Combination of Photodynamic Therapy and Specific Immunotherapy Efficiently Eradicates Established Tumors

Home , Cancer immunotherapy, Photodynamic therapy

Published OnlineFirst November 6, 2015; DOI: 10.1158/1078-0432.CCR-15-0515

Cancer Therapy: Preclinical Clinical Cancer Research Combination of Photodynamic Therapy and Speciﬁc Immunotherapy Efﬁciently Eradicates Established Tumors Jan Willem Kleinovink1, Pieter B. van Driel2,3, Thomas J. Snoeks2, Natasa Prokopi1, Marieke F. Fransen1, Luis J. Cruz2, Laura Mezzanotte2, Alan Chan3, Clemens W. Lowik€ 2, and Ferry Ossendorp1

Abstract

Purpose: The efficacy of immunotherapy against advanced cured one third of mice. Importantly, all cured mice were fully cancer may be improved by combination strategies. Photody- protected against subsequent tumor rechallenge, and combina- namic therapy (PDT) is a local tumor ablation method based on tion treatment of primary tumors led to eradication of distant localized activation of a photosensitizer, leading to oxygen rad- secondary tumors, indicating the induction of a systemic antitu- ical-induced tumor cell death. PDT can enhance antitumor mor immune response. Indeed, PDT by itself induced a significant þ immune responses by release of antigen and danger signals, CD8 T-cell response against the tumor, which was increased supporting combination protocols of PDT with immunotherapy. when combined with SLP vaccination and essential for the ther- Experimental Design: We investigated the local and systemic apeutic effect of combination therapy. immune effects of PDT after treatment of established tumors. In Conclusions: We show that immunotherapy can be efficiently two independent aggressive mouse tumor models, TC-1 and RMA, combined with PDT to eradicate established tumors, based on we combined PDT with therapeutic vaccination using synthetic strong local tumor ablation and the induction of a robust systemic long peptides (SLP) containing epitopes from tumor antigens. immune response. These results suggest combination of active Results: PDT of established tumors using the photosensitizer immunotherapy with tumor ablation by PDT as a feasible novel Bremachlorin resulted in significant delay of tumor outgrowth. treatment strategy for advanced cancer. Clin Cancer Res; 1–10. 2015 Combination treatment of PDT with therapeutic SLP vaccination AACR.

Introduction epitopes of tumor antigens (1–4). Besides widely shared tumor antigens such as those expressed by virally induced tumors, this A major challenge in medical oncology is the development of approach can also be applied to individual patient-specific neo- efficient treatment options for advanced cancer, which current- antigens (5, 6). Clinical studies using therapeutic SLP vaccination ly are limited. The clinical situation of advanced primary against cancer are ongoing based on encouraging results in tumors with possible metastases asks for therapeutic protocols preclinical tumor models (7–9). For instance, clinical phase I/II that combine a strong anti-tumor effect to eradicate known studies using a set of overlapping peptides covering the E6 and E7 tumors with the induction of a systemic antitumor immune oncoproteins of human papillomavirus 16 (HPV16) have been response to eliminate distant metastases. As the immune sys- successful in patients with HPV16-induced premalignant disease tem can strongly and specifically attack targets based on the (10). This peptide vaccine formulation induced HPV16-specific principle of antigen-specificity, cancer immunotherapy aims to T cell responses in all 20 patients and resulted in clinical responses employ these characteristics of the immune system to attack in about 80% of patients and nearly 50% complete remissions and eradicate tumors. correlating with robust effector T cell immunity. However, thus A promising approach of cancer immunotherapy is therapeutic far this vaccine was not clinically effective against established vaccination using synthetic long peptides (SLP) covering T cell þ HVP16 cancer despite detectable vaccine-induced T cell responses (11, 12). This is one of the examples illustrating that 1Department of Immunohematology and Blood Transfusion, Leiden successful treatment of advanced cancer requires combination University Medical Center, Leiden, the Netherlands. 2Department of protocols, as single-treatment modalities are insufficiently effec- Radiology, Leiden University Medical Center, Leiden, the Netherlands. tive. Therapies causing immunogenic cell death are of particular 3Percuros BV, Enschede, the Netherlands. interest for combination with immunotherapy, as the reduction Note: Supplementary data for this article are available at Clinical Cancer of tumor burden and the immunogenic effects can enhance the Research Online (http://clincancerres.aacrjournals.org/). efficacy of immunotherapy. Combinations of immunotherapy Corresponding Author: Ferry Ossendorp, Leiden University Medical Center, with conventional cancer therapies such as chemotherapy or Albinusdreef 2, 2300 RC Leiden, the Netherlands. Phone: 31-71-526-3843; Fax: radiotherapy are already under investigation. In this study, we 31-71-526-5267; E-mail: [email protected] examine the use of photodynamic therapy (PDT), a tumor abla- doi: 10.1158/1078-0432.CCR-15-0515 tion method that is widely clinically applied for various prema- 2015 American Association for Cancer Research. lignant and malignant lesions.

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petent C57BL/6 mice) were bred in the animal breeding facility Translational Relevance of the Leiden University Medical Center, the Netherlands. All Cancer immunotherapy has shown promising results experiments were approved by the animal ethical committee of although a significant proportion of patients respond poorly the University of Leiden. The TC-1 mouse tumor cell line (a gift or relapse at a later stage, therefore more potent combination from T.C. Wu, Johns Hopkins University, Baltimore, MD) expres- therapies are required. Tumor ablation by photodynamic sing HPV16 E6 and E7 oncoproteins was generated as previously therapy (PDT) can strongly reduce tumor mass and induce described (32). RMA is a mutagenized derivative of RBL-5, a the release of tumor antigen and pro-inflammatory mediators, Rauscher MuLV-induced T-cell lymphoma line of C57BL/6 origin therefore being an attractive option for combination with (34). Cell lines were assured to be free of rodent viruses and immunotherapy. In this preclinical study, we show that Mycoplasma by regular PCR analysis. Authentication of the cell tumor-specific immunotherapy by synthetic long peptide lines was done by antigen-specific T-cell recognition and cells of (SLP) vaccination can be efficiently combined with PDT, low passage number were used for all experiments. TC-1 cells were leading to eradication of established tumors based on strong cultured as previously described (35). RMA cells were cultured in þ local tumor ablation and the induction of a CD8 T cell IMDM (Lonza) containing 8% FCS (Greiner), 100 IU/mL peni- response. PDT and SLP vaccination are independently already cillin/streptomycin (Gibco), 2 mmol/L glutamin (Gibco), and applied in the clinic, allowing a swift translation for poten- 25 mmol/L 2-mercaptoethanol. For tumor inoculation, 100,000 tially a large group of cancer patients. TC-1 or 1,000 RMA tumor cells in 100 mL PBS were injected subcutaneously in the right flank of the mice. For tumor rechallenge, the identical injection was given in the left flank to distin- guish possible outgrowth from regrowth of the original tumor. In PDT, an inactive light-sensitive molecule called photosen- For double-tumor experiments, an identical TC-1 inoculation was fl sitizer is administered and subsequently activated by irradiation given in the left ank 3 days after primary tumor inoculation, to of the target area with visible light of a specific wavelength. The mimic the clinical situation of a large primary tumor with smaller activated photosensitizer reacts with oxygen to form reactive metastases. Tumors were measured 3 times per week with a caliper oxygen species, which induce tumor cell death and vascular and the volume was calculated by multiplying the tumor dia- shutdown (13, 14). Besides direct cytotoxic effects on tumor cells, meters in three dimensions. Survival curves are based on the moment of sacrificing the mice upon reaching the maximally PDT has been shown to cause the release of antigen and immu- 3 nogenic factors such as damage-associated molecular patterns allowed tumor volume of 2,000 mm . (DAMP) from dying tumor cells (15–25). These immunologic in vitro effects make PDT an attractive option for combinations with Photosensitizer uptake and irradiation In vitro immunotherapy in the treatment of advanced tumors. Here, we Bremachlorin uptake by tumor cells was analyzed by use Bremachlorin, also known as Radachlorin, a novel photosen- incubating TC-1 tumor cells with Bremachlorin at the dose and sitizer that benefits from improved pharmacokinetics and high- time as indicated, washing the cells in PBS, and measuring the fl fl wavelength irradiation reaching deeper tissue. Bremachlorin is Bremachlorin uorescence compared with control cells by ow In vivo currently being tested in clinical trials for basal cell carcinoma and cytometry (BD Calibur, emission channel FL4). Brema- non–small cell lung carcinoma (26–31). chlorin uptake by tumors was visualized using a Pearl Impulse in vitro In this study we investigated the combination of Bremachlorin- imager (Li-cor). For photodynamic treatment ,TC-1 m based PDT with therapeutic peptide vaccination in two mouse tumor cells were incubated with 1 g/mL Bremachlorin for 3 models of highly aggressive subcutaneous tumors. The tumor line hours in 24-well plates, then the cells were washed with PBS to TC-1 expresses the E6 and E7 oncoproteins of HPV16 as a model remove all free photosensitizer, and fresh medium was added. Irradiation of the whole well followed immediately for 2 for human HPV16-induced tumors, and has previously been 2 2 shown to be sensitive for Bremachlorin-PDT (32, 33). RMA is an minutes at 116 mW/cm (14 J/cm ) using a 662 nm Milon aggressive T-cell lymphoma cell line induced by Rauscher murine Lakhta laser. leukemia virus (MuLV; ref. 34). We show that PDT strongly ablated established fast-growing tumors, leading to a significantly PDT þ longer survival and specific CD8 T cell responses against the Tumors were treated 9 days (TC-1) or 14 days (RMA) after tumor. Combining PDT with therapeutic peptide vaccination inoculation, both at an average tumor diameter of 5 mm. First, 20 efficiently eradicated established tumors, which was dependent mg/kg Bremachlorin photosensitizer (RadaPharma International, þ m on the presence of CD8 T cells. Importantly, combination 0.35% in SWFI, average 100 L) was injected intravenously in the treatment of primary tumors led to subsequent eradication of tail vein, followed by irradiation of the tumor 6 hours later using a distant established secondary tumors and provided protection 662 nm Milon Lakhta laser. The 6-hour interval was selected based – against repeated tumor challenge. Therefore, this successful com- on drug light timing experiments, and has been described to bination of PDT and therapeutic vaccination, resulting in robust result in a predominantly vascular localization of Bremachlorin (29). A continuous irradiation protocol of 1,000 seconds at 116 anti-tumor response and immunologic memory, suggests a novel 2 2 therapeutic combination strategy for advanced cancer. mW/cm (116 J/cm ) was used based on optimization experiments (data not shown). For irradiation, the skin in the tumor area was shaved and the mice were anaesthetized by inhalation of Materials and Methods isoflurane and positioned horizontally on a heat mat. Precision Mice and cell lines irradiation of the tumor was ensured by using a fiber fixed Wild-type C57BL/6 mice were obtained from Charles River vertically above the mouse, and the exposed area was precisely Laboratories. Albino B6 mice (tyrosinase-deficient immunocom- adjusted using a diaphragm.

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þ Serum analysis for HMGB1 CD8 T cell depletion Serum was obtained from blood samples taken 1 hour Hybridoma cells producing depleting CD8 mAb (clone 2.43) after PDT treatment, or at the same time for untreated con- were cultured in Protein-Free Hybridoma Medium (Gibco), and þ trols. The HMGB1 serum level was determined by a sandwich mAbs were purified using a Protein G column. To deplete CD8 T ELISA kit (IBL International) following the manufacturer's cells, mice received an i.p. injection of 150 mg anti-CD8 antibodies protocol. on day 8 after tumor inoculation, followed by periodical depletion by 50 mg antibody every 5 days until day 30 after tumor Ex vivo lymph node analysis inoculation. All control mice received in parallel similar amounts TC-1 tumor–bearing animals received the standard PDT treat- of isotype control rat IgG. Efficient T cell depletion was assured by ment as described above, and were sacrificed after 6 days and the flow cytometry analysis of blood lymphocytes stained for cell tumor-draining inguinal lymph node was obtained, together surface expression of CD8. with the contralateral inguinal lymph node. The lymph nodes were incubated with 2.5 mg/mL Liberase TL (Roche) for 20 Statistical analysis minutes at 37 C and single-cell suspensions were made using Statistical analysis was performed using the GraphPad Prism 70-mm cell strainers (BD Biosciences). The cells were then version 5.0 software. Data are shown as mean SEM for each stained with fluorescently labeled antibodies against CD3e, group, and comparison of groups was performed by two-tailed CD8a, CD11c, and with 7-AAD- and APC-labeled tetramer for Student t test, with the exception of survival curves, which were flow cytometry analysis. compared using the LogRank Mantel–Cox test. Statistical differ- ences were considered significant at P value of <0.05. Flow cytometry All flow cytometry analyses were performed by suspending cells in FACS buffer (PBS with 0.5% BSA and 0.02% sodium azide) and Results analysis on a BD FACS Calibur. Antibodies against CD3, CD8, or Efficient photosensitizer uptake allows strong tumor ablation CD11c and the dyes Annexin V and 7-AAD were purchased from For effective PDT, sufficient photosensitizer uptake by tumor BD, eBioscience, and BioLegend. The APC-labeled H-2Db RAHY- cells is required to ensure irradiation-induced cell death. Both TC- NIVTF tetramer was own production. 1 and RMA tumor cells showed a dose-dependent uptake after incubation with Bremachlorin (Supplementary Fig. S1a). Irradi- SLP vaccination ation of Bremachlorin-treated TC-1 cells using visible light The SLP vaccine for TC-1 (sequence GQAEPDRAHY- resulted in >98% cell death based on Annexin V and 7-AAD NIVTFCCKCDSTLRLCVQSTHVDIR), including both a CD4 and analysis, which was completely dependent on the presence of a CD8 epitope from the HPV16 E7 oncoprotein, was given on both the photosensitizer and the irradiation (Supplementary Fig. days 7 and 21 after tumor inoculation by injecting 150 mg S1b). Photosensitizer uptake in established tumors was shown by peptide subcutaneously in the left flank of the mouse (35). The intravenously injecting mice bearing subcutaneous TC-1 or RMA peptide was dissolved in 100 mL PBS and mixed 1:1 with tumors with Bremachlorin, which after 6 hours accumulated in Incomplete Freund's Adjuvant (IFA), which was then emulsi- the tumor area (Supplementary Fig. S2). To analyze whether this fied for 30 minutes on a vortex. Subsequent experiments photosensitizer accumulation is sufficient for photodynamic included only one dose of peptide vaccination, based on our ablation, growing TC-1 tumors with a diameter of 5 mm were unpublished data that the second dose has no additional effect. irradiated with a focused laser beam 6 hours after injection of In the experiments of Fig. 3 where a secondary tumor was Bremachlorin. After a clear inflammatory reaction in the treated inoculated on day 3, peptide vaccination was delayed by 1 day area in the first days after PDT, a strongly flattened tumor lesion to allow the secondary tumor to become established. As both remained with a necrotic appearance. This resulted in a significant flanks were occupied by tumors, the vaccine was administered delay in tumor growth of at least 7 days, after which tumor in the tail-base region, which was found to be an excellent outgrowth resumed with a growth rate similar to untreated vaccination site (unpublished data, Kleinovink and collea- tumors (Fig. 1A). gues). The peptide vaccine for RMA tumors contains epitopes from Rauscher MuLV and existed of a single vaccination on day PDT induces an anti-tumor immune response 14 containing 20 nmole of the Env-encoded CD4 epitope As we aimed to use Bremachlorin-based PDT in combination EPLTSLTPRCNTAWNRLKL and 50 nmole of the Gag-encoded with immunotherapy, we analyzed the immunologic effects of CD8 epitope CCLCLTVFL (36) complemented with 20 mgCpG PDT in our model. It has previously been shown that PDT can (ODN 1826, Invivogen), in 100 mL PBS subcutaneously in the contribute to antitumor immune responses through the release of tail-base region. DAMPs, such as HMGB1 (17, 18). Serum analysis of TC-1 tumor- bearing mice 1 hour after PDT showed a significant increase in þ Systemic blood analysis for specific CD8 T cell response HMGB1 compared with untreated mice (Fig. 1B). To investigate þ The systemic tumor-specific CD8 T cell response was deter- the immunologic consequences of the massive tumor cell death mined by taking venous blood samples from the tail vein 8 days induced by PDT, we analyzed the tumor-draining lymph nodes after peptide vaccination or on the same day for nonvaccinated 6 days after PDT treatment of TC-1 tumors and compared them animals. After erythrocyte lysis of the blood samples, the tumor- with contralateral lymph nodes not draining the irradiated tumor þ þ specific CD8 T cell response was determined by flow cytometry area. PDT induced a strong tumor antigen-specificCD8 T cell analysis after staining of the cells with CD3e, CD8b, and APC- response in the tumor-draining lymph nodes, accompanied by a þ conjugated tetramers for the relevant peptide–MHC complex on significant increase in the total number of CD8 T cells, which þ the CD8 T cell. was not increased in the nondraining nodes of the same animals

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A B 80 2,000 ** 1,800 Control ) 3 1,600 PDT 60 Figure 1. PDT strongly delays tumor outgrowth 1,400 and induces an immune response 1,200 against the tumor. A, tumor outgrowth 40 curves of subcutaneous TC-1 tumors in 1,000 BL/6 mice treated with PDT on day 9 800 (arrow) after tumor inoculation, compared with untreated control Tumor volume (mm 600 20 tumors. Pooled data of two 400 independent experiments, n ¼ 10 to HMGB1 serum level (ng/mL) 12 mice. B, ELISA serum analysis for 200 HMGB1 in 9 mice at 1 hour after PDT 0 0 versus untreated control mice. Pooled 5 10 15 20 25 30 35 data of two independent experiments, Days after tumor inoculation PDT n ¼ 9 mice. C, flow cytometry analysis Control of TC-1 tumor-draining lymph nodes (dLN) or contralateral nondraining lymph nodes (ndLN) of 4 mice at 6 days + + + C CD8 T cells Tumor-specific CD8 T cells CD11c cells after PDT in comparison to untreated 160,000 1,000 4,000 control mice (Ctrl). Single-cell ** ** * suspensions from lymph nodes were 140,000 stained for CD3e, CD8a, CD11c, and the 800 120,000 3,000 Db-RAHYNIVTF Tetramer (Tm) for fi 100,000 the tumor antigen-speci c T cell 600 receptor. Y-axes show absolute 80,000 2,000 numbers of total CD8þ T cells þ þ 60,000 400 (CD3 CD8 ), tumor-antigen-specific þ þ þ þ Number of cells

Number of cells CD8 T cells (CD3 CD8 Tm ), or

40,000 Number of cells 1,000 þ 200 CD11c cells. Statistical analysis by 20,000 Student t test, signiﬁcance is indicated by asterisks: , P < 0.05; , P < 0.01. 0 0 0 Ctrl PDT Ctrl PDT Ctrl PDT Ctrl PDT Ctrl PDT Ctrl PDT ndLN dLN ndLN dLN ndLN dLN

(Fig. 1C). Untreated tumor-bearing mice mounted only a minimal cells injected at a distant location 2 to 3 months after primary þ CD8 T cell response against the tumor, quantitatively similar to curative treatment, indicating the induction of protective systemic nondraining lymph nodes of PDT-treated mice. Strikingly, also the immunity (Supplementary Fig. S4A). To investigate whether þ numbers of CD11c dendritic cells (DC) were strongly increased in combination therapy can also eradicate existing established dis- the draining nodes of the PDT-treated tumor, suggesting that the tant tumors, mice were inoculated with TC-1 tumors in both DC facilitate cross-presentation of tumor-associated antigen to T flanks followed by combination therapy where PDT was only cells in local lymphoid organs to stimulate antitumor responses. applied on the primary tumor in the right flank, as depicted in Supplementary Fig. S4B. The outgrowth of secondary tumors was Combination of PDT and therapeutic vaccination eradicates not delayed by PDT of the contralateral primary tumor (Fig. 3A). established tumors Mice treated by peptide vaccination showed an initial regression Altogether, the strong tumor ablation and beneficial immuno- of both primary and secondary tumors, after which nearly all logic effects of Bremachlorin-PDT make it an attractive candidate tumors resumed to grow out. In contrast, combination treatment for combination with immunotherapy. As we have previously of PDT and peptide vaccination caused definite cure from both shown that the TC-1 tumor model is susceptible to therapeutic primary and secondary tumors in over 30% of mice, significantly SLP vaccination (7), we combined Bremachlorin-PDT with SLP higher than peptide vaccination alone (Fig. 3B). vaccination following the experimental setup depicted in Sup- þ plementary Fig. S3. Single treatments of PDT or peptide vaccina- Treatment-induced anti-tumor CD8 T cells are essential for tion of established TC-1 tumors each resulted in a significant delay therapeutic efficacy in tumor outgrowth and increased survival, but neither treatment As we found that PDT induces a local immune response in was curative. However, when PDT was combined with SLP vac- lymph nodes and that combination therapy using local PDT is cination, overall survival was strongly increased and over one also able to cure distant secondary tumors, we analyzed the þ third of mice were cured (Fig. 2). systemic CD8 T cell response against the tumor. Using specific þ MHC tetramer staining to identify tumor antigen-specific CD8 T Combination treatment protects against tumor rechallenge and cells, we could show that SLP vaccination raised the levels of þ eradicates established secondary tumors CD8 T cells specific for the HVP16 E7 epitope used for vacci- All mice cured from their TC-1 tumor after combination ther- nation in circulating blood as we have reported previously apy of PDT and SLP vaccination subsequently rejected TC-1 tumor (Fig. 4A; ref. 7). Importantly, also PDT significantly increased

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A Control PDT 2,000 2,000 ) ) 1,800 1,800 3 3 1,600 0/10 1,600 0/12 1,400 1,400 1,200 1,200 1,000 1,000 800 800 600 600 400 400 Tumor volume (mm Tumor volume (mm 200 200 0 0 0 10 20 30 40 50 60 70 80 90 100110120 0 10 20 30 40 50 60 70 80 90 100110120 Days after tumor inoculation Days after tumor inoculation

Figure 2. Peptide PDT + Peptide Curative combination treatment of 2,000 2,000 ) 3 ) established TC-1 tumors by PDT and 1,800 3 1,800 synthetic long peptide vaccination. A, 1,600 0/16 1,600 5/15 tumor outgrowth curves of TC-1 tumor- 1,400 1,400 bearing mice treated with PDT, peptide 1,200 1,200 vaccination, or combined treatment, 1,000 1,000 compared with untreated control 800 800 tumors. PDT was done on day 9 after tumor inoculation (arrows), peptide 600 600 Tumor volume (mm was administered subcutaneously in 400 Tumor volume (mm 400 IFA in the contralateral ﬂank on day 7 200 200 and 21. Pooled data of two independent 0 0 experiments, 10 to 16 mice. The 0 10 20 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60 70 80 90 100110120 fractions of mice that cleared the tumor Days after tumor inoculation Days after tumor inoculation are indicated. B, corresponding survival curves. All long-term surviving B mice had cleared their tumor on day 32 100 Control or earlier, and remained tumor-free throughout the experiment. Survival 90 PDT curve statistics by LogRank x2 test. 80 Statistical signiﬁcance is indicated by Peptide asterisks: , P < 0.001. 70 PDT + peptide 60 *** 50

40 % Survival 30 *** 20

0 0 10 20 30 40 50 60 70 80 120 Days after tumor inoculation

þ þ percentage of tumor antigen-specific CD8 T cells circulating in treatment was abrogated, suggesting a crucial role of CD8 T cells blood, supporting the immunogenic effects of PDT described in this combination treatment protocol (Fig. 4B). earlier. Moreover, PDT even further increased the SLP-induced þ CD8 T cell response, reflecting the efficacy of combination Efficient PDT–vaccination combination in virally induced treatment in tumor control. To analyze whether these tumor- lymphoma tumors þ specific CD8 T cells are responsible for the observed tumor Next, we applied PDT and peptide vaccination in another control, TC-1 tumor-bearing mice treated with PDT and SLP aggressive tumor system, the RMA lymphoma model for which þ vaccination were depleted of all CD8 cells using an anti-CD8 we have previously described efficient prophylactic peptide vac- antibody. Periodical screening of systemic venous blood con- cination, which prevented tumor outgrowth through the effects of þ þ þ firmed a persisting reduction in the number of CD8 T cells of both CD4 and CD8 T cells (36). Previous attempts in our group over 98% during the experiment (data not shown). In the absence to treat established RMA tumors by therapeutic peptide vaccina- þ of CD8 T cells, the curative effect of PDT and SLP combination tion have never been successful. Here, we show that combination

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A Day 0 3 8 9

TC-1 tumor inoculation Peptide vaccination PDT

Control PDT Peptide PDT + peptide

2,000 2,000 2,000 2,000

) 1,800 1,800 1,800 1,800 3 Primary tumor 1,600 1,600 1,600 1,600 1,400 Secondary tumor 1,400 1,400 1,400 1,200 1,200 1,200 1,200 1,000 1,000 1,000 1,000 800 800 800 800 600 600 600 600 400 0/14 400 0/16 400 6/35 400 11/34 Tumor volume (mm 200 200 200 200 0 0 0 0 0203040506070809010010 0203040506070809010010 0203040506070809010010 0203040506070809010010 Days after tumor inoculation Days after tumor inoculation Days after tumor inoculation Days after tumor inoculation

B 100 Control 90 PDT 80 Peptide 70 PDT + peptide 60

40 % Survival 30

20 *

0 0 10 20 30 40 50 60 70 80 90 100 Days after tumor inoculation

Figure 3. Combination treatment of primary tumors leads to durable eradication of distant tumors. A, tumor outgrowth curves of mice bearing established subcutaneous TC-1 tumors in both flanks, treated with systemic peptide vaccination on day 8 followed by PDT of only the primary tumor in the right flank on day 9 (arrows). Primary tumors (gray lines) were inoculated on day 0 in the right flank, secondary tumors (black lines) on day 3 in the left flank. The fractions of mice that cleared both tumors are indicated. B, corresponding survival curves. All long-term surviving mice had cleared both tumors on day 35 or earlier, and remained tumor-free throughout the experiment. Statistical analysis by LogRank x2 test, statistical significance is indicated by asterisks: , P < 0.05.

of Bremachlorin-PDT and therapeutic peptide vaccination in mice of established tumors using Bremachlorin-PDT strongly reduces bearing subcutaneous RMA tumors resulted in significantly pro- tumor burden and at the same time induces antitumor T cell longed survival compared with either single treatment alone, responses, which was significantly enhanced when combined similar to our observations in the TC-1 model (Fig. 5). All mice with therapeutic long peptide vaccination. Importantly, the sys- þ cured of their primary tumor were able to reject RMA tumor cells temic antitumor CD8 T cell response induced by combination upon rechallenge at a distant location over 2 months after treatment was essential for the therapeutic effect, and likely treatment (data not shown), suggesting that also in this model provided long-term protection because all cured mice did not PDT and peptide vaccination induced systemic immunity against develop a tumor after renewed injection of tumor cells at a the tumor. different body site. The relevance of the systemic immune response was emphasized by the eradication of distant secondary tumors after combination therapy of primary tumors. Our com- Discussion bination protocol therefore meets the requirements of an efficient In this study we suggest a novel therapeutic combination treatment strategy for advanced cancer that we discussed earlier: a strategy for advanced metastatic cancer, consisting of PDT-medi- strong anti-tumor effect to eradicate known tumors, and a lasting ated tumor ablation and tumor-specific peptide vaccination. In systemic immune response to identify possible metastatic tumor two independent aggressive tumor models we show that ablation sites.

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A B 100 0.4 Control 90 PDT + peptide α * 80 PDT + peptide + CD8 0.3 70

T cells T 60 + *** ***

0.2 50 ** 40 % Survival 30

% Specific CD8 0.1 20

0.0 0 T 0 15 20 25 30 35 40 45 50 55 80 PD Days after tumor inoculation Control Peptide + peptide T PD

Figure 4. þ The strong effect of combination treatment is dependent on a treatment-induced systemic CD8 T cell response against the tumor. A, Tetramer þ staining showing the percentage of CD8 T cells in tail vein blood that is specific for the HPV16 E7 epitope expressed by TC-1 tumor cells, 8 days after þ treatment. B, survival curves of TC-1 tumor-bearing mice treated with PDT and peptide vaccination during antibody-mediated depletion of CD8 T cells. PDT was performed on day 9 after tumor inoculation, peptide was administered subcutaneously in IFA in the contralateral flank on day 7 and 21. Depleting antibody was administered i.p. regularly from day 8 to day 45, resulting in >98% depletion of CD8þ cells in the blood within 24 hours after injection. Pooled data of individual experiments with a total of 16 to 24 mice per group. All long-term surviving mice had cleared their tumor on day 25 or earlier, and remained tumor-free throughout the experiment. Statistical analysis of (A) by the Student t test and of B by the LogRank x2 test. Statistical significance is indicated by asterisks: , P < 0.05; , P < 0.01; , P < 0.001.

PDT, like other tumor ablation therapies, aims to strongly around 15% to 20% of human cancer is estimated to be virally affect tumor cells although minimizing damage to healthy induced (38, 39). Importantly, the application of combination tissue. The nontoxic nature of the two individual components therapy using PDT and SLP vaccination may theoretically be of PDT, the photosensitizer and the irradiation with visible extended to virtually any type of cancer, as was illustrated by light, allows precise restriction of the photodynamic effect to recent studies identifying neo-epitopes, which are mutated the target region. Pharmacokinetic optimization of photosen- sequences in "self" proteins acquired by tumor cells, allowing sitizers has been aimed at a better accumulation in tumors and "nonself"-peptide/MHC recognition by specificTcells.Ther- a faster clearance from other tissues. This has led to new apeutic vaccination with long peptides containing these generations of photosensitizers, which moreover are opti- tumor-specific neo-epitopes has proven successful in preclin- mized for the use of higher wavelength irradiation light. This ical studies (5, 6). increases the effect range of PDT, as a higher wavelength of Our findings in the TC-1 mouse model for HPV16-induced light penetrates deeper through tissue. The use of flexible human tumors are of particular interest as PDT is currently interstitial optical fibers allows both precision irradiation of already clinically studied in the treatment of HPV16-induced complexly localized tumors and the treatment of bulky tumor gynecologic lesions (40–42). These studies used topical admin- masses (13). istration of the second-generation photosensitizer 5-ALA, and Severalanimaltumormodelsexpressingknowntumor reported inefficient photosensitizer distribution through the antigens are available to study the immunologic effects of target tissue leading to incomplete responses. The use of novel PDTonanantigen-specific level; however, many concern photosensitizers such as Bremachlorin may help to resolve this artificially introduced model antigens such as chicken ovalbu- issue. Combination treatments of PDT and immunotherapy to min which have no clinical relevance (19). To overcome this improve the therapeutic effect are being investigated preclini- limitation, a recent study used a murine mastocytoma tumor cally and clinically using nonspecific immunostimulatory expressing P1A, the mouse homologue of human MAGE agents (43–45). However, tumor-specific immunotherapy such cancer/testis antigens, and showed antigen-specific immune as therapeutic peptide vaccination with HPV antigens may be responses against this clinically relevant tumor antigen and preferred to ensure a stronger and target-specific effect. Alter- corresponding effects on tumor growth (37). In this study, we natively, to overcome the tumor-mediated immune suppres- used two mouse tumor models expressing known epitopes sion, T cell checkpoint blocking antibodies form an attractive from oncogenic viruses as a model for HPV- or leukemia virus– therapeutic option for combination with PDT in order to boost induced cancer in humans, and showed successful combina- the antitumor T cell response and relieve the immune system tion treatment of these tumors by PDT and peptide vaccina- from suppression (46, 47). Taken together, this successful tion. These tumor models expressing viral epitopes are of combination of systemic immunotherapy and local tumor clinically relevance to a large group of cancer patients as ablation, which are independently already clinically applied,

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Control PDT A 2,000 2,000 1,800 1,800 ) ) 3 3 0/17 3/15 1,600 1,600 1,400 1,400 1,200 1,200 1,000 1,000 800 800 600

600 Tumor volume (mm Tumor volume (mm 400 400 200 200 0 0 10 15 20 25 30 35 40 45 50 55 60 10 15 20 25 30 35 40 45 50 55 60 Days after tumor inoculation Days after tumor inoculation

Peptide PDT + peptide 2,000 2,000 1,800 1,800 ) ) 3 1,600 1/16 3 1,600 7/18 Figure 5. 1,400 1,400 Therapeutic treatment of murine 1,200 1,200 leukemia virus-induced lymphoma by 1,000 1,000 PDT and tumor-speciﬁc peptide 800 800 vaccination. A, tumor outgrowth curves – 600 of RMA tumor bearing mice treated

Tumor volume (mm 600 Tumor volume (mm with PDT, peptide vaccination, or 400 400 combined therapy, compared with 200 200 untreated control tumors. PDT was 0 0 given on day 14 after tumor inoculation, 10 15 20 25 30 35 40 45 50 55 60 10 15 20 25 30 35 40 45 50 55 60 the peptide vaccine was mixed with Days after tumor inoculation Days after tumor inoculation CpG and administered subcutaneously in the tail-base in PBS on day 12. The fractions of mice that cleared the tumor B 100 Control are indicated. B, corresponding survival curves. All long-term surviving mice had 90 PDT cleared their tumor on day 38 or earlier, Peptide and remained tumor-free throughout the experiment. Survival curve statistics 80 2 *** PDT + peptide by LogRank x test, statistical signiﬁcance is indicated by asterisks: 70 , P < 0.05; , P < 0.01; , P < 0.001.

60 **

% Survival 40 ** * 30

0 0 15 20 25 30 35 40 45 60 Days after tumor inoculation

proposes an attractive clinical treatment strategy for advanced Acquisition of data (provided animals, acquired and managed patients, cancer. provided facilities, etc.): J.W. Kleinovink, P.B. van Driel, T.J. Snoeks, N. Prokopi, M.F. Fransen ﬂ Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Disclosure of Potential Con icts of Interest computational analysis): J.W. Kleinovink, P.B. van Driel, T.J. Snoeks, N. ﬂ No potential con icts of interest were disclosed. Prokopi, M.F. Fransen, L. Mezzanotte, F. Ossendorp Writing, review, and/or revision of the manuscript: J.W. Kleinovink, P.B. van Authors' Contributions Driel, M.F. Fransen, L.J. Cruz, L. Mezzanotte, A. Chan, C.W. Lowik,€ F. Ossendorp Conception and design: J.W. Kleinovink, P.B. van Driel, L.J. Cruz, F. Ossendorp Administrative, technical, or material support (i.e., reporting or organizing Development of methodology: J.W. Kleinovink, P.B. van Driel, T.J. Snoeks, L.J. data, constructing databases): J.W. Kleinovink, A. Chan, F. Ossendorp Cruz, C.W. Lowik,€ F. Ossendorp Study supervision: L.J. Cruz, C.W. Lowik,€ F. Ossendorp

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Combined Photodynamic Therapy–Immunotherapy against Advanced Cancer

Acknowledgments advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate The authors would like to thank A. Reshetnikov and H. Vink for expertise this fact. and supply of Bremachlorin photosensitizer; W. Benckhuijsen, N. Dolezal, and J.W. Drijfhout for providing synthetic peptides; and K. Franken for providing MHC–peptide tetramers. The costs of publication of this article were defrayed in part by the Received March 3, 2015; revised October 15, 2015; accepted October 16, payment of page charges. This article must therefore be hereby marked 2015; published OnlineFirst November 6, 2015.

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Combination of Photodynamic Therapy and Specific Immunotherapy Efficiently Eradicates Established Tumors

Jan Willem Kleinovink, Pieter B. van Driel, Thomas J. Snoeks, et al.

Clin Cancer Res Published OnlineFirst November 6, 2015.

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