dana-Farber Cancer Institute) were grown s.c. in 6–10-week female C57BL/6 mice [2,6]. Lung metastases were established as described [7]. Lewis lung cells expressing OVA were provided by Nelson [8] and grown as described [9]. CT26 colon carcinoma cells (CRL-2638) were provided by Dr M. Smyth (Peter MacCallum Cancer Centre) and grown s.c. in 6–10-week female Balb/c mice. Animals were supplied by the Animal Resource Centre (Perth, Australia). All experiments were reviewed and approved by the QIMR animal ethics committee.

3054

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

2.2. PEP005 treatment Tumours were treated by i.t. injection (30 gauge needle) of PEP005 dissolved in acetone then RPMI1640 (final acetone concentration 4%), or PEP005 dissolved in 100% PEG400. PEP005 (ingenol mebutate) was supplied by Peplin Inc.

2.3. Dendritic cell vaccination DC2.4 was developed by superinfecting GM-CSF-transduced bone marrow cells with myc and raf oncogenes [10]. DC2.4 cells (≈30 × 106 ) (provided by Dr K. Rock) were simultaneously treated with 1 ␮g/ml LPS (E. coli 055:B5, Sigma), 200 U/ml murine IFN␥ (Chemicon International, Temecula, CA, USA) or 20 ␮g/ml PEP005, the peptides SVYDFFVWL (SVY) [7] (30 ␮g/ml), KVPRNQDWL (KVP) [11] (30 ␮g/ml) or SIINFEKL (SII) (10 ␮g/ml) as indicated (Auspep Pty Ltd., Parkville, Australia), and OVA (Sigma) as a source of CD4 T cell help (10 ␮g/ml). The cells were incubated for 3 h at 37 ◦ C in ≈3 ml of medium with occasional mixing, were irradiated (3000 rad), washed twice and delivered i.v. ((3–5) × 106 cells/mouse) in 200 ␮l RPMI1640.

2.4. Cancer vaccine The vaccine comprised SPSYVYHQF (SPS) peptide [12] and OVA (as a source of CD4 T cell help) dissolved in RPMI1640 emulsified with Montanide ISA 51 VG (Seppic, Paris, France) (3:7, v/v), and injected s.c. Mice received 100 ␮l of vaccine containing 50 ␮g peptide and 10 ␮g OVA.

2.5. T cells for adoptive immunotherapy Balb/c mice (n = 3) were inoculated s.c. with CT26 tumour cells (105 ). When tumours reached 36–48 mm2 (day 7) splenocytes were restimulated in vitro [13] for 7 days with SPS peptide (2 ␮g/ml) and pooled. Mice received 106 of these cells i.v., which gave 9643 SPSspecific IFN␥ ELISPOT spots per 106 splenocytes prior to transfer (data not shown).

2.6. ELISPOT assay Ex vivo IFN␥ ELISPOT assays were conducted as described [14], but using MultiScreen-IP plates (Millipore), 10 IU/ml of recombinant human IL-2 (Cetus), and 2 ␮g/ml of peptide or OVA. Cultured ELISPOT assays were performed by first culturing the splenocytes for 6 days [13], using SII (2 ␮g/ml) pulsed and irradiated EL4 cells, or 10 ␮g/ml OVA, prior to the ELISPOT assay.

2.7. PEP005 as a vaccine adjuvant OVA in PBS (20 mg/ml) was mixed with PEP005 (500 ␮g/ml PEP005 in 100% PEG400) or 100% PEG400 (1:8, v/v) and each C57BL/6 mouse injected s.c. with 45 ␮l, which contained 100 ␮g OVA with or without 20 ␮g PEP005. For DC-based immunisations, OVA (20 mg/ml) was mixed with PEG400 (1:9, v/v) and added (50 ␮l) to DC2.4 cells (3 × 107 ) in 150 ␮l of medium for 1 h at room temperature with gentle rotation. LPS and IFN␥ were then added in 2 ␮l (final 1 ␮g/ml and 200 U/ml, respectively) and cells incubated 1 h at 37 ◦ C. After a wash in RPMI1640 cells were injected i.v. in 200 ␮l (3 × 106 cells/mouse). Complete Freund’s Adjuvant (CFA) (Sigma) was formulated (1:1, v/v) with OVA in PBS and injected s.c. (100 ␮g OVA in 50 ␮l of vaccine/mouse).

Table 1 Cure rates following i.t. injection of PEP005 into different 15–20 mm2 tumours grown s.c. in C57BL/6 or Balb/c mice. Tumour

PEP005 dose (␮g)

Number cured/number treated Expt. 1, Expt. 2, etc.

B16a

16 25 2 × 25b

5/10, 12/15 14/14, 13/14, 15/18, 17/24 12/12, 18/18, 12/12

B16-OVAa

16 25 2 × 25b

12/15 11/11 17/17, 16/16

Lewis Lung-OVAa

8 16 25

6/14 7/11 27/27

CT26c

25 25

17/17 11/11

a b c

C57BL/6 mice Daily for 2 days. Balb/c mice.

2.8. PEP005 activity on dendritic cells in vivo C57BL/6 mice (n = 3 per group) were injected with LPS (10 ␮g) or PEP005 (20 ␮g) s.c. on the lower back. After 12 h inguinal lymph node splenocytes were harvested and DCs enriched by nycodenz density centrifugation (Optiprep, Vital Diagnostic) (1.077 g/cm3 ) [15]. Cells were blocked with Rat IgG (Sigma) (2 ␮g/ml) and stained with anti-CD11c Alexa Fluor (MCD11c20, Invitrogen), CD80 (1610A1), CD86 (GL1), isotype control (R35-95) (BD Pharmingen) and analysed by FACSCalibur (Becton Dickinson) using CellQuest Pro software. 2.9. Statistics Statistical analysis was performed using SPPS for Windows (Version 15.0, 2007, Chicago: SPSS Inc.). 3. Results 3.1. PEP005-mediated cure of Lewis Lung-OVA tumours protected against challenge with Lewis Lung-OVA We established that a single i.t. injection of 25 ␮g of PEP005 was capable of curing most s.c. tumours with an area of 15–20 mm2 , with lower doses resulting in a reduction in the percentage of tumours cured (Table 1). The course of regression, erythema, eschar formation and excellent cosmesis of cured tumours was very similar (data not shown) to that described previously for topical PEP005 treatment [2,3]. Given the proinflammatory nature of PEP005 treatment, we sought to determine whether PEP005 treatment would induce cellular immune responses capable of rejecting a subsequent challenge with the same tumour. We first chose to use a suboptimal dose of PEP005 in order also to examine the effect of PEP005-mediated regression as well as cure of primary tumours. Primary Lewis Lung-OVA tumours were treated on day 0 with 8 ␮g of i.t. PEP005. On day 4 the animals were challenged with Lewis Lung-OVA on the opposite flank. In mice where PEP005 treatment had cured the primary tumour, emergence of these secondary tumours was significantly delayed compared to controls (log-rank test p = 0.002), with secondary tumours failing to appear in 50% of animals (Fig. 1A, PEP005 cured). In the control group, which had received neither primary tumours nor PEP005 treatment, secondary tumours emerged in all animals by days 6–11 (Fig. 1A, Controls). In mice where PEP005 treatment regressed, but failed to cure the primary tumours (with tumours re-emerging days 11–13), the growth of the secondaries was slightly but not significantly

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

3055

Fig. 1. (A) Protection against Lewis Lung-OVA challenge following PEP005 treatment of primary Lewis Lung-OVA tumours. C57BL/6 mice received 2 × 105 Lewis Lung-OVA s.c. on the back on day −7. When these primary tumours reached an average 13.6 ± S.E. 1.8 mm2 (range 10–25 mm2 ) on day 0, 13 mice were treated once with 8 ␮g of PEP005 delivered i.t. In 8 mice the primary tumours were cured, and these mice received 2 × 105 Lewis Lung-OVA s.c. on day 4 on the opposite flank from the primary tumours (PEP005 cured). In the remaining animals (n = 5) PEP005 treatment regressed the primary tumours, but after 11–13 days the primary tumours regressed. These animals also received secondary tumours as above (PEP005 reg.). Two control groups were included. One group (n = 6) received no primary tumours, but was treated s.c. with PEP005 day 0 and received secondary tumours on the opposite flank day 4 (PEP005 s.c.). The second control group (n = 8) received no primary tumours, was not treated with PEP005, and received secondary tumours as above (Controls). Secondary tumours were monitored over time and were deemed to have emerged when they had reached 4 mm2 . See B for legend. (B) PEP005-mediated cure of Lewis Lung-OVA primary tumours did not reduce the growth of secondary tumours in Foxn1nu mice. The experiment described in A was repeated in Foxn1nu mice; PEP005 cured (n = 4), PEP005 regressed (n = 7), Control (n = 7). A PEP005 s.c. group was not included. (C) Treatment of primary Lewis Lung-OVA tumours with PEP005-induced SII-specific CD8 T cell responses. Day 13; a parallel group of animals were set up as for A except that on day 13 they were euthanased and splenocytes analysed for SII-specific CD8 T cell responses using an ex vivo IFN␥ ELISPOT assay (primaries cured n = 6; primaries regressed n = 8, PEP005 s.c. n = 5, Controls n = 5). Days 34–54; the animals described in A were euthanased between days 34 and 54, and splenocytes analysed by ex vivo IFN␥ ELISPOT. (D) Mice whose primary Lewis Lung-OVA tumours were cured with PE005 were resistant to challenge with Lewis Lung-OVA, B16-OVA, but not B16 tumours. Groups of C57BL/6 mice (n = 8 mice per group) were inoculated day −3 with Lewis Lung-OVA cells (106 ) and on day 0 when the tumours had reached 19.3 ± S.E. 1.2 mm2 they were cured with an i.t. injection of 20 ␮g PEP005. Control mice with no primary tumours received the same PEP005 treatment s.c. On day 6 the two groups of mice were challenged with Lewis Lung-OVA (LL-OVA) (5 × 105 ), B16-OVA (105 ) or B16 (105 ) cells on the opposite flank. Growth of these secondary tumours was monitored over time and when they reached 100 mm2 the animal was euthanased.

(p = 0.15) delayed (Fig. 1A, PEP005 reg.). In animals without primary tumours, injection of PEP005 s.c. on day 4 at a site distant from the secondary tumours had no effect on the growth of secondary tumours (Fig. 1A, PEP005 s.c.). Cure of primary tumours with PEP005 was thus able to mediate significant protection against a subsequent challenge with the same tumour type at a distant site, with PEP005-mediated regression being significantly less effective. 3.2. The role of T cells The role of CD8 T cells in mediating protection against tumour challenge in the Lewis Lung and Lewis Lung-OVA models is well established [8,9,16], suggesting that PEP005 treatment might induce anti-cancer CD8 T cells. When the experiment shown in Fig. 1A was repeated in T cell defective Foxn1nu mice, no protective effect against secondary challenge was evident (Fig. 1B), indicating that the protective effects against secondary tumours observed in Fig. 1A required the generation of anti-cancer T cells. We have

previously shown that neutrophil-mediated ADCC in Foxn1nu mice prevents relapse of PEP005-treated tumours [3]. The data in Fig. 1B also illustrated that this mechanism was not responsible for regression of secondary tumours. To analyze the anti-cancer CD8 T cell responses generated by the treatments described in Fig. 1A, groups of animals were established as in Fig. 1A except that on day 13 splenocytes were analysed for SIIspecific responses using an ex vivo IFN␥ ELISPOT assay (Fig. 1C, Day 13). The mice whose primary tumours were cured by PEP005 treatment had significantly more SII-specific CD8 T cells than animals treated with PEP005 s.c. (p = 0.01, unpaired t-test, PEP005 cured versus PEP005 s.c.). Animals in which PEP005-treatment regressed the tumours also had elevated SII-specific CD8 T cells (PEP005 reg.), but this only approached significance (p = 0.06, PEP005 regressed versus PEP005 s.c.). PEP005 injected s.c. (rather than i.t.) (PEP005 s.c.) failed to increase the number of SII-specific CD8 T cell numbers compared to untreated controls (Controls). A similar pattern of SII-specific responses was seen when this analysis was repeated

3056

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

between days 34 and 54 (Fig. 1C, Day 34–54. PEP005 cured versus PEP005 s.c. p = 0.008, PEP005 regressed versus PEP005 s.c. p = 0.07). PEP005 treatment thus promoted the generation of tumour-specific CD8 T cells. 3.3. Mice whose primary tumours were cured with PEP005 were resistant to challenge with Lewis Lung-OVA, B16-OVA but not B16 To illustrate that resistance to challenge (Fig. 1A) was antigen specific, Lewis Lung-OVA tumours on a group of mice were treated with a curative dose of i.t. PEP005 (20 ␮g). Control mice with no primary tumours received 20 ␮g PEP005 s.c. Six days later the animals were challenged with Lewis Lung-OVA, B16OVA or B16 tumours. PEP005-mediated cure of primary Lewis Lung-OVA tumours induced resistance to challenge with Lewis Lung-OVA tumours (Fig. 1D, LL-OVA) (log-rank test p < 0.001). PEP005-mediated cure of primary Lewis Lung-OVA tumours also resulted in resistance to challenge with B16-OVA (Fig. 1D, B16OVA) (log-rank test p < 0.001), but importantly, not resistance to B16 challenge (Fig. 1D, B16). The role of SII-specific CD8 T cells in regression of B16-OVA tumours is well established [6,17] (see also Figs. 3B and 4B), indicating that the effect on secondaries of the PEP005-mediated cure of primaries was antigen specific. 3.4. PEP005-mediated cure of B16-OVA tumours protected against challenge with B16-OVA lung metastases To determine whether PEP005-mediated cure of primary tumours could inhibit the growth of secondary tumours in a different model, the B16-OVA lung metastasis model was used [7]. Established primary s.c. B16-OVA tumours were treated with a suboptimal dose of PEP005 on day 0. On day 6 mice were challenged i.v. with B16-OVA cells. Mice in which primary tumours had been cured by the PEP005 treatment showed a significant reduction in the numbers of lung metastases when compared to (i) control animals that had no primary tumours and received no treatment, and (ii) animals that had no primary tumours and received PEP005 s.c. (Fig. 2A, Mann–Whitney test p = 0.001 and p = 0.002, respectively). Animals whose primary tumours were regressed, but not cured by PEP005 showed no significant reduction (p = 0.182) in the number of lung metastases (Fig. 2A, PEP005 reg.). This again illustrated that curing the primary tumours was important for detecting a significant effect on secondary tumours in these models, presumably because anti-cancer CD8 T cells induced by PEP005-mediated cure of primaries are more effective against secondaries when they are not also called upon to regress an incompletely regressed primary tumour. 3.5. B16 lung metastases in a therapeutic setting and the effect of tumour burden Regression of tumours is substantially easier in prophylactic settings than it is in therapeutic settings [17,18]. We thus sought to test the ability of PEP005-mediated cure of primary tumours to regress secondary tumours in a therapeutic setting. Mice received the primary s.c. tumour and the i.v. lung metastases on day −2. On day 0, when the s.c. tumours had reached 21.8 ± S.E. 2.4 mm2 , they were treated with a curative dose of i.t. PEP005. A parallel control group neither received primary tumours nor received PEP005 treatment, but received the i.v. lung metastases. On day 18 the animals were euthanased and lung weights determined (lung metastases could not be counted in this B16 model as large numbers of the metastases merged into large tumour masses). The lung weights were significantly higher in control animals than animals in which the primary tumours had been cured with PEP005 (Fig. 2B. p = 0.023, unpaired t-test, PEP005 cured versus Controls). After subtracting

Fig. 2. (A) Protection against B16-OVA lung metastases challenge following PEP005 treatment of primary B16-OVA tumours. C57BL/6 mice (n = 13) received 5 × 105 B16-OVA s.c. on the back on day −6. When these primary tumours had reached 10–20 mm2 on day 0, they were treated once with 18 ␮g of PEP005 delivered i.t., which cured the tumours in 8 mice (PEP005 cured) and regressed the tumours in 5 mice (PEP005 regressed). Controls were as above. Secondary B16-OVA (105 ) were injected i.v. on day 6 and animals were sacrificed on day 27 and lung metastases counted. (B) PEP005-mediated cure of primary B16 tumours reduced the growth of established B16 lung metastases. C57BL/6 mice (n = 7) were injected with 106 B16 cells s.c. and 5 × 104 B16 cell i.v. on day −2. On day 0 the s.c. tumours had reached an average 21.8 ± S.E. 2.4 mm2 and were cured with 25 ␮g of i.t. PEP005 (PEP005). Controls received B16 cells i.v. only. The PEP005 s.c group received no s.c. tumours and PEP005 was injected s.c. into the skin (n = 5). Naïve mice received no tumours (n = 6 per group). On day 18 the mice were euthanased and lung weights determined (PEP005). (C) The effect of increasing metastatic tumour burden. The experiment shown in B was repeated except mice received 105 B16 cells i.v. On day 0 the s.c. tumours had reached an average of 26.2 ± S.E. 3.4 mm2 and were treated as in A. (D) DC2.4 therapy of B16 lung metastases. C57BL/6 mice (n = 7) were given 105 B16 cells i.v. on day −6, and on days 0 and 7 the mice were treated with DC2.4 cells (3 × 106 cells/mouse i.v.) that had been sensitized with SVY and KVP peptides, matured with LPS and IFN␥, irradiated and washed. The mice were also given 6 i.p. injections of rIL-2 (5 × 104 U) over 4 days immediately following each DC treatment (DC). Control mice received i.v. B16 (n = 6) and naïve mice received no tumours (n = 4 per group). Seven days after the final DC2.4 treatment, mice were sacrificed and lungs were removed and weighed.

the mean weight of lungs from tumour-free animals (Fig. 2B, Naïve, dotted line), this represented a reduction in lung tumour burden of over 50%. PEP005 delivered s.c. again showed no significant anticancer activity (Fig. 2B, PEP005 s.c.). Thus in this therapeutic setting PEP005-mediated cure of primary B16 tumours was able to regress significantly established B16 lung metastases. CD8 T cell-based anti-cancer strategies usually work better when the tumour burden is low [17,19–22]. To assess the effect of secondary tumour burden on the ability of PEP005-mediated cure

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

3057

of primary tumours to regress the lung metastases, the experiment in Fig. 2B was repeated except that the i.v. dose of B16 cells was increased twofold to 105 . On day 0 the primary s.c. tumours had reached 26.2 ± S.E. 3.4 mm2 and were again treated with a curative dose of i.t. PEP005. The lung weights were determined on day 18, and the increased i.v. dose of B16 cells led to an average 1.7fold increase in lung tumour burden, from a mean weight (after subtracting the weight of naïve tumour-free lungs) of 0.17 ± S.E. 0.08 g in Fig. 2B to 0.28 ± S.E. 0.06 g in Fig. 2C. Although the primary tumours were of a comparable size (a mean of 26.2 mm2 compared with 21.8 mm2 ), their cure with PEP005 did not significantly reduce the weight of lung metastases in this experiment (Fig. 2C, compare PEP005 with Controls). Thus the anti-cancer activity mediated by PEP005-mediated cure of primary tumours was less evident when the secondary tumour burden was increased. 3.6. Dendritic cell therapy The DC2.4 cell line has been used for DC therapy in several mouse models [23]. To assess DC2.4 therapy for B16 lung metastases, DC2.4 cells were pulsed with two CD8 T cell epitopes known to stimulate anti-B16 CD8 T cell responses; SVY from Trp-2 [7] and the human gp100 epitope KVP [11]. The DC2.4 cells were matured with lipopolysaccharide (LPS) and IFN␥ [24] as this significantly enhanced their ability to induce CD8 T cell responses, and upregulates expression of CD40, CD80, CD86 and MHC II (data not shown). The peptide pulsed and matured DC2.4 cells were then injected i.v. into animals with the same B16 lung metastasis burden as described in Fig. 2C and were able to reduce significantly the lung tumour load (Fig. 2D, unpaired t-test, p = 0.009 for DC versus Controls). When the mean weight of tumour-free lungs was subtracted, a > 70% reduction in lung tumour burden was observed. These results confirmed that DC2.4 cells can be used for DC therapy, and also illustrated that when tumour burden is higher, DC therapy, perhaps not surprisingly, can out-perform PEP005-mediated cure of primary tumours for therapy of secondary tumours. 3.7. Synergy between PEP005-mediated cure of primary tumours and DC therapy to reduce growth of secondaries in the B16-OVA model

Fig. 3. (A) Synergistic effects between PEP005-mediated cure of primaries and DC therapy on the growth of established secondary s.c. B16-OVA tumours. On day −3 C57BL/6 mice were inoculated with 2 × 106 B16-OVA cells on the right flank (primary tumours) and 2 × 104 B16-OVA cells on the left flank (secondary tumours). On days 0 and 1 the primary B16-OVA tumours, which had reached 30.4 ± S.D. 6 mm2 were cured with i.t. injections of 25 ␮g of PEP005. On days 4 and 11 mice received DC therapy using DC2.4 cells (3 × 106 ) pulsed with SVY, KVP and SII peptides (n = 12, PEP005 + DC). A second group of mice were treated in the same way (primaries measuring 30 ± S.D. 5.1 mm2 on day 0), but did not receive the DC therapy (n = 13, PEP005). A third group was inoculated with secondary tumours only and received the DC therapy (n = 14, DC), and the control group was given only secondary tumours and received no treatments (n = 18, Control). Animals were euthanased when the secondary tumours reached 100 mm2 . (B) Correlation with CD8 T cell induction. Animal groups were established and treated as in A and on day 10 mice were sacrificed and splenocytes analysed in an ex vivo ELISPOT assay for responses to SVY, KVP and SII (n = 5 per group). The responses following PEP005 + DC were significantly greater than the sum of the responses seen after PEP005 plus those seen after DC alone, p = 0.02 by ANOVA, which included a term for each epitope. (C) Synergistic effects between PEP005-mediated cure of primaries and DC therapy on the growth of established secondary B16 tumours. The experiment in A was repeated except that B16 cells were used (n = 6 per group). On day −2 C57BL/6 mice were inoculated with

To determine whether the immunostimulatory activity of PEP005-mediated cure of primary tumours would synergize with DC therapy to reduce the growth of secondaries, four groups of C57BL/6 mice were established. In the first group 2 × 106 B16-OVA cells were injected s.c. on the right flank (primary tumour) and 2 × 104 B16-OVA cells s.c. on the left flank (secondary tumour) on day −3. On day 0, when the primary tumours had reached a mean of 30 ± S.D. 5.1 mm2 , the primary tumours were treated and cured with PEP005. On days 4 and 11 the mice also received DC therapy, which comprised DC2.4 cells sensitized with peptides representing three major epitopes recognized by B16-OVA-specific CD8 T cells (Fig. 3A, PEP005 + DC). The second group was like the first except that DC therapy was omitted (Fig. 3A, PEP005). In the third group only secondary tumours were established and mice were given the same DC therapy as the first group (Fig. 3A, DC). The fourth group

106 B16 cells on the right flank (primary tumours) and 5 × 104 cells on the left flank (secondary tumours). On day 0 the primary tumours reached 20 ± S.D. 1.9 mm2 and were treated on days 0 and 1 with PEP005. On days 5 and 12 mice received DC2.4 cells pulsed with SVY and KVP (PEP005 + DC). A second group of mice was treated like the first but without DC therapy (PEP005). A third group was inoculated with secondary tumours only and received DC therapy (DC). The control group was given only secondary tumours and received no treatment (Controls). Animals were euthanased when secondary tumours reached 100 mm2 .

3058

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

only had secondary tumours and received no treatment (Fig. 3A, Control). The combination of curing the primary tumours with PEP005 plus DC therapy showed significantly greater anti-cancer activity against the secondary tumours than PEP005-mediated cure of primaries alone or DC therapy alone (log-rank test p = 0.02 and 0.049, respectively). Individually, PEP005-mediated cure of primaries and DC therapy both also significantly reduced the growth of secondaries when compared to the no treatment controls (log-rank test p = 0.002 and <0.001, respectively) (Fig. 3A). Tumour growth curves for this experiment (Supporting Fig. 1A) illustrate that all the secondary tumours (implanted on day −3) continue to grow at a similar rate until about day 4, after which the growth of tumours in the control animals was clearly faster. 3.8. Synergistic activity against secondaries of combined PEP005 and DC therapies correlates with anti-cancer CD8 T cell induction in the B16-OVA model To determine whether the synergistic effect of PEP005mediated cure of primaries and DC therapy (Fig. 3A) correlated with anti-cancer CD8 T cell induction, a group of animals were treated as in Fig. 3A except that splenocytes were analysed for ex vivo T cell responses specific for the three dominant CD8 T cell epitopes presented by B16-OVA (Fig. 3B). The magnitude of CD8 T cell responses (Fig. 3B) correlated with regression of secondaries (Fig. 3A), and total CD8 T cell responses following PEP005 + DC treatment was significantly greater than the combined responses of PEP005-mediated cure and DC therapy (p = 0.02). These data illustrated that PEP005 cure of primary tumours can synergize with DC therapy for induction of anti-cancer CD8 T cells, and suggested that the synergistic anti-cancer activity seen in Fig. 3A was due to synergistic induction of CD8 T cells. 3.9. Synergy between PEP005-mediated cure of primary tumours and DC therapy to reduce growth of secondaries in the B16 model Using the more aggressive B16 model, a comparable experiment to that shown in Fig. 3A was undertaken except that the SII peptide was omitted from the DC therapy. Again curing of primary tumours with PEP005 combined with DC therapy significantly delayed growth of secondaries compared to either treatment alone (p = 0.001 and 0.046, respectively) (Fig. 3C). DC therapy significantly increased survival compared to the no treatment controls (p = 0.009), whereas DCs without peptide antigen did not (p = 0.1). In this setting PEP005 cure of primary tumours did not show any detectable activity against secondary tumours. Growth curves for this experiment are shown in Supporting Fig. 1B. The experiment shown in Fig. 3A and C illustrated that PEP005mediated cure of larger primary tumours synergized with DC therapy to regress smaller secondary tumours. Said in a different way, DC therapy was significantly more effective against secondaries in animals where the primary tumours had been cured with PEP005 treatment, than it was in animals where there was no primary tumour. A lower tumour burden in itself can often improve the outcome of CD8 T cell-based immunotherapies [17,19–22]. However, PEP005-cure of primary tumours was not simply improving the DC-based regression of secondary tumours by reducing the overall tumour burden in the animal, but was actively contributing to improved regression of the secondary tumours. 3.10. Combining PEP005-mediated cure of primary tumours with peptide vaccination reduces the growth of CT26 colon carcinoma secondaries To further illustrate the broad utility of combining PEP005mediated cure of primaries with CD8 T cell-based immunother-

apies, an experiment similar in set up to that shown in Fig. 3A was undertaken except that the CT26 colon carcinoma model was used [12]. CT26 expresses an endogenous tumour antigen gp70, which contains a known H2-Ld restricted CD8 T cell epitope (AH1). The vaccine used herein comprised this epitope SPSYVYHQF (SPS) [12] formulated with Montanide ISA 51, an adjuvant used to deliver CD8 epitopes in human cancer immunotherapy trials [25]. The combination of curing the primary tumours with PEP005 plus vaccine (Fig. 4A, PEP005 + Vaccine) showed significantly greater anti-cancer activity against the secondary tumours than PEP005-mediated cure of primaries alone (Fig. 4A, PEP005) or vaccine alone (Fig. 4A, Vaccine) (log-rank test p = 0.003 and <0.001, respectively). Individually, PEP005-mediated cure of primaries and vaccine alone both significantly reduced the growth of secondaries when compared to the no treatment controls (Fig. 4A, Control) (logrank test p = 0.011 and 0.013, respectively). Tumour growth curves of this experiment (Supporting Fig. 1C), again illustrated that secondary tumours implanted on day −3 continued to grow at a similar rate until days 3–4, after which the faster growth of tumours in the control group could be clearly seen. Thus in a second tumour model (CT26), in a different mouse strain (Balb/c), and using a different CD8 T cell-based vaccination modality targeting an endogenous tumour antigen, PEP005-mediated cure of primaries was again able to synergize with the vaccine therapy to decrease the growth of secondary tumours.

3.11. Synergistic activity against secondaries of combined PEP005 and peptide vaccination correlates with anti-cancer CD8 T cell induction in the CT26 model To determine whether the synergistic effect of PEP005mediated cure of primaries and peptide vaccination (Fig. 4A) correlated with anti-cancer CD8 T cell induction, a group of animals were treated as in Fig. 4A except that splenocytes were analysed for ex vivo T cell responses specific for the dominant CD8 T cell epitope presented by CT26 tumours, SPS. The anti-cancer CD8 T cell responses (Fig. 4B) again correlated well with regression of metastases (seen in Fig. 4A). The combination of vaccine and PEP005-mediated cure induced significantly more SPS-specific T cells than vaccine alone, and PEP005-mediated cure induced significantly more T cells than the Control (Fig. 4B). Vaccine therapy alone also induced more T cells than Control, and this approached significance (p = 0.057).

3.12. Synergy between PEP005-mediated cure of primary tumours and adoptive immunotherapy with anti-cancer T cells to regress secondary CT26 tumours To determine whether PEP005-mediated curing of primary tumours could promote activity against secondary tumours following adoptive T cell immunotherapy, the experiment shown in Fig. 4A was repeated but instead of a cancer vaccine, mice received SPSspecific T cells. The combination of curing the primary tumours with PEP005 plus adoptive immunotherapy showed significantly greater anti-cancer activity against the secondary tumours than PEP005-mediated cure of primaries alone or T cells alone (log-rank test p = 0.001 and 0.008, respectively) (Fig. 4C). PEP005 treatment of primaries and adoptive immunotherapy individually also significantly slowed the growth of the secondaries compared with controls (p = 0.003 for both) (Fig. 4C). Growth curves are shown in Supporting Fig. 1D. Thus PEP005-mediated cure of primary tumours also synergized with adoptive immunotherapy using anticancer T cells.

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

3059

3.13. Adjuvant activity of PEP005 To explore whether PEP005 has adjuvant activity, OVA was mixed with PEP005 and injected s.c. into mice. Ten days later splenocytes were assayed by ELISPOT for (i) T cell responses to the CD8 T cell epitope, SIINFEKL (SII), and (ii) for T cell responses specific for OVA (predominantly CD4 T cells). Ex vivo ELISPOT did not show significant responses (data not shown). Cultured ELISPOT assays [26] illustrated that OVA delivered with PEP005 induced significantly more SII-specific responses (Fig. 5A, PEP005 + OVA, black bar) than OVA alone (Fig. 5A, OVA, black bar) (p = 0.02, t-test). OVA formulated with PEP005 also induced small responses specific for OVA (Fig. 5A, PEP005 + OVA, white bar), but these were not significantly different from those seen after vaccination with OVA alone (Fig. 5A, OVA, white bar). For comparison the following were tested in parallel; (i) OVA loaded DC2.4 cells (Fig. 5A, DC + OVA), which represents an efficient method of inducing SII-specific CD8 T cell responses [27], and (ii) OVA formulated in CFA (Fig. 5A, CFA + OVA, black bar), which represents an efficient method for inducing OVAspecific CD4 T cells [28]. OVA formulated with PEP005 induced about 20% of the SII-specific responses seen for DC + OVA (Fig. 5A, black bars), and about 20% of the OVA-specific responses seen after CFA + OVA (Fig. 5A, white bars). These experiments illustrate that PEP005 has adjuvant activity, and importantly was able to promote CD8 T cell induction when co-delivered with whole protein antigen (OVA) in vivo. 3.14. Dendritic cell stimulation by PEP005

Fig. 4. (A) PEP005-mediated cure of primaries synergized with a peptide vaccine to regress established secondary CT26 colon carcinoma tumours. On day −3 C57BL/6 mice were inoculated with 105 CT26 cells on the left flank (primary tumour) and 5 × 103 CT26 cells on the right flank (secondary tumour). On day 0 primary CT26 tumours, which had reached 16.8 ± S.D. 3.2 mm2 , were cured with i.t. injections of 25 ␮g of PEP005 formulated in PEG400. On days 3 and 10 mice received a peptide vaccine (Montanide ISA 51) containing the SPS epitope (n = 9, PEP005 + Vaccine). A second group of mice were treated in the same way (primaries measuring 17.5 ± S.D. 2.1 mm2 on day 0), but did not receive the vaccine (n = 8, PEP005). A third group was inoculated with secondary tumours only and received the vaccine (n = 11, Vaccine), and the control group was given only secondary tumours and received no treatment (n = 12, Controls). The growth of the secondaries was monitored over time and animals euthanased when tumours reached 100 mm2 . (B) Correlation with CD8 T cell induction. Animal groups were established and treated as in Fig. 4A and on day 7 mice were sacrificed and splenocytes analysed in an ex vivo ELISPOT assay for responses to SPS (n = 5 per group). (C) PEP005-mediated cure of primaries synergized with adoptively transferred T cells to regress established secondary CT26 tumours. Primary and secondary tumours were established as for A and on day −1 mice received an i.v. injection of SPS-specific T cells. On day 0 the primary CT26 tumours, which had reached 11 ± S.D. 2 mm2 were cured with PEP005 as above. Secondary tumours were monitored over time as above (n = 7, PEP005 + T cells). A second group of mice

A common feature of most adjuvants is their ability to stimulate dendritic cells. To determine whether PEP005 was able to promote DC maturation, mice were injected s.c. with PEP005 and draining lymph node DCs analysed for expression of the maturation markers CD80 and CD86. PEP005 treatment induced a 3.3- and 1.4fold increase in the mean fluorescent intensity (MFI) of CD80 and CD86 over control mice, respectively (Fig. 5B). (MHC II and CD40 expression were not significantly increased – data not shown). LPS, which is well known efficiently to upregulate CD80 and CD86 on DCs [29], showed a 6- and 2.6-fold increase for CD80 and CD86, respectively (Fig. 5B). Therefore, although less potent then LPS, PEP005 treatment clearly upregulated maturation markers on DCs in vivo. Treatment in vitro of purified splenic CD11chigh DCs [15] with non-toxic doses of PEP005 (20–500 ng/ml) failed to induce IL-12 p40 (as measured by real-time RT-PCR), whereas CpG oligonucleotide treatment showed a > 30 fold induction of this cytokine (data not shown). Thus in vitro PEP005 alone was unable to induce IL-12. Other factors may operate in vivo to stimulate secretion of this cytokine (see Section 4). To determine whether direct exposure of DCs to PEP005 contributes to improved CD8 T cell induction, SII-pulsed DCs were treated with PEP005 or diluent in vitro and were then injected i.v. into naïve mice. Animals vaccinated with PEP005-treated DCs generated significantly more CD8 T cells than animals vaccinated with DCs treated with diluent (Fig. 5C), suggesting that direct exposure of DCs to PEP005 can contribute to their activation. As might be expected, SII-pulsed DCs matured with LPS and IFN␥ outperformed PEP005-treated DCs, consistent with the data show in Fig. 5A and B.

were treated in the same way (primaries measuring 15 ± S.D. 2 mm2 on day 0), but did not receive T cells (n = 4, PEP005). A third group was inoculated with secondary tumours only and received the T cells (n = 4, T cells). The control group was given only secondary tumours and received no treatment (n = 6, Controls).

3060

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

4. Discussion

Fig. 5. (A) CD8 T cell induction following co-delivery of PEP005 with a whole protein antigen. Groups of C57BL/6 mice (n = 3) were injected s.c. with either OVA mixed with PEP005 (PEP005 + OVA), OVA alone, OVA loaded and matured DC2.4 cells (DC + OVA), OVA formulated with CFA (CFA + OVA), or nothing (Naive). After 10 days splenocytes were harvested and analysed by cultured ELISPOT for T cell responses to the CD8 T cell epitope, SII (black bars) and OVA (white bars). (B) Upregulation of CD80 and CD86 on DCs by PEP005 in vivo. PEP005, LPS or PBS (Control) were injected s.c. into the lower backs of C57BL/6 mice. After 12 h inguinal lymph node lymphocytes from 5 mice were pooled for each group, and DC populations enriched using Optiprep gradients. The cells were then analysed by FACS for CD80 and CD86 expression after gating on the CD11chigh DC population. Isotype control antibody staining was performed on lymphocytes from LPS treated mice (left panel) and PEP005-treated mice (right panel). The mean fluorescent intensity is indicated for each graph. (C) PEP005treated DCs induced increased levels of CD8 T cell responses. DC2.4 cells were pulsed with SII/OVA in the presence of either 20 ␮g/ml PEP005 (+PEP005), diluent (No PEP005) or LPS and IFN␥, and were injected i.v. (5 × 106 ) (n = 5 per group). Ten days later splenocytes were analysed by ex vivo ELISPOT assays.

Here we show for the first time that curing established primary tumours with local PEP005 treatment generated anti-cancer CD8 T cell activity that was able to regress distant secondary tumours. This was illustrated in (i) prophylactic settings where challenge with secondary tumours occurred after PEP005-mediated curing of primaries (Figs. 1A and 2A), and (ii) in more realistic therapeutic settings, where both the primary and the secondary tumours were established before PEP005 treatment (Figs. 2B, 3A and 4A & C). PEP005-mediated cure of primary tumours was also shown to synergize with a number of CD8 T cell-based immunotherapies to improve regression of secondary tumours; (i) a DC vaccine (Fig. 3A and C), (ii) a peptide vaccine (Fig. 4A), and (iii) adoptive T cell immunotherapy (Fig. 4C). Finally, PEP005 was shown to have adjuvant activities (Fig. 5). PEP005 thus emerges as a novel chemotherapeutic agent, which is not only effective at regressing PEP005-treated tumours, but also adjuvants the antigens within the treated tumour thereby promoting generation of anti-cancer CD8 T cells. PEP005 treatment may thus find utility as an immunostimulatory debulking agent that can be used in conjunction with a number of CD8 T cell-based immunotherapeutic interventions. There are a number of genetic, cellular and protein-based i.t. immunotherapeutic approaches that induce cellular immune responses that are capable of regressing the treated tumour, and these responses have the potential to regress distant metastases [30,31]. However, with some notable exceptions [12,32,33], the effect of such treatments on secondary tumours has not been widely reported. Apart from manufacturing and effective delivery issues associated with such biological i.t. anti-cancer modalities, these approaches also rely on the T cells to regress both the treated and the secondary tumours. Certain i.t. chemotherapies that do directly regress the treated tumours can also induce anticancer cellular immunity, and such responses can be shown to reject subsequent tumour challenges, i.e. they can provide prophylactic protection [30,34–36]. However, we are unaware of studies that demonstrate therapeutic regression of pre-existing secondary tumours (established prior to treatment initiation) by such chemotherapy-induced immune responses in the absence of additional manipulations. Such therapeutic activity is clearly evident after PEP005 treatment (Fig. 2B, and black triangles in Figs. 3A and 4A & C). Combining immuno- and chemotherapies in certain settings can promote anti-cancer T cell immunity that is capable of curing primary tumours and rejecting subsequent tumour challenges [37,38]. However, only a limited number of reports [39,40] of combination therapies have demonstrated that treatment of one tumour can lead to regression of distant pre-existing (secondary) tumours. Here we show PEP005-mediated cure of primaries was able to synergize with a number of different CD8 T cell-based immunotherapies to provide therapeutic activity against secondary tumours (Fig. 2B, and black squares in Figs. 3A & C and 4A & C). Thus local PEP005 treatment (i) can regress the treated tumour thereby reducing the tumour burden, which in itself is likely to improve CD8 T cell-based immunotherapy [17,19–22] and (ii) can adjuvant the tumour debris thereby promoting therapeutic anti-cancer CD8 T cell activity. Few chemotherapeutic modalities have adjuvant activity, in fact most are considered to be immunosuppressive. The adjuvant activity of PEP005 is also noteworthy as it was able to induce CD8 T cell responses when codelivered with a whole protein antigen (Fig. 5A). Proteins delivered extracellularly are generally poor at inducing CD8 T cell responses. One might speculate that the membranolytic properties of PEP005 [2] may facilitate delivery of tumour antigens into the class I processing pathway [41]. The saponin adjuvant QuilA also has membranolytic properties [42] and can induce CD8 T cells when co-delivered with whole protein antigens [43].

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

PEP005 was able to upregulate CD80 and CD86 in vivo (Fig. 5B), an activity likely due to PKC stimulation by PEP005, since other PKC activators also upregulate these markers on DCs [44,45]. Although direct exposure of DCs to PEP005 appeared able to activate DC to some extent (Fig. 5C), the inability of PEP005 to induce IL-12 in vitro suggests that addition factors are provided in vivo to stimulate DCs. PEP005 has been shown to both recruit and activate neutrophils [3] and activated neutrophils have been shown to induce DC IL-12 production [46] and to help maintain Th1 responses [47]. Induction of primary tumour cell necrosis by PEP005 treatment [2] may also contribute, as treatments that induce primary necrosis of tumour cells have been shown to promote T cell responses [16,36,48]. In contrast to cryotherapy [49], i.t. co-administration of the Toll-like receptor (TLR) agonists, LPS, poly I:C or CpG oligonucleotides with PEP005 did not increase the activity against secondary tumours (data not shown), suggesting that in vivo the inflammatory responses induced by PEP005 treatment [3] provided sufficient “danger signals” for CD8 T cell generation. Use of i.t. chemotherapy might be viewed as limited to visible and/or palpable tumours positioned near the skin surface. However, this is likely to change with the recent advances in imaging technology that should make drug delivery to deep-seated tumours a viable option in the future. These systems include physically integrated single photon emission computed tomography (SPECT)/computed tomography (CT), Positron emission tomography (PET)/CT and PET/magnetic resonance imaging (MRI), which combine the high anatomical resolution of CT and MRI with the high sensitivity of SPECT and PET [50–52]. Acknowledgements We wish to thank Sandy Tall (Tall Bennett, Mona Vale, Australia) for Montanide ISA 51, Paula Hall (QIMR) for help with the Flow Cytometry, and Xueqin Liu and Dr M. Wykes for help with DC preparation. The work was funded by the Queensland Cancer Fund and the National Health and Medical Research Council, Australia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2009.03.025. References [1] Ogbourne SM, Hampson P, Lord JM, Parsons P, De Witte PA, Suhrbier A.Proceedings of the first international conference on PEP005. Anticancer Drug 2007;18:357–62. [2] Ogbourne SM, Suhrbier A, Jones B, Cozzi SJ, Boyle GM, Morris M, et al. Antitumor activity of 3-ingenyl angelate: plasma membrane and mitochondrial disruption and necrotic cell death. Cancer Res 2004;64:2833–9. [3] Challacombe JM, Suhrbier A, Parsons PG, Jones B, Hampson P, Kavanagh D, et al. Neutrophils are a key component of the antitumor efficacy of topical chemotherapy with ingenol-3-angelate. J Immunol 2006;177:8123–32. [4] Cozzi SJ, Parsons PG, Ogbourne SM, Pedley J, Boyle GM. Induction of senescence in diterpene ester-treated melanoma cells via protein kinase C-dependent hyperactivation of the mitogen-activated protein kinase pathway. Cancer Res 2006;66:10083–91. [5] Kedei N, Lundberg DJ, Toth A, Welburn P, Garfield SH, Blumberg PM. Characterization of the interaction of ingenol 3-angelate with protein kinase C. Cancer Res 2004;64:3243–55. [6] Anraku I, Harvey TJ, Linedale R, Gardner J, Harrich D, Suhrbier A, et al. Kunjin virus replicon vaccine vectors induce protective CD8 + T-cell immunity. J Virol 2002;76:3791–9. [7] Zeh 3rd HJ, Perry-Lalley D, Dudley ME, Rosenberg SA, Yang JC. High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy. J Immunol 1999;162:989–94. [8] Nelson DJ, Mukherjee S, Bundell C, Fisher S, van Hagen D, Robinson B. Tumor progression despite efficient tumor antigen cross-presentation and effective “arming” of tumor antigen-specific CTL. J Immunol 2001;166:5557–66. [9] Lenarczyk A, Le TT, Drane D, Malliaros J, Pearse M, Hamilton R, et al. ISCOM based vaccines for cancer immunotherapy. Vaccine 2004;22:963–74.

3061

[10] Shen Z, Reznikoff G, Dranoff G, Rock KL. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J Immunol 1997;158:2723–30. [11] Lou Y, Wang G, Lizee G, Kim GJ, Finkelstein SE, Feng C, et al. Dendritic cells strongly boost the antitumor activity of adoptively transferred T cells in vivo. Cancer Res 2004;64:6783–90. [12] Ali SA, Lynam J, McLean CS, Entwisle C, Loudon P, Rojas JM, et al. Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulating factor, correlates with antigenspecific adaptive immunity. J Immunol 2002;168:3512–9. [13] Elliott SL, Pye S, Le T, Mateo L, Cox J, Macdonald L, et al. Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems. Vaccine 1999;17:2009–19. [14] Le TT, Drane D, Malliaros J, Cox JC, Rothel L, Pearse M, et al. Cytotoxic T cell polyepitope vaccines delivered by ISCOMs. Vaccine 2001;19:4669–75. [15] MacDonald KP, Rowe V, Filippich C, Thomas R, Clouston AD, Welply JK, et al. Donor pretreatment with progenipoietin-1 is superior to granulocyte colonystimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation. Blood 2003;101:2033–42. [16] Machlenkin A, Goldberger O, Tirosh B, Paz A, Volovitz I, Bar-Haim E, et al. Combined dendritic cell cryotherapy of tumor induces systemic antimetastatic immunity. Clin Cancer Res 2005;11:4955–61. [17] McAllister A, Arbetman AE, Mandl S, Pena-Rossi C, Andino R. Recombinant yellow fever viruses are effective therapeutic vaccines for treatment of murine experimental solid tumors and pulmonary metastases. J Virol 2000;74:9197–205. [18] Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA, et al. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8 + T cells. J Exp Med 2003;198:569–80. [19] Mocellin S, Mandruzzato S, Bronte V, Lise M, Nitti D. Part I: vaccines for solid tumours. Lancet Oncol 2004;5:681–9. [20] Berinstein N. Overview of therapeutic vaccination approaches for cancer. Semin Oncol 2003;30:1–8. [21] Mukherjee S, Nelson D, Loh S, van Bruggen I, Palmer LJ, Leong C, et al. The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor. Cancer Gene Ther 2001;8:580–8. [22] Jager D, Knuth A. Antibodies and vaccines—hope or illusion? Breast 2005;14:631–5. [23] Ni HT, Spellman SR, Jean WC, Hall WA, Low WC. Immunization with dendritic cells pulsed with tumor extract increases survival of mice bearing intracranial gliomas. J Neurooncol 2001;51:1–9. [24] Huttner KG, Breuer SK, Paul P, Majdic O, Heitger A, Felzmann T. Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells. Cancer Immunol Immunother 2005;54:67–77. [25] Chianese-Bullock KA, Pressley J, Garbee C, Hibbitts S, Murphy C, Yamshchikov G, et al. MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma. J Immunol 2005;174:3080–6. [26] McKinnon LR, Ball TB, Wachihi C, McLaren PJ, Waruk JL, Mao X, et al. Epitope cross-reactivity frequently differs between central and effector memory HIVspecific CD8 + T cells. J Immunol 2007;178:3750–6. [27] Imai J, Hasegawa H, Maruya M, Koyasu S, Yahara I. Exogenous antigens are processed through the endoplasmic reticulum-associated degradation (ERAD) in cross-presentation by dendritic cells. Int Immunol 2005;17:45–53. [28] Creusot RJ, Thomsen LL, van Wely CA, Topley P, Tite JP, Chain BM. Early commitment of adoptively transferred CD4 + T cells following particle-mediated DNA vaccination: implications for the study of immunomodulation. Vaccine 2001;19:1678–87. [29] Sheng KC, Pouniotis DS, Wright MD, Tang CK, Lazoura E, Pietersz GA, et al. Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells. Immunology 2006;118:372–83. [30] Goldberg EP, Hadba AR, Almond BA, Marotta JS. Intratumoral cancer chemotherapy and immunotherapy: opportunities for nonsystemic preoperative drug delivery. J Pharm Pharmacol 2002;54:159–80. [31] Crittenden MR, Thanarajasingam U, Vile RG, Gough MJ. Intratumoral immunotherapy: using the tumour against itself. Immunology 2005;114:11–22. [32] Yu P, Lee Y, Liu W, Chin RK, Wang J, Wang Y, et al. Priming of naive T cells inside tumors leads to eradication of established tumors. Nat Immunol 2004;5:141–9. [33] Hu JC, Coffin RS, Davis CJ, Graham NJ, Groves N, Guest PJ, et al. A phase I study of OncoVEXGM-CSF, a second-generation oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor. Clin Cancer Res 2006;12:6737–47. [34] Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med 2005;202:1691–701. [35] Luo L, Qiao H, Meng F, Dong X, Zhou B, Jiang H, et al. Arsenic trioxide synergizes with B7H3-mediated immunotherapy to eradicate hepatocellular carcinomas. Int J Cancer 2006;118:1823–30. [36] Castano AP, Mroz P, Hamblin MR. Photodynamic therapy and anti-tumour immunity. Nat Rev Cancer 2006;6:535–45. [37] Lake RA, Robinson BW. Immunotherapy and chemotherapy—a practical partnership. Nat Rev Cancer 2005;5:397–405. [38] Terando A, Mule JJ. On combining antineoplastic drugs with tumor vaccines. Cancer Immunol Immunother 2003;52:680–5.

3062

T.T.T. Le et al. / Vaccine 27 (2009) 3053–3062

[39] Saji H, Song W, Furumoto K, Kato H, Engleman EG. Systemic antitumor effect of intratumoral injection of dendritic cells in combination with local photodynamic therapy. Clin Cancer Res 2006;12:2568–74. [40] Tanaka F, Yamaguchi H, Ohta M, Mashino K, Sonoda H, Sadanaga N, et al. Intratumoral injection of dendritic cells after treatment of anticancer drugs induces tumor-specific antitumor effect in vivo. Int J Cancer 2002;101:265–9. [41] Moron G, Dadaglio G, Leclerc C. New tools for antigen delivery to the MHC class I pathway. Trends Immunol 2004;25:92–7. [42] Kamstrup S, San Martin R, Doberti A, Grande H, Dalsgaard K. Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A. Vaccine 2000;18:2244–9. [43] Fernando GJ, Stewart TJ, Tindle RW, Frazer IH. Th2-type CD4 + cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model. J Immunol 1998;161:2421–7. [44] Do Y, Hegde VL, Nagarkatti PS, Nagarkatti M. Bryostatin-1 enhances the maturation and antigen-presenting ability of murine and human dendritic cells. Cancer Res 2004;64:6756–65. [45] Davis TA, Saini AA, Blair PJ, Levine BL, Craighead N, Harlan DM, et al. Phorbol esters induce differentiation of human CD34 + hemopoietic progenitors to dendritic cells: evidence for protein kinase C-mediated signaling. J Immunol 1998;160:3689–97.

[46] Bennouna S, Denkers EY. Microbial antigen triggers rapid mobilization of TNFalpha to the surface of mouse neutrophils transforming them into inducers of high-level dendritic cell TNF-alpha production. J Immunol 2005;174:4845– 51. [47] Maletto BA, Ropolo AS, Alignani DO, Liscovsky MV, Ranocchia RP, Moron VG, et al. Presence of neutrophil-bearing antigen in lymphoid organs of immune mice. Blood 2006;108:3094–102. [48] den Brok MH, Sutmuller RP, van der Voort R, Bennink EJ, Figdor CG, Ruers TJ, et al. In situ tumor ablation creates an antigen source for the generation of antitumor immunity. Cancer Res 2004;64:4024–9. [49] den Brok MH, Sutmuller RP, Nierkens S, Bennink EJ, Frielink C, Toonen LW, et al. Efficient loading of dendritic cells following cryo and radiofrequency ablation in combination with immune modulation induces anti-tumour immunity. Br J Cancer 2006;95:896–905. [50] Seemann MD. Whole-body PET/MRI: the future in oncological imaging. Technol Cancer Res Treat 2005;4:577–82. [51] Blodgett TM, Meltzer CC, Townsend DW. PET/CT: form and function. Radiology 2007;242:360–85. [52] Ellis RJ, Kaminsky DA. Fused radioimmunoscintigraphy for treatment planning. Rev Urol 2006;8(Suppl 1):S11–9.