CD27 Agonism Plus PD-1 Blockade Recapitulates

Published OnlineFirst March 28, 2016; DOI: 10.1158/0008-5472.CAN-15-3130

Cancer Microenvironment and Immunology Research

CD27 Agonism Plus PD-1 Blockade Recapitulates CD4þ T-cell Help in Therapeutic Anticancer Vaccination Tomasz Ahrends1, Nikolina Baba˛ ła1, Yanling Xiao1, Hideo Yagita2, Hans van Eenennaam3, and Jannie Borst1

Abstract

While showing promise, vaccination strategies to treat cancer helper epitopes, more so than combined PD-1 and CTLA-4 require further optimization. Likely barriers to efficacy involve blockade. Combining CD27 agonism with CTLA-4 blockade cancer-associated immunosuppression and peripheral tolerance, improved vaccine-induced CTL priming and tumor infiltration, which limit the generation of effective vaccine-specific cytotoxic T but only combination with PD-1 blockade was effective at þ lymphocytes (CTL). Because CD4 T cells improve CTL respon- eradicating tumors, thereby fully recapitulating the effect of þ siveness, next-generation vaccines include helper epitopes. Here, CD4 T-cell help on vaccine efficacy. PD-1 blockade alone did þ we demonstrate in mice how CD4 T-cell help optimizes the CTL not affect CTL priming or tumor infiltration, so these results þ response to a clinically relevant DNA vaccine engineered to implied that it cooperated with CD4 T-cell help by alleviating combat human papillomavirus–expressing tumors. Inclusion of immune suppression against CTL in the tumor. Helper epitope tumor-unrelated helper epitopes greatly increased CTL priming, inclusion or CD27 agonism did not stimulate regulatory T cells, þ effector, and memory T-cell programming. CD4 T-cell help and vaccine efficacy was also improved by CD27 agonism in þ optimized the CTL response in all these aspects via CD27/CD70 the presence of CD4 T-cell help. Our findings provide a costimulation. Notably, administration of an agonistic CD27 preclinical rationale to apply CD27 agonist antibodies, either antibody could largely replace helper epitopes in promoting alone or combined with PD-1 blockade, to improve the ther- þ primary and memory CTL responses, acting directly on CD8 T apeutic efficacy of cancer vaccines and immunotherapy gener- cells. CD27 agonism improved efficacy of the vaccine without ally. Cancer Res; 76(10); 2921–31. 2016 AACR.

Introduction self-antigens, with the aid of natural (n)regulatory T cells (Treg) and their coinhibitory receptor CTLA-4 (4, 5). Therapeutic vaccination with protein or DNA, aimed at raising A key goal in therapeutic vaccination is therefore to activate DCs. a cytotoxic T lymphocyte (CTL) response to pre-existing cancer, is This can be done with adjuvants that bind to pattern-recognition an attractive form of immunotherapy. It is easy to apply, poten- þ receptors (PRR; ref. 6). CD4 T cells can also activate DCs and thus tially cheap, and can be designed to avoid autoimmune responses compensate for deﬁcient "danger" signaling (7, 8). Therefore, (1). Up to now, however, therapeutic vaccines have not been very vaccines can be optimized by inclusion of MHC Class II–binding successful, not even against immunogenic virus-induced cancers epitopes (1). Upon priming with an MHC Class I–binding epitope (1, 2). In therapeutic vaccination, dendritic cells (DC) are a key þ only, CD8 T cells are nonresponsive or even tolerized (9). How- target for antigen delivery (3). DCs are expert at cross-presenting þ ever, when (a) helper epitope(s) is included, CD4 T cells will antigens, i.e., loading peptides derived from the extracellular þ optimize ("license") DCs for CTL priming. In this way, the DC vaccine into MHC Class I molecules for presentation to CD8 þ þ translates instructions from the CD4 T cell to the CD8 T cell. T cells. Moreover, activated DCs are optimal for T-cell priming, þ CD4 T-cell help not only promotes the primary CTL response, but because they produce the relevant costimulatory molecules and also instills memory capacity into responder CTLs (7, 8). Helper cytokines. Resting DCs, however, maintain peripheral tolerance to epitope inclusion was successful in therapeutic vaccination against premalignant human papillomavirus (HPV)–induced disease (10), 1 but it has not yet delivered its promise in cancer patients (1, 11). Division of Immunology,The Netherlands Cancer Institute-Antoni van þ Leeuwenhoek, Amsterdam, the Netherlands. 2Department of Immu- Costimulation is important in the delivery of CD4 T-cell help. 3 þ nology, Juntendo University School of Medicine,Tokyo, Japan. Aduro When the CD4 T cell recognizes peptide/MHC Class II complex on Biotech Europe, Oss, the Netherlands. the DC, it upregulates CD40 ligand (L). This triggers CD40 on the Note: Supplementary data for this article are available at Cancer Research DC, which "licenses" the DC to induce a CTL response (12–14). It is Online (http://cancerres.aacrjournals.org/). not known how exactly the DC changes as a result of CD40 Corresponding Author: Jannie Borst, Netherlands Cancer Institute, Plesman- triggering and how this subsequently optimizes priming, effector, þ laan 121, Amsterdam 1066 CX, the Netherlands. Phone: 31205122056; Fax: and memory differentiation of the CD8 T cell. However, it is clear 31205122057; E-mail: [email protected] that the DC acquires costimulatory capacity and thereby promotes þ doi: 10.1158/0008-5472.CAN-15-3130 the CD8 T-cell response. Upon PRR- or CD40 stimulation, the DC 2016 American Association for Cancer Research. upregulates CD80 and CD86, the ligands of the T-cell costimulatory

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receptor CD28. The DC also starts to express CD70, the ligand of the were injected i.p. at 100 mg per mouse in 100 mL HBSS on each costimulatory receptor CD27. DC activation thus allows costimula- day of primary vaccination and in case of FR70 mAb also at day 9. þ þ tion via CD28 and CD27 that promote CD8 and CD4 T-cell Control mice were injected with equal amounts of rat IgG2b responses by complementary mechanisms (15). CD27 is a member (LTF-2) or IgG2a (2A3) isotype controls (Bio X Cell). Depleting of the TNF receptor family that also includes CD134 (OX40), anti–CD4-mAb (GK1.5; Bio X Cell) was injected i.p. at 200 mg per CD137 (4-1BB), and CD357 (GITR). All these receptors support mouse in 100 mL HBSS on day –2 and on each day of primary the T-cell response—in a large part by survival signaling (16). vaccination. Agonistic mAbs to all these receptors are in clinical trials in cancer patients (17), in the expectation that they may improve results In vivo cytotoxicity assay achieved by current CTLA-4 and PD-1 blockade (18). Splenocytes from na€ve mice were labeled ex vivo with 0.1 Thus, changing the vaccine formulation and/or adding immu- mmol/L carboxyfluorescein succinimidyl ester (CFSE; Life Tech- nomodulatory antibodies to overcome deficient DC activation nologies) and pulsed with 5 mmol/L E749-57 peptide (specific could improve CTL priming. The CTLs raised must furthermore target), or labeled with 1 mmol/L CFSE and left unpulsed overcome suppression in the tumor microenvironment, as (control). Subsequently, 5 106 cells of each population were imposed by tumor cells, regulatory Tregs, and other cell types. injected in the retro-orbital plexus of the same recipient mouse, Blocking PD-1 with therapeutic mAb can alleviate one of these and 16 hours later, spleens were analyzed by flow cytometry. suppressive mechanisms (18, 19). It has not been investigated Percent killing was calculated as follows: 100 – ([(% specific þ whether CD4 T-cell help also addresses this bottleneck in the targets in vaccinated recipients/% control targets in vaccinated CTL response. recipients)/(% specific targets in control recipients/% control þ Here, we examined by which mechanisms CD4 T-cell help targets in control recipients)] x 100). improves the CTL response, using a protocol of intraepidermal DNA vaccination. This protocol proved superior to intramuscular Tumor challenge vaccination in raising a CTL response to human immunodefi- At day 0, mice were injected s.c. with 1 105 TC-1 tumor cells ciency virus in monkeys and is under clinical development (20, that express HPV16 E6 and E7 proteins (26). Vaccination was þ 21). We identified CD27 costimulation of CD8 T cells as the key performed on the indicated days after inoculation. Tumor growth þ effector pathway of CD4 T-cell help for the CTL response. We was measured by caliper in two dimensions. Tumor volume was demonstrate that antibody-based CD27 agonism is a very pow- calculated as: volume ¼ (width2 x length)/2. Mice were sacrificed erful combination to optimize therapeutic vaccination that acts when the tumor diameter reached 15 mm or when the tumor synergistically with PD-1 blockade. Our study provides the guide- volume exceeded 1,000 mm3. The TC-1 cell line was received in lines for rational optimization of cancer immunotherapy with 2015 from Leiden University Medical Center, and further authen- immunomodulatory antibodies alone or in combination with tication was not performed by the authors. therapeutic vaccines. Tissue preparation and flow cytometry Materials and Methods Peripheral blood cells were obtained by tail bleeding; spleen, inguinal lymph nodes, and tumors were passed through a 70 mm Mice cell strainer (BD Falcon). After erythrocyte lysis, cell suspensions Gender matched, 7- to 8-week-old C57BL/6J mice from The were washed and stained with relevant mAbs and phycoerythrin- Jackson Laboratory were used in accordance with national guide- conjugated H-2Db/E7 tetramers (22). Fluorochrome-labeled lines and as approved by the institutional Experimental Animal 49-57 mAbs employed were as follows: anti-CD8a (53-6.7), anti-CD4 Committee (DEC) and maintained in HEPA-filtered isolators in a (RM4-5), anti-IFNg (XMG1.2), anti-TNFa (TN3-19.12), all specific pathogen-free facility. from BD Biosciences or eBioscience, and anti-GZMB (CLB-GB11; Enzo Life Sciences). Prior to cytokine detection with the BD Vaccination Cytofix/Cytoperm Kit (BD Biosciences), cells were incubated for HELP-E7SH and E7SH DNA vaccines were generated as 16 hours with 1 mg/mL PADRE (AKFVAAWTLKAAA), E749-57 described (22). The amino acid sequences they encode are pro- (RAHYNIVTF), or no peptide (control) in IMDM with 8% FCS. vided in Supplementary Fig. S1. For DNA "tattoo" vaccination, the In calculating the frequency of responder cells, the signal observed hair on a hind leg was removed using depilating cream (Veet; in corresponding nonstimulated control samples was subtracted. Reckitt Benckiser) on day –1. On days 0, 3, and 6, mice were Intracellular Foxp3 and granzyme B (GZMB) staining was per- anesthetized, and 15 mL of a 2 mg/mL DNA solution in 10 mmol/L formed after cell fixation and permeabilization using the Foxp3/ Tris and 1 mmol/L EDTA, pH 8.0, was applied to the hairless skin transcription factor staining buffer set (eBioscience). Flow cyto- with a Permanent Make Up tattoo machine (MT Derm GmbH), metry was performed using the LSRFortessa (BD Biosciences), and using a sterile disposable 9-needle bar with a needle depth of data were analyzed with FlowJo software (Tree Star Inc.). Live cells 1 mm and oscillating at a frequency of 100 Hz for 45 seconds were selected based on propidium iodide- or near-infra red dye (20, 23). For rechallenge, the hair removal step was repeated at (Life Technologies) exclusion. day –1, and mice received a single DNA tattoo and an i.p. injection with 5 mg lipopolysaccharide (LPS) from E. coli 055:B5 (Sigma) in Statistical analysis 100 mL Hank's Buffered Salt Solution (HBSS) on day 0. Data were analyzed with GraphPad Prism software using unpaired two-tailed Student t test or a log-rank test. Error bars Antibody treatments in figures indicate SD. A P value < 0.05 was considered statis- Blocking mAbs to CD70 (FR70; ref. 24), CTLA-4 (9D9), and tically significant; , P < 0.05; , P < 0.005; , P < 0.001; and PD-1 (RMP1-14) or agonistic mAb to CD27 (RM27-3E5; ref. 25) , P < 0.0001.

2922 Cancer Res; 76(10) May 15, 2016 Cancer Research

CD4þ T-cell Help in Therapeutic Cancer Vaccination

A B C

E7SH (no help) HELP E7SH × ×

P30 PADRE NEF

Non-dLN Spleen Primary vaccination Days post-vaccination D E Blood dLN Non-dLN Spleen 4 10 15.2 3.9 1.1 12.3 103

102 Help 101

100 100 101 102 103 104 5.4 0.6 0.5 4.4 49-57 /E7 b No help H-2D

CD8 Days post-vaccination

Figure 1. CD4þ T-cell help optimizes the CD8þ T-cell response upon intraepidermal DNA vaccination. A, vaccine design: both "Help" and "No Help" vaccines encode a "gene-shuffled" version of HPV-16 E7. The "Help" vaccine additionally encodes human tetanus toxoid p30, PADRE, and HIV NEF epitopes (Supplementary þ Fig. S1). B–D, mice (n ¼ 10 per group) received "Help" or "No Help" vaccine on days 0, 3, and 6. The CD8 T-cell response was followed in time by flow cytometric b b þ analysis of peripheral blood with H-2D /E749-57 tetramers and anti-CD8 mAb and is depicted as percentage of H-2D /E749-57 tetramer cells among total þ b þ þ CD8 T cells (B). On day 10, 3 mice per group were sacrificed, and absolute numbers of H-2D /E749-57 tetramer CD8 T cells in dLN, non-dLN, and spleen were enumerated (C). The remaining 7 mice per group were rechallenged once with "No Help" vaccine and injected i.p. with LPS on day 50, and the CD8þ T-cell b response was subsequently followed in blood (B). Representative staining of cells from indicated organs with H-2D /E749-57 tetramer and anti-CD8 mAb at day b þ þ 10 is depicted in D. Numbers in quadrants indicate the percentage of H-2D /E749-57 tetramer cells within total CD8 T cells. Results are representative of three experiments. E, mice (n ¼ 5 per group) were challenged with "Help" or "No Help" vaccine and treated with control mAb or depleting aCD4 mAb. Percentages of b þ þ H-2D /E749-57 tetramer cells were measured among total CD8 T cells in blood over time. Results are representative of two experiments.

To validate the system, we first examined effects of helper Results þ þ epitope inclusion on the primary CD8 T-cell response. "Help" Intraepidermal DNA vaccination reveals the effects of CD4 b þ clearly increased the magnitude of the primary H-2D /E749-57- T-cell help for the CD8 T-cell response þ specific CD8 T-cell response, as measured in blood up to day 50 In our vaccination strategy, naked DNA encoding the antigen of (Fig. 1B and D). At the peak of the response at day 10, "Help" had þ interest is "tattooed" into the skin (20). This results in keratinocyte also significantly increased the total numbers of E7-specific CD8 transfection and delivery of antigen to the draining lymph nodes T cells in dLNs, non-dLNs, and spleen (Fig. 1C and D). Thus, þ þ (dLN; ref. 23). The two DNA vaccines used do or do not contain CD4 T-cell help benefitted the priming of E7-specific CD8 T helper epitopes, which offered us a unique window to read out the cells and their systemic dissemination. þ effects of CD4 T-cell help on the CTL response (Fig. 1A; Sup- To test whether the "Help" vaccine could program memory þ plementary Fig. S1; ref. 22). The vaccines encode a "gene shuffled" function into CD8 T cells, mice that had been primed with version of the HPV type 16 E7 protein, including the immuno- "Help" or "No Help" vaccine were rechallenged with "No Help" b– dominant H-2D restricted epitope E749-57. In the "Help" ver- vaccine. In this setting, mice primed with "Help" vaccine had a þ sion, the vaccine additionally encodes unrelated epitopes binding much higher secondary E7-specific CD8 T-cell response than to all human MHC Class II alleles (Supplementary Fig. S1), of mice primed with "No Help" vaccine, even though neither which the p30 (27) and PADRE (28) epitopes also bind to mouse received help during rechallenge (Fig. 1B). Thus, the model þ MHC Class II. revealed the effect of CD4 T-cell help delivered during priming

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þ þ in the form of memory programming. Note that we needed to key effector pathway of CD4 T-cell help for the CD8 T-cell inject LPS i.p. as "danger signal" in conjunction with the second- response. þ ary "No Help" vaccine to reveal the effect of CD4 T-cell help on þ the memory CD8 T-cell response. The CD27/CD70 pathway dictates the generation of CTL and Next, we confirmed that increased CTL responsiveness to the þ Th1 cell responses "Help" vaccine was due to engagement of CD4 T cells. Depletion þ þ Next, we assessed the contribution of CD4 T-cell help and of CD4 cells by infusion of anti-CD4 mAb nullified the effects of CD27/CD70 pathway to the quality of the primary T-cell helper epitope inclusion on the CTL response, while it did not response. To monitor CTL differentiation, the cytolytic effector affect the response to the "No Help" vaccine (Fig. 1E). We þ molecule GZMB was detected in CD8 T cells in blood, dLN, and conclude that the intraepidermal DNA vaccination model robust- þ spleen at day 10 after vaccination. Comparison of "Help" versus ly reveals the effects of CD4 T-cell help in terms of magnitude of þ þ "No Help" vaccination indicated that CD4 T-cell help greatly the primary and memory CD8 T-cell response. þ þ increased the frequency of GZMB CD8 T cells raised (Fig. 3A þ þ and E). CD70 blocking reduced the CTL response to "Help" CD4 T-cell help increases the magnitude of the CD8 T-cell vaccination to the level seen after "No Help" vaccination. More- response via the CD27/CD70 pathway over, CD27 agonism strongly promoted the CTL response to "No We next determined the importance of CD27/CD70 costimu- þ þ Help" vaccination (Fig. 3A and E). The frequency of CD8 T cells lation as downstream effector of CD4 T-cell help. To test the þ coexpressing IFNg and TNFa was also greatly increased as a result requirement of CD27/CD70 costimulation for delivery of CD4 þ of CD4 T-cell help, reduced by CD70 blocking after "Help" T-cell help, the CD27/CD70 pathway was inhibited. Mice were vaccination and increased by CD27 agonism after "No Help" given "Help" vaccine combined with either control mAb or an vaccination (Fig. 3B and F). anti-CD70 mAb that blocks interaction with CD27 (24). At day 10 þ IFNg and TNFa expressions were also assessed in CD4 T cells. after vaccination, when the primary response in the "Help" The "Help" vaccine raised a Th1 response that was dependent on control situation was at its peak, anti-CD70 mAb treatment CD27/CD70 costimulation (Fig. 3C). CD27 agonism did not lowered the magnitude of the response to the level in the "No þ engage a CD4 T-cell response after "No Help" vaccination, as Help" situation (Fig. 2A). At later time points, the effect was less þ þ þ assessed by either single IFNg or TNFa CD4 T cells (results not pronounced, but peak response levels remained significantly þ þ þ shown) or double IFNg TNFa CD4 T cells, although we lower than in the "Help" control situation. CD70 blocking also þ þ þ þ observed a slight increase of IFNg TNFa CD4 T cells in the significantly reduced the E7-specific CD8 T-cell response in spleen (Fig. 3C). The inclusion of helper epitopes in the vaccine dLNs, non-dLNs, and spleen at day 10 (Fig. 2B; primary flow þ did not elicit the expansion of CD4 Tregs, as identified by Foxp3 cytometric data in Supplementary Fig. S2). Interestingly, blocking expression, nor did deliberate CD27 costimulation (Fig. 3D). the closely related TNF ligands 4-1BBL or OX40L did not or only þ Taken together, the data validate the effects of helper epitope marginally impede the effects of CD4 T-cell help for the CTL inclusion on the Th1 and CTL response and show that these response (Supplementary Fig. S3). responses are greatly dependent on the CD27/CD70 pathway. Next, we tested whether deliberate CD27 costimulation þ could overcome the requirement for CD4 T-cell help for the þ þ CD8 T-cell response. For this purpose, mice were given "No CD4 T-cell help and the CD27/CD70 pathway dictate Help" vaccine combined with either a control mAb or a CD27 antitumor efficacy of therapeutic vaccination agonist mAb that stimulates CD27 function in the absence of CTL function was tested by in vivo cytotoxicity assays. In the first CD70 binding (25). CD27 agonism significantly increased the assay, we injected syngeneic splenocytes as target cells that had þ magnitude of the CD8 T-cell response to "No Help" vaccina- been loaded with control or E749-57 peptide and labeled with tion as monitored in the blood, throughout the entire kinetics either a high or low dose of CFSE (29). This assay clearly revealed þ of the primary response (Fig. 2C). At the peak of the response the effect of CD4 T-cell help for the CTL response: About 90% of þ on day 10, E7-specificCD8 T-cell numbers in dLN and spleen the E749-57 peptide-loaded target cells were killed in the group that were also greatly increased as a result of CD27 agonism had received "Help" vaccine, whereas less than 10% of the target (Fig. 2D). After CD4-cell depletion with specificmAb,CD27 cells were killed in mice that had received "No Help" vaccine þ agonism elevated the CD8 T-cell response to the "No help" (Fig. 4A and B). Furthermore, CD70 blockade upon "Help" vaccine even more (Fig. 2E). We conclude therefore that delib- vaccination reduced CTL activity to the levels seen in the "No erate CD27 costimulation largely bypasses the requirement of Help" control group, whereas CD27 agonism upon "No Help" þ þ CD4 T-cell help for CD8 T-cell priming. vaccination raised CTL activity to the level seen in the "Help" To determine the effect of CD27/CD70 costimulation on control group (Fig. 4A and B). These data prove that the CD27/ þ memory programming, mice were rechallenged at day 50 with CD70 costimulatory pathway is the key effector pathway of CD4 "No Help" vaccine. In the mice that had been primed with T-cell help for the CTL response. þ "Help" vaccine and treated with blocking mAb to CD70, the We next examined the contribution of CD4 T-cell help and the memory response did not exceed the response to "No Help" CD27/CD70 pathway to the antitumor effect of the "Help" vaccine, even at its peak. Thus, "memory programming" as a vaccine. For this purpose, TC-1 tumor cells expressing HPV E7 þ result of CD4 T-cell help greatly depends on CD27/CD70 protein (26) were implanted 5 days prior to vaccination. The costimulation (Fig. 2F). Strikingly, in mice that had been tumor reached a palpable size while the response to vaccination primed with "No Help" vaccine and treated with agonist mAb was ongoing, which allowed us to evaluate tumor rejection to CD27, the memory response was comparable in magnitude (Fig. 4C). In this setting, all mice that had received the "Help" with that in mice primed with "Help" vaccine (Fig. 2F). The vaccine experienced complete tumor regression (Fig. 4C) and combined data indicate that CD27/CD70 costimulation is the survived long-term (Fig. 4D), whereas all mice that had received

2924 Cancer Res; 76(10) May 15, 2016 Cancer Research

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Ahrends et al.

A B

No help No help

Blood Spleen Blood Spleen C D

No help No help

Blood Spleen Blood Spleen E F

Figure 3. þ þ Generation of effector CD4 T cells and CD8 T cells depends on CD27/CD70 costimulation. Mice (n ¼ 3 per group) received "Help" or "No Help" vaccine on days 0, 3, and 6 in combination with isotype control, blocking aCD70, or agonistic aCD27 mAb. On day 10, ﬂow cytometric analyses were done in cells from þ þ blood, dLN, and spleen. GZMB and Foxp3 expressions were determined directly ex vivo.IFNg and TNFa expressions in CD8 or CD4 T cells were determined after 16-hour in vitro stimulation of total blood, LN, or spleen cells with MHC Class I or MHC Class II restricted peptides or no peptide (control). A, frequencies þ þ þ þ þ þ þ þ of GZMB cells within total CD8 T cells. B, frequencies of IFNg TNFa cells within total CD8 T cells. C, frequencies of IFNg TNFa cells within total CD4 T cells. þ þ D, frequencies of Foxp3 cells within total CD4 T cells. E and F, representative ﬂow cytometric analyses. Results are representative of two experiments.

þ the "No Help" vaccine had to be sacrificed due to tumor outgrowth the effects of CD4 T-cell help on antitumor efficacy. Treatment (Fig. 4C and D). CD70 blockade almost abolished the therapeutic with aCTLA-4 or aPD-1 mAb alone did not improve tumor effect of the "Help" vaccine. Conversely, the "No Help" vaccine control by "No Help" vaccination, whereas combined treatment was rendered more curative upon combination with agonistic had a modest beneficial effect that was less significant than that of CD27 mAb treatment: improved tumor control resulted in long- CD27 agonism alone (Fig. 5A and B). These data suggested that term survival of 60% of the animals (Fig. 4C and D). CD27 costimulation was unique in its effects on CTL priming. Thus, helper epitope addition converts the vaccine from being To examine this, we tested the effects of CTLA-4 or PD-1 nonfunctional to therapeutically effective, leading to 100% cura- blockade alone or in combination with CD27 agonism on CTL tion of tumor-bearing mice in this experimental setting. This priming in response to "No help" vaccination. As shown before, þ curative effect greatly depends on CD27/CD70 costimulation, as CD4 T-cell help and CD27 agonism promoted CTL priming demonstrated by CD70 blockade. Engagement of CD27 with (Fig. 6A and B). In contrast, CTLA-4 or PD-1 blockade did not. agonistic mAb renders the vaccine that lacks helper epitopes Interestingly though, combined CTLA-4 blockade and CD27 therapeutically effective, albeit less so than the vaccine with helper agonism improved CTL priming, even to a greater magnitude þ epitopes. than did CD4 T-cell help, whereas PD-1 blockade did not add to the effect of CD27 agonism (Fig. 6A and B). Despite the superior Combined CD27 agonism and PD-1 blockade recapitulates the CTL response in case of combined CTLA-4 blockade and CD27 þ therapeutic effects of CD4 T-cell help agonism, CTLA-4 blockade did not improve tumor control as Combined CTLA-4 and PD-1 blockade constitutes the current compared with CD27 agonism alone, whereas combined PD-1 breakthrough in cancer immunotherapy (18). Therefore, we blockade and CD27 agonism was 100% curative (Fig. 6C and D). examined whether CTLA-4 and/or PD-1 blockade could bypass This was remarkable, because combined CTLA-4 blockade and

2926 Cancer Res; 76(10) May 15, 2016 Cancer Research

CD4þ T-cell Help in Therapeutic Cancer Vaccination

A B

Naïve control Help No help No help

48.2 51.8 10.1 89.9 33.1 66.9 42.3 57.7 24.8 75.2 % of Max of % % of Specific killling CFSE

No help

Naïve control C No help No help No help 1,250 Help 1,250 1,250 1,250 1,000 1,000 1,000 1,000 750 750 750 750 500 500 500 500 250 250 250 250

Days after tumor challenge Days after tumor challenge Days after tumor challenge Days after tumor challenge D

No help No help % Survival

Time (days)

Figure 4. þ CD4 T-cell help increases generation of functional CTL with antitumor efficacy via the CD27/CD70 pathway. A and B, in vivo cytotoxicity assay. Mice (n ¼ 3 per group) received "Help" or "No Help" vaccine on days 0, 3, and 6 in combination with isotype control, blocking aCD70, or agonistic aCD27 mAb. On day 10, mice were injected i.v. with a 1:1 ratio of E749-57-peptide–loaded and nonloaded splenocytes that had been labeled with a low or high dose of CFSE, respectively. After 16 hours, the percentage of specific in vivo killing was determined by flow cytometry based on the ratio of CFSEhigh versus CFSElow target cells detected in spleen. A, bar diagram depicting all results. B, representative flow cytometry histograms of gated CFSEþ cells. Results are representative of two experiments. C and D, tumor challenge. Cumulative data of two experiments with n ¼ 5 per group each. Mice were injected s.c. with 1 105 TC-1 tumor cells on day 0 and received "Help" or "No Help" vaccine on days 5, 8, and 11 in combination with isotype control, blocking aCD70, or agonistic aCD27 mAb. C, tumor sizes as measured by caliper. D, overall survival.

CD27 agonism was also superior in regards to numbers of tumor- improved the therapeutic efficacy of the vaccine with helper specific CTLs in the tumor as compared with combined PD-1 epitopes. blockade and CD27 agonism (Fig. 6E and F). We conclude that CD27 agonism and PD-1 blockade together recapitulate the þ effects of CD4 T-cell help on the CTL response against the tumor. Discussion To test the effect of CD27 agonism on tumor control upon The intraepidermal DNA "tattoo" strategy is very potent as "Help" vaccination, we created a therapeutic window by vacci- compared with intramuscular vaccination (21). The vaccine DNA nating 12 instead of 5 days after tumor inoculation. CD27 agon- is expressed in keratinocytes (22). How antigen is subsequently ism significantly increased the generation of HPV E7–specific and delivered to antigen-presenting cells (APC) is not known, but þ effector phenotype CD8 T cells as compared with "Help" vac- most likely part of the antigen passively drains to the lymph node cination only and did not induce Treg expansion (Fig. 7A–D). In and many APCs that receive antigen are not activated for lack of this setting, the "Help" vaccine was not curative, whereas com- contact with "danger" signal. This follows from the fact that helper þ bined CD27 agonism reduced tumor outgrowth and significantly epitope inclusion and consequent CD4 T-cell help robustly improved survival (Fig. 7E and F). Thus, CD27 agonism also promotes the CTL response to the vaccine. We here make use of

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Ahrends et al.

A Help No help No help No help No help 1,250 1,250 1,250 1,250 1,250 1,000 1,000 1,000 1,000 1,000 750 750 750 750 750 500 500 500 500 500 250 250 250 250 250 T

Days after tumor challenge Days after tumor challenge Days after tumor challenge Days after tumor challenge Days after tumor challenge B No help No help No help No help % Survival

Time (days)

Figure 5. Effects of CTLA-4, PD-1, or combined blockade on antitumor immunity. Mice were injected s.c. with 1 105 TC-1 tumor cells on day 0 and subsequently challenged with "Help" or "No Help" vaccine. The "No Help" vaccinated mice were treated with isotype control antibody or with aPD-1 mAb alone, aCTLA-4 alone, or in combination on days 5, 8, and 11. Tumor sizes (A) and overall survival of the mice (B) were monitored. Cumulative data of two experiments with n ¼ 5 and n ¼ 6 per group are shown.

þ exogenous helper epitopes, and the CD4 T cells raised can cells, since CD70 expression is under control of CD40 signaling therefore not recognize the tumor, whereas they can deliver help (35, 36). In this way, "licensed" DCs can exert CD27 costimula- þ for CTL priming and memory programming. tion of CD8 T cells (36, 38). þ Intravital imaging of T cells and APCs in the lymph node is CD27/CD70 costimulation is also important for CD4 T cells. þ elucidating how CD4 T-cell help for the CTL response is orches- It promotes Th1 differentiation in mouse and human (32, 39, 40). trated. T-cell priming involves successive interactions between T In our model, CD27 agonism did not induce a Treg expansion. cells and different APCs in the lymph node, as guided by che- This is in agreement with a study in human where transient CD70 þ mokines (30). In a mouse model of virus infection, CD4 and costimulation of Tregs inhibited their suppressive function and þ CD8 T cells were seen to independently contact XCR1 DCs. converted them into Th1 cells (39). Tregs can respond to thera- þ However, they next contacted the same XCR1 DCs. This second peutic vaccination, as has been observed upon vaccination with priming event promoted memory precursor formation and recall long peptides in human patients (41). However, we did not þ capacity of CD8 T cells and thus proved to be the platform for observe any effect of exogenous helper epitope inclusion on Treg þ delivery of CD4 T-cell help (31). expansion after vaccination. CD4-cell depletion improved the þ We have identified CD27/CD70 costimulation of CD8 T cells effect of CD27 agonism, suggesting that natural Tregs did restrain þ þ as a key effector pathway of CD4 T-cell help. CD70 blocking CD8 T-cell priming to the "No help" vaccine. Using tumor- þ strongly reduced the effects of CD4 T-cell help, whereas 4-1BBL intrinsic helper epitopes in vaccines has the advantage that the þ or OX40L blocking had none or only modest effects. Moreover, effector CD4 T cells raised can recognize the tumor and directly þ CD27 agonism could largely replace CD4 T-cell help in the act upon the tumor or indirectly by promoting CTL function (42). generation and memory responsiveness of CTL. Findings in However, exogenous helper epitopes can be useful when tumor- mouse and human have already indicated the importance of specific helper epitopes are unknown. CD27 signaling for primary and memory CTL responses (32). In Strikingly, the CTL response raised upon combined CD27 þ CD8 T-cell priming, CD27 signaling promotes clonal expansion agonism and CTLA-4 blockade was greater in magnitude to the þ by antiapoptotic and prometabolic effects (33). It broadens the T one raised after CD4 T-cell help, also in the tumor, but this did cell receptor repertoire of the effector T-cell pool, by maintaining not improve tumor control. In contrast, combined CD27 agonism þ low-affinity clones (34). CD70 can be induced on DCs by PRR and PD-1 blockade recapitulated the effects of CD4 T-cell help þ þ stimulation (35, 36), so that both CD4 and CD8 T cells in the on tumor control, even though the combination did not signif- þ dLN may profit from CD27/CD70 costimulation. Notably, CD8 icantly increase CTL priming or tumor infiltration as compared T cells make XCL1 upon CD27 costimulation, which may pro- with CD27 agonism alone. PD-1 blockade most likely overruled þ þ mote their subsequent interaction with XCR1 DCs (37). XCR1 suppression exerted on the CTLs in the tumor site, so that they þ DCs may upregulate CD70 expression when they contact CD4 T could kill their targets. This agrees with clinical data, where the

2928 Cancer Res; 76(10) May 15, 2016 Cancer Research

CD4þ T-cell Help in Therapeutic Cancer Vaccination

A B S

No help No help

CD1,250 No help 1,000 No help No help No help 750

500 % Survival T 250

Days after tumor challenge Time (days)

EF No help No help No help No help No help No help No help No help S

Figure 6. Combined effects of CD27 agonism and CTLA-4 or PD-1 blockade on the CTL response and antitumor immunity. A and B, mice (n ¼ 6 per group) were challenged with "Help" or "No Help" vaccine on days 0, 3, and 6. The "Help" vaccinated mice were treated with isotype control antibody. The "No Help" vaccinated mice were treated with isotype control, agonistic aCD27, or blocking aCTLA-4 or aPD-1 mAb alone or with agonistic aCD27 mAb in combination with aCTLA-4 or b þ þ þ þ aPD-1 mAb. A, percentage of H-2D /E749-57 tetramer cells among total CD8 T cells in blood over time. B, frequencies of GZMB CD8 T cells in blood at day 10 after vaccination. C and D, in independent experiments, mice (n ¼ 6 to 10 per group) were injected s.c. with 1 105 TC-1 tumor cells on day 0 and subsequently challenged with "Help" vaccine and isotype control mAb, with "No Help" vaccine and isotype control mAb, or agonistic aCD27 mAb alone, or in combination with aCTLA-4 or aPD-1 mAb on days 5, 8, and 11. C, tumor sizes. D, overall survival. E and F, mice (n ¼ 4 per group) were injected s.c. with 1 105 TC-1 b þ þ tumor cells on day 0 and subsequently vaccinated and treated with mAb as indicated in C and D. E and F, frequencies of H-2D /E749-57 tetramer (E) and GZMB (F) þ cells among total CD8 T cells in tumors at day 15 after tumor inoculation. Results are representative of two independent experiments. patient's response to PD-1 blockade correlates with PD-L1 expres- immune suppression. CD27 agonism could also enhance the sion in the tumor (43). If this is indeed the case, we conclude that efﬁcacy of vaccine that contained helper epitopes. This can easily þ CD4 T-cell help also aids in overruling tumor-associated be understood, because not all APCs that present the vaccine

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Ahrends et al.

AB CD S

Blood dLN Spleen Blood dLN Spleen Blood dLN Spleen Blood dLN Spleen E F 1,500

1,000

500 % Survival

Days after tumor challenge Days after tumor challenge

Figure 7. þ CD27 agonism improves CTL responses in the presence of CD4 T-cell help. A–D, nontumor-bearing mice (n ¼ 5 per group) received "Help" vaccine in combination with isotype control or agonistic aCD27 mAb on days 0, 3, and 6. The indicated analyses were performed at day 10 after vaccination. Results are representative of two independent experiments. E and F, tumor challenge. Cumulative data of two experiments with n ¼ 4 per group each. Mice were injected s.c. with 1 105 TC-1 tumor cells on day 0 and received "Help" vaccine in combination with control or agonistic aCD27 mAb on days 5, 8, and 11. D, tumor sizes. E, overall survival.

þ þ antigen may have benefitted from CD4 T-cell help. CD4 T-cell Disclosure of Potential Conflicts of Interest help or antibody-based immunomodulation did not alter the low H. van Eenennaam and J. Borst are inventors on a patent claiming CD27 NK-cell numbers in the tumor (results not shown), but we do not agonist antibodies. H. van Eenennaam is shareholder of Aduro Inc. No potential fl exclude that NK-cell activity may have contributed to the observed con icts of interest were disclosed by the other authors. treatment effects. Our data highlight that CD27 agonism, CTLA-4 blockade, and Authors' Contributions PD-1 blockade all optimize the CTL response by distinct, nonre- Conception and design: T. Ahrends, J. Borst dundant mechanisms. Combined CTLA-4 and PD-1 blockade was Acquisition of data (provided animals, acquired and managed patients, previously shown to improve the efficacy of cellular vaccines provided facilities, etc.): T. Ahrends, N. Baba˛ ła, Y. Xiao against s.c. implanted melanoma and colon cancer cell lines Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Ahrends, N. Baba˛ ła, Y. Xiao, J. Borst (44, 45). However, in our setting, this combination was much Writing, review, and/or revision of the manuscript: T. Ahrends, H. Yagita, less potent than combined CD27 agonism and PD-1 blockade. H. van Eenennaam, J. Borst We have used a setting of overtly immunogenic cancer with viral epitopes. However, the same principles apply in case of cancers that do not have pathogen-derived antigens, but to which central Acknowledgments tolerance is incomplete, such as cancers bearing neoantigens or The authors thank Drs. K. Oosterhuis and A. Bins for advice on the vaccination model, professors T.N. Schumacher and J.B.A.G. Haanen for provision of immunogenic cancer-testis antigens. For example, CD27 agonism the DNA vaccines, and M. Toebes for assistance with MHC tetramer production. can stimulate a CTL response to endogenous antigens in mouse melanoma and T-cell lymphoma (46, 47). We believe that exploiting the division of labor between key costimulatory and Grant Support coinhibitory receptors provides a rational guideline to improve This work was supported by grant NKI 2012-5397 of the Dutch Cancer the efficacy of cancer therapy. This includes therapeutic vaccina- Society (J. Borst). The costs of publication of this article were defrayed in part by the payment of tion, but also radiation- or drug-based tumor cell destruction, page charges. This article must therefore be hereby marked advertisement in wherein the tumor acts as endogenous vaccine. A CD27 agonist accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mAb (48) is currently in clinical trials, and it will be of interest to observe how it performs, especially in combination with PD-1 Received November 20, 2015; revised March 7, 2016; accepted March 11, blockade. 2016; published OnlineFirst March 28, 2016.

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www.aacrjournals.org Cancer Res; 76(10) May 15, 2016 2931

CD27 Agonism Plus PD-1 Blockade Recapitulates CD4+ T-cell Help in Therapeutic Anticancer Vaccination

Tomasz Ahrends, Nikolina Babala, Yanling Xiao, et al.

Cancer Res 2016;76:2921-2931. Published OnlineFirst March 28, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-3130

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