Combination OX40 agonism/CTLA-4 blockade PNAS PLUS with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

Stefanie N. Lincha, Melissa J. Kasiewicza, Michael J. McNamaraa, Ian F. Hilgart-Martiszusa, Mohammad Farhada,b, and William L. Redmonda,1

aRobert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, OR 97213; and bCell, Developmental, and Cancer Biology Department, Oregon Health and Science University, Portland, OR 97239

Edited by Laurence Zitvogel, Institut Gustave Roussy-France, Villejuif Cedex, France, and accepted by the Editorial Board December 7, 2015 (received for review June 26, 2015) Immunotherapy is gathering momentum as a primary therapy for of preclinical models. Treatment with an agonist aOX40 mAb cancer patients. However, monotherapies have limited efficacy in directly stimulated CD4 and CD8 T cells and induced their improving outcomes and benefit only a subset of patients. Combina- expansion, differentiation, and up-regulation of prosurvival mole- tion therapies targeting multiple pathways can augment an immune cules (7–12). Moreover, OX40 ligation promoted the generation response to improve survival further. Here, we demonstrate that dual of long-lived memory CD8 T cells and enhanced their function. aOX40 (anti-CD134)/aCTLA-4 (anti–cytotoxic T-lymphocyte–associated Recent data indicate that combined aOX40/aCTLA-4 therapy 4) immunotherapy generated a potent antigen-specific CD8 induced a robust effector CD4 and CD8 T-cell response necessary T-cell response, enhancing expansion, effector function, and memory for tumor regression and significantly improved the survival of T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 tumor-bearing hosts relative to therapy with either agent alone T cells was critical for promoting their maximal expansion following (13). Despite this success, the response to combination aOX40/ combination therapy. Animals treated with combination therapy aCTLA-4 treatment was greatly diminished in more established – and vaccination using anti DEC-205 (dendritic and epithelial cells, tumors. This therapy may be unable to overcome T-cell anergy – 205 kDa) HER2 (human epidermal receptor 2) had sig- in more established immunosuppressive tumor microenviron- nificantly improved survival in a mammary carcinoma model. Vacci- ments, possibly because it is ineffective at specifically targeting nation with combination therapy uniquely restricted Th2- and expanding tumor-reactive T cells and relies on limited or production by CD4 cells, relative to combination therapy alone, and defective endogenous priming by dendritic cells (14). Currently, enhanced IFNγ production by CD8 and CD4 cells. We observed an in- clinically tested vaccines targeting cross-presenting dendritic cells crease in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemo- [i.e., anti–DEC-205 (dendritic and epithelial cells, 205 kDa) mAb kine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on conjugated to tumor antigens] have demonstrated promise by activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF – production by CD8 and CD4 T cells following treatment. Furthermore, priming a more robust cytotoxic T-cell response (15 19). The this therapy was associated with extensive tumor destruction and possibility remains that increased Th2-cytokine production by T-cell infiltration into the tumor. Notably, in a spontaneous model of CD4 T cells following combination therapy may reduce its prostate adenocarcinoma, vaccination with combination therapy re- versed anergy and enhanced the expansion and function of CD8 T cells Significance recognizing a tumor-associated antigen. Collectively, these data dem- onstrate that the addition of a vaccine with combined aOX40/aCTLA-4 Several immunotherapies are approved for treating cancer pa- immunotherapy augmented antitumor CD8 T-cell function while limit- tients, including aCTLA-4 (anti–cytotoxic T-lymphocyte–asso- ing Th2 polarization in CD4 cells and improved overall survival. ciated protein 4; ipilimumab) and anti–PD-1 (anti-programmed cell death protein 1; nivolumab; pembrolizumab), but the best clinical CD8 T cell | costimulation | OX40 | CTLA-4 | anti–DEC-205/HER2 results are coming from combination immunotherapy. Our re- search demonstrates that aOX40 (anti-CD134)/aCTLA-4 immuno- romoting a robust CD8 T-cell response is vital for the gen- therapy can lead to a potentially tumor-promoting Th2-cytokine Peration of an effective antitumor immune response. How- milieu (IL-4, IL-5, IL-13) when relying on endogenous antigen ever, immunosuppressive mechanisms used by the tumor result presentation. However, aOX40/aCTLA-4 treatment combined – in the exhaustion of tumor-infiltrating lymphocytes (TIL), lead- with a tumor antigen-specific vaccine, DEC-205 (anti dendritic and – ing to cytotoxic T-cell anergy and dysfunction. To overcome this epithelial cells, 205 kDa) HER2 (human epidermal growth factor dysfunction, investigators have had considerable success using receptor 2), promoted robust tumor infiltration by effector CD8 immune checkpoint inhibitors, such as aCTLA-4 (cytotoxic T cells and restored Th1 polarization of CD4 T cells, leading to T-lymphocyte–associated protein 4) mAbs, to promote T-cell improved overall survival in a mammary carcinoma model. This function. CTLA-4, a negative regulatory molecule on the surface study has direct relevance for the design of combination therapy of T cells, is indispensable for preventing the expansion and trials in patients. activation of autoreactive T cells. Inhibition of this surface re- – Author contributions: S.N.L. and W.L.R. designed research; S.N.L., M.J.K., M.J.M., I.F.H.-M., ceptor using antagonist aCTLA-4 mAb augmented effector CD4 and M.F. performed research; S.N.L. and W.L.R. analyzed data; and S.N.L. and W.L.R. and CD8 T-cell responses and inhibited or reduced the sup- wrote the paper. pressive function of Treg cells (1–5). However, only a subset of The authors declare no conflict of interest. patients treated with aCTLA-4 mAb exhibit an objective clinical This article is a PNAS Direct Submission. L.Z. is a guest editor invited by the Editorial response (6). Board. Checkpoint blockade targets T-cell coinhibitory molecules, but Freely available online through the PNAS open access option. other strategies targeting costimulatory molecules, such as the 1To whom correspondence should be addressed. Email: [email protected].

TNF receptor family member OX40 (CD134), have demon- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. INFLAMMATION IMMUNOLOGY AND strated success in promoting tumor regression in a wide variety 1073/pnas.1510518113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1510518113 PNAS | Published online January 4, 2016 | E319–E327 Downloaded by guest on September 29, 2021 − − therapeutic efficacy, because inhibition of IL-4 with aOX40/ The expansion of wild-type or OX40 / OT-I cells in animals re- aCTLA-4 treatment significantly improved survival (13). Others ceiving combination therapy was analyzed by flow cytometry. OX40 have shown that a dominant Th2 cytokine response reduced the expression on CD8 T cells was required for optimal expansion, and + + efficacy of treatment, whereas a Th1-skewed immune response its expression enhanced the total number of Ki-67 ,granzymeB , + + + resulted in more favorable outcomes (20–23). IFNγ ,TNFα ,andIL-2 cells (Fig. 2A); we observed minimal We hypothesized that dendritic cell-targeted vaccination against change in the percentage of these populations relative to controls a tumor-associated antigen in conjunction with combination (Fig. 2B). We also confirmed OX40 expression on CD8 T cells from aOX40/aCTLA-4 mAb immunotherapy would be sufficient to the TIL of mice bearing TUBO mammary carcinomas (Figs. S1B promote a cytotoxic antitumor T-cell response, redirect a Th2 bias and S2A) and of TRAMP (transgenic adenocarcinoma of the in CD4 T cells, and improve survival in mice with established mouse prostate) mice with spontaneous prostate adenocarcinoma tumors. Here, we report that vaccination using anti–DEC-205/ (Fig. S3B). HER2 (human epidermal growth factor receptor 2) mAb along Previous studies demonstrated that CTLA-4 expression on CD4 with combination aOX40/aCTLA-4 mAbs significantly expanded T cells is required to augment CD8 T-cell function indirectly (1, 2). effector CD8 T cells, resulting in a more favorable Th1 cytokine To assess the role of CTLA-4 expression on CD8 T cells, we used profile and inducing a critical accumulation of effector T cells in humanized CTLA-4 knock-in (huCTLA-4) mice engineered to the tumor, with increased tumor-free survival relative to either express only the extracellular portion of the human CTLA-4 re- therapy alone. Moreover, combination therapy with vaccination ceptor, thereby preventing them from responding to mouse reversed T-cell anergy and promoted a robust effector T-cell aCTLA-4 mAb (2). Animals were treated with combination ther- response to a tumor-associated antigen in a spontaneous ade- apy following adoptive transfer of OT-I cells; as expected, nocarcinoma model. huCTLA-4 mice treated with mouse aCTLA-4 mAb had no change in OT-I expansion relative to IgG controls (Fig. 2C). Although Results we still observed an additive effect of combination therapy over Combined aOX40 and aCTLA-4 Therapy Significantly Enhanced monotherapy alone in huCTLA-4 mice, OT-1 expansion and Antigen-Specific CD8 T-Cell Expansion, Function, and Persistence. proliferation were blunted compared with the wild-type controls We have shown previously that combining OX40 stimulation given combination therapy (Fig. 2C). We also confirmed the ex- with CTLA-4 blockade effectively enhanced tumor regression and pression of CTLA-4 on CD8 T cells from the TIL of tumor-bearing survival in several tumor models (13). However, the mechanisms by mice (Fig. S1). These data indicate that mAb binding to the CTLA-4 which combination therapy augments antigen-specific CD8 T-cell receptor on CD4 T cells, or other accessory cells, mediates only a responses remain unknown. To address this question, we transferred portion of the CD8 T-cell expansion induced by combination purified OT-I CD8 T cells into wild-type mice and treated them with therapy and that CD8 T-cell–specific CTLA-4 blockade is critical in controlIgG,aCTLA-4,aOX40,or dual aOX40/aCTLA-4 therapy in the context of combination therapy to induce maximal efficacy. the presence of antigen. Combination therapy resulted in a signifi- It has been shown that CD4 T cells are required for optimal cant expansion of circulating antigen-specific T cells (∼85% of all efficacy of aOX40 or aCTLA-4 mAb (1, 2, 29). To assess whether CD8 T cells) compared with either monotherapy alone (38.9% for CD4 T cells are necessary to achieve maximal CD8 T-cell ex- aCTLA-4 and 62.1% for aOX40) (Fig. 1A). Furthermore, com- pansion following combination therapy, we depleted CD4 T cells bined aOX40/aCTLA-4 therapy augmented CD8 T-cell prolifer- before treatment. Antigen-specific CD8 T-cell expansion was ation and effector function over controls, as evidenced by increased significantly reduced in CD4-depleted animals compared with levels of Ki-67 (cellular marker of proliferation) and granzyme B, control mice, with an approximately fivefold difference in total respectively (Fig. 1 B and C). Dual therapy also significantly enhanced cell numbers (Fig. 2D). Together, these data indicate that OX40 the frequency of memory T cells: We observed expansion at day 7 that andCTLA-4expressiononCD8Tcells,inadditiontoCD4 persisted 10 wk later at an elevated frequency compared with either T-cell help, is necessary to promote maximal CD8 T-cell expansion monotherapy alone (Fig. 1D). and effector function following combination therapy. To investigate the extent to which combination therapy skewed antigen-specific CD8 T-cell differentiation toward an effector ver- Combination Therapy with Vaccination-Induced Tumor Regression sus memory phenotype, we examined surface expression of KLRG-1 and Enhanced Survival in Tumor-Bearing Mice. It has been shown (killer cell lectin-like receptor subfamily G, member 1), CD127, that combination therapy drives Th2 cytokine production and that and CD62L. The surface expression of these receptors changes IL-4 expression limits the efficacy of treatment (13). Moreover, this based on differentiation status; central memory cells are typi- treatment relies on endogenously primed T cells and is likely to be cally CD127hi and CD62Lhi, and effector memory cells are CD127lo limited by suboptimal dendritic cell priming against tumor antigens − and CD62L /lo (24, 25). We observed an increase in KLRG-1 (14). We hypothesized that the presence of a vaccine to drive cross- and CD127 expression on CD8 T cells in mice given combi- presentation of a tumor-associated antigen would enhance T-cell nation therapy, with no change in CD62L expression (Fig. 1E). priming and diminish the Th2 bias of combination therapy. HER2 CD127hi and KLRG-1hi dual-positive cells were increased, and is a tumor-associated antigen that is overexpressed in a subset of CD127loKLRG-1lo cells were reduced (Fig. 1 F and G). There breast cancers and is associated with disease recurrence and poor was no change in the percentage of CD62L/KLRG-1 dual-positive prognosis (30). Mice bearing established HER2-expressing TUBO cells following combination therapy (Fig. 1F). These data demon- mammary carcinomas were immunized with anti–DEC-205/HER2 strate that combination therapy enhanced the expansion, function, mAb and adjuvant [poly(I:C)] and were treated with combination and persistence of antigen-specific memory T cells in vivo. therapy (Fig. S2A). Combination therapy plus HER2 vaccination significantly enhanced tumor regression (Fig. 3A) and survival (Fig. Maximal Expansion of CD8 T Cells Following Combination Therapy 3B) over combination therapy or vaccine and adjuvant. There was Requires OX40 and CTLA-4 Expression on CD8 T Cells, and the Presence no difference among between groups treated with anti–DEC-205– of CD4 T Cells. Prior studies have shown that OX40 expression on Mock/poly(I:C) plus aOX40/aCTLA-4 or with poly(I:C) plus CD8 T cells contributes to polyclonal expansion and function fol- aOX40/aCTLA-4 relative to combination aOX40/aCTLA-4 alone lowing aOX40 therapy (7). However, OX40 is constitutively ex- (Fig. S2 C and D). We observed that although combination pressed on Treg cells, and engagement on this subset could enhance therapy drove Th2 cytokine production from CD4 T cells, com- or limit their suppressive capacity (26–28). Therefore, we in- bination therapy plus HER2 vaccination significantly reduced the vestigated whether engagement of OX40 directly on CD8 T cells production of IL-4, IL-5, and IL-13 (Fig. 3 C and D). Further- was necessary for their expansion following combination therapy. more, combination therapy with vaccination significantly increased

E320 | www.pnas.org/cgi/doi/10.1073/pnas.1510518113 Linch et al. Downloaded by guest on September 29, 2021 PNAS PLUS A E 100 40 * 100 rat IgG rat IgG/aCTLA-4 aOX40 aOX40/aCTLA-4 * 80 80 17.6% 38.9% 62.1% 85.1% 30 60 60 20 40 40 %CD62L+ %KLRG-1+ %CD127+

CD8 10 20 20 Thy1.1 0 0 0 rat IgG rat IgG/aCTLA-4 aOX40 aOX40/aCTLA-4 B rat IgG rat IgG/aCTL A-4 aOX40 aOX4 0/aCTL A-4 39.5% 64.1% 63.9% 82% F rat IgG rat IgG/aCTLA-4 aOX40 aOX40/aCTLA-4 Ki-67 32.1% 51.2% 62.1% 85.1% IL-7Ra (CD127) GrzB Thy1.1 CD62L

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Fig. 1. Combination therapy boosts the expansion, function, and persistence of memory CD8 T cells. Naive OT-I CD8 T cells (3 × 106) were purified by negative selection and adoptively transferred into wild-type C57BL/6 mice. Donor OT-I cells were stimulated with soluble OVA (day 0), along with mono- or combi- nation therapy: 50 μg aOX40 or control rat IgG on days 0 and 1 and 200 μg aCTLA-4 on days 0, 2, and 4. Mice were bled on days 7, 14, 28, and 78, and donor + + + cell phenotype was analyzed by flow cytometry. Donor OT-I cells were gated on live CD8 Thy1.1 events. Graphs depict (A and D) the frequency of OT-I cells + + + + + of all CD8 T cells, (B and C) the percentage of Ki-67 and granzyme B OT-I cells, and (E and G) the percentage of KLRG-1 , CD62L , and CD127 OT-I cells. Data shown are for one representative mouse (A, B, and F) or all mice in each cohort (C–E and G) at day 7. Graphs depict the mean ± SEM for one of three in- dependent experiments (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

+ IFNγ,TNFα, and IL-2 production by CD8 T cells (Fig. 3D); there CD3 lymphocyte infiltration throughout the tumors relative to was no difference in TGF-β expression in the tumor following mice treated with combination therapy or controls (Fig. 4 A–H). treatment (Fig. S2B). Production of MIP-1α (macrophage regu- We further quantified this finding using flow cytometry and ob- latory protein-1α)/CCL3 [ (C-C motif) ligand 3], MIP- served a significant increase in the frequency of tumor-infiltrating 1β/CCL4, RANTES (regulated on activation, normal T-cell ex- CD4 and CD8 T cells in mice receiving combination therapy with pressed and excreted)/CCL5, and GM-CSF also was significantly anti–DEC-205/HER2 vaccination (Fig. 4I). This increase coincided enhanced by both CD4 and CD8 T cells in mice receiving com- with a significant increase in the CD8:Treg ratio (Fig. 4J), which has bination therapy with HER2 vaccination (Fig. 3E). been associated with the sensitivity of a tumor to a given therapy To understand the impact of this therapy on the tumor micro- and also with improved overall survival in patients with breast environment, we examined tumor immune infiltration following cancer and other cancer subtypes (3, 31–33). Collectively, these treatment. Immunohistochemistry revealed that vaccination alone data indicate that directing the CD8 T-cell response using anti– was insufficient to drive tumor destruction and T-cell infiltration. DEC-205/HER2 vaccination enhances the efficacy of combination

However, mice treated with combination immunotherapy plus therapy, reverses Th2 cytokine production by CD4 T cells, and INFLAMMATION HER2 vaccination had extensive tumor destruction and increased enhances effector CD8 T-cell infiltration into the tumor. IMMUNOLOGY AND

Linch et al. PNAS | Published online January 4, 2016 | E321 Downloaded by guest on September 29, 2021 *** 7 6 ** WT A 1.5 10 5.0 10 C 2.0 107 * OX40-/- ** * 4.0 106 1.5 107 rat IgG 1.0 107 * 6 aCTLA-4 huCTLA-4 3.0 10 mice

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Fig. 2. OX40 and CTLA-4 expression on CD8 T cells and CD4 T cells is required for maximal CD8 T-cell expansion and function following combination therapy. − − (A and B) Naive OT-I or OX40 / OT-I CD8 T cells were purified by negative selection and adoptively transferred into wild-type C57BL/6 mice. (C) Naive wild- type OT-I CD8 T cells were adoptively transferred into humanized CTLA-4 knock-in mice. (D) OT-I CD8 T cells were adoptively transferred into wild-type mice. Donor OT-I cells were stimulated with soluble OVA on day 0 along with mono- or combination therapy: aOX40 or control rat IgG on days 0 and 1 and aCTLA-4 on days 0, 2, and 4. Splenocytes were harvested on day 7, and donor cell phenotype was analyzed by flow cytometry. Graphs depict the mean ± SEM for three independent experiments (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Combination Therapy with Vaccination Reversed T-Cell Anergy and to drive prostatic expression of membrane-bound ovalbumin Augmented Effector Function to a Tumor-Associated Antigen in Mice (OVA) (36). OT-I CD8 T cells were adoptively transferred into with Spontaneous Prostate Cancer. To determine whether combi- TRAMP/POET-1 mice and rested for 28 d to induce anergy (37). nation therapy with vaccination was more effective at inducing Mice were treated with combination therapy and anti–DEC-205– tumor-specific CD8 T-cell expansion, we used the B16F10 model OVA vaccination (model shown in Fig. S3A). TRAMP/POET-1 with adoptive transfer of tumor-specific Pmel (premelanosome mice treated with combination therapy plus vaccination had a protein) CD8 T cells (hereafter, “Pmels”), which are insufficient significant boost in CD8 T-cell expansion, proliferation, and ef- on their own to overcome peripheral tolerance and induce tumor fector function in the spleen (Fig. 5 B–D) and lymph nodes (Fig. regression (34). B16F10 tumor-bearing mice given Pmels were S3C). Combination therapy with vaccination induced a signifi- immunized with gp100 peptide and adjuvant along with combi- cantly greater number of IFNγ-, TNFα-, and IL-2–producing nation therapy. Peripheral blood was analyzed by flow cytometry CD8 T cells compared with vaccination and either monotherapy to assess the expansion of tumor-specific Pmels. We observed an (Fig. 5 C and D). These data indicate that combined aOX40/ increase in Pmel expansion in animals treated with combination aCTLA-4 therapy with vaccination was uniquely capable of re- therapy and vaccination, specifically an increase in the proportion versing CD8 T-cell anergy to a tumor-associated antigen in vivo of Pmels to CD8 T cells and Pmels to peripheral blood mono- and promoting robust expansion and function of these cells. nuclear cells (Fig. 5A). It has been shown that antigen-specific T-cell anergy is an early Discussion event in tumor development and poses a significant barrier to Although aOX40 or aCTLA-4 monotherapy improves an antitumor therapeutic vaccination. To test the efficacy of combination ther- immune response, monotherapy alone is insufficient to eliminate apy with vaccination on T-cell anergy in a spontaneous tumor tumor burden in the majority of patients. Our previous studies model, we used TRAMP/POET-1 (probasin ovalbumin expressing demonstrated that combined aOX40/aCTLA-4 therapy signifi- transgenic) mice. TRAMP mice express SV40 T antigen under cantly increased the survival of animals with TRAMP-C1 prostate control of the rat probasin promoter, resulting in antigen expres- or MCA-205 sarcoma tumors compared with monotherapy alone sion in the prostate epithelium upon sexual maturity. These mice (13).Weelaborateonthesedatato show that combination therapy develop prostate intraepithelial neoplasia (PIN) by roughly age significantly boosted an antigen-specific CD8 T-cell response that 12 wk and adenocarcinoma by age 24 wk (35). We crossed TRAMP persisted for many weeks following treatment (Fig. 1). Our previous mice with POET-1 mice in which the probasin promoter was used studies also suggested that combined therapy affected T-cell

E322 | www.pnas.org/cgi/doi/10.1073/pnas.1510518113 Linch et al. Downloaded by guest on September 29, 2021 A PNAS PLUS rat IgG Vaccine alone aOX40/aCTLA-4 aOX40/aCTLA-4/vaccine

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20 ** **** 200 **** **** Percent survival 0 0 50 100 150 200 0 Time (Days) IL-5 IL-6 IL-10 IL-17 IL-22 TNFa

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Fig. 3. Combination therapy with vaccination improves survival and inhibits Th2-polarization in CD4 T cells in a mammary carcinoma model. TUBO mammary carcinoma cells were implanted on day 0 into the flank of female BALB/c mice. Tumor-bearing mice were given control rat IgG on days 10 and 14, vaccine [anti– DEC-205/HER2/poly(I:C)] on days 10 and 14, combination aOX40 on days 10 and 14/aCTLA-4 on days 10, 12, and 14, or both vaccine and combination immu- notherapy. (A and B)Tumorgrowth(A) and survival (B) were monitored over time. Graphs depict the mean ± SEM for three independent experiments combined (n = 15–25 per group). (C–E) CD4 (C)andCD8(D and E) T cells were purified and sorted from the lymph nodes of tumor-bearing mice 7 d after the end of therapy (day 21). Cells were stimulated in vitro with anti-CD3 for 4 h, and supernatants were collected. Cytokine and chemokine levels were determined by multiplex ELISA. Graphs depict the mean ± SEM for two independent experiments (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

differentiation, in terms of transcriptional regulation. In the cur- a memory phenotype or potentially could target those cells rent study, we demonstrate an increase in dual CD127hiKLRG-1hi specifically. The precise reason for this effect is currently unclear, antigen-specific CD8 T cells and a decrease in CD127loKLRG-1lo but this skewing may prove beneficial when targeting a rare cells (Fig. 1E). For virus-specific CD8 T cells, CD127hiKLRG-1hi population of tumor-associated antigen-specific CD8 T cells CD8 T cells typically represent memory cells, whereas CD127lo in vivo. lo

KLRG-1 cells represent early effectors (24, 38). Combination OX40 ligation can induce the formation of long-lived memory INFLAMMATION therapy may preferentially skew a CD8 T-cell population toward CD4 T cells and boost an effector CD8 T-cell response through IMMUNOLOGY AND

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0 0

Fig. 4. Combination therapy with vaccination induces robust tumor destruction and effector T-cell infiltration into the tumor. (A–H) TUBO mammary car- cinoma cells were implanted into the flanks of female BALB/c mice on day 0. Tumor-bearing mice were given control rat IgG on days 10 and 14 (A and E), vaccine [anti–DEC-205/HER2/poly(I:C)] on days 10 and 14 (B and F), combination immunotherapy with aOX40 on days 10 and 14/aCTLA-4 on days 10, 12, and 14 (C and G), or both vaccine and combination immunotherapy (D and H). Tumors were harvested on day 21 and were analyzed by immunohistochemistry. Images depict sections of paraffin-embedded slides stained using H&E (A–D) or anti-CD3 plus DAPI (E–H). (I and J) Tumors were harvested and analyzed by flow cytometry. Graphs depict the mean ± SEM for one of two independent experiments (n = 4 or 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

both direct and indirect ligation of the respective T-cell subsets. that in the context of a strong costimulatory signal, as provided by Here, we demonstrate that, in the context of dual aOX40/aCTLA-4 OX40 ligation, and in the absence of other inflammatory signals to therapy, OX40 deficiency on CD8 T cells significantly reduced drive T-cell differentiation, CTLA-4 blockade provides an added + their ability to become IFNγ-producing, granzyme B effector boost in CD8 T-cell expansion and effector function. cells. OX40 expression on CD8 T cells is required to boost CD8 The efficacy of combination therapy relies on endogenous anti- T-cell expansion and survival following dual therapy, as has been genpresentationtoTcellstodriveanantitumorimmuneresponse. demonstrated previously in the context of OX40 agonism by itself However, it has been shown that dendritic cell function is di- (7, 9, 39). These data demonstrate the need for OX40 engagement minished in tumor-bearing hosts, and the increase in Th2 polari- on CD8 T cells in the context of combined therapy, indicating that, zation in CD4 T cells limited the efficacy of dual immunotherapy although antibody-dependent, cell-mediated cytotoxicity is one (13, 14, 42). Targeting cross-presenting dendritic cells using anti– mechanism by which aOX40 improves tumor eradication (40), DEC-205/HER2 with combination therapy reduced the generation OX40 expression on CD8 T cells is still an essential component of of Th2-polarized CD4 T cells, promoted a robust cytotoxic CD8 the efficacy of aOX40 therapies. T-cell response and infiltration into the tumor, and enhanced overall We also investigated the requirement for CTLA-4 expression on survival (Figs. 3 and 4). One probable mechanism for the efficacy of CD8 T cells following dual therapy, because previous work has vaccination plus combination therapy is the promotion of better demonstrated that CTLA-4 blockade indirectly augments effector T-cell receptor (TCR) stimulation and Th1-polarized T-cell priming. CD8 T-cell function through cell-extrinsic effects on CD4 T cells Evidence for this notion comes from studies showing that lower- (1, 4, 41). Surprisingly, in the context of combined OX40 agonism/ affinity stimulation, which is thought to be the majority of endog- CTLA-4 blockade, the expression of CTLA-4 on CD8 T cells is enous antigen presentation in the periphery because of central and required to promote maximal CD8 T-cell expansion, because we peripheral tolerance, tends to promote Th2 CD4 T-cell responses observed twofold greater CD8 T-cell expansion in mice treated with preferentially (43, 44). In particular, anti–DEC-205/HER2 vacci- dual aOX40/aCTLA-4 therapy than mice treated with aOX40 nation was shown to contain epitopes specifically capable of elic- alone. In contrast, Pedicord et al. (1) established that in studies iting a strong CD4 T-cell response (17). In the absence of a specific combining CTLA-4 blockade with Listeria infection, CTLA-4 ex- antigen, administration of aOX40/aCTLA-4 therapy likely pro- pression on CD8 T cells was dispensable for the boost in CD8 motes the expansion of T cells receiving suboptimal TCR stimu- T-cell expansion following treatment with aCTLA-4 mAb. lation from endogenous peptides on dendritic cells, hence the However, Listeria infection provides other potent costimulatory increase in Th2 cytokine production. By providing tumor-specific molecules, such as CD40, and the production of proinflammatory antigen to cross-presenting dendritic cells, we were able to promote , which may overshadow any effect of CTLA-4 expression a robust Th1 response, as evidenced by increased IFNγ,TNFα,and on CD8 T cells. Along these lines, Gattinoni et al. (41) demon- IL-2 production by CD8 T cells (Fig. 3). Furthermore, we observed strated that CTLA-4 expression on CD8 T cells had a modest an increase in MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 impact on tumor growth in the B16 melanoma model. It appears production by CD4 and CD8 T cells following combination therapy

E324 | www.pnas.org/cgi/doi/10.1073/pnas.1510518113 Linch et al. Downloaded by guest on September 29, 2021 PNAS PLUS AC0.3 13.9 0.8 1.1 0.3 3 30 6 Vaccine CD8 20 4 alone

CD8/Total 10 2 1.3 35.4 0.9 6.7 0.7 11.6

Pmel Vaccine/ % 0 %Pmel CD8/Live PBMC 0 aOX40/ rat IgG/vaccine aOX40/vaccine aCTLA-4 aCTLA-4/vaccine aOX40/aCTLA-4/vaccine IL-2 TNF-a IFN-g B Thy1.1 D ** 7 ** 7 vaccine alone 2.5×10 vaccine alone 2.5×10 * * vacc/aCTLA-4 ** vacc/aCTLA-4 2.0×107 vacc/aOX40 * 2.0×107 vacc/aOX40 vacc/aOX40/aCTLA-4 vacc/aOX40/aCTLA-4 1.5×107 ** 1.5×107 * *** 1.0×107 1.0×107 ** ** * ** × 6 × 6 * Total cellnumber Total cellnumber 5.0 10 * 5.0 10 0 0 OT-I Ki-67 GrzB KLRG-1 CD62L CD25 IFNg+ TNFa+ IL-2+ OT-I CD8 T cells OT-I CD8 T cells

Fig. 5. Combination therapy with vaccination reverses CD8 T-cell anergy and restores T-cell function in a spontaneous adenocarcinoma model. (A) Naive Pmel TCR transgenic CD8 T cells were purified by negative selection and adoptively transferred on day 6 into B16F10 tumor-bearing wild-type C57BL/6 mice. Mice were treated with IgG or aOX40 on days 7 and 11; with aCTLA-4 on days 7, 9, and 11; or with monotherapy or combination therapy and vaccine (gp100/ anti-CD40) on day 7. Blood was analyzed by flow cytometry on day 14. (B–D) Naive OT-I CD8 T cells (5 × 105) were purified by negative selection and adoptively transferred into TRAMP/POET-1 mice on day 0. Mice were stimulated with 5 μg anti–DEC-205–OVA and 50 μg anti-CD40 on day 28 along with mono- or combination therapy (50 μg aOX40 or control rat IgG on days 28 and 29 and 200 μg aCTLA-4 on days 28, 30, and 32). Splenocytes were analyzed on day 35, and + + donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8 Thy1.1 events. Graphs depict (B) the total number of + + + + + + OT-I cells; the total number of Ki-67 , granzyme B (GrzB), KLRG-1 , CD62L , and CD25 OT-I; (C and D) and the percentage (C) and total number (D) of IFNγ-, TNFα-, and IL-2–producing OT-I cells. Data are shown for one representative mouse (C) or for all mice in each cohort for donor OT-I cells (B and D). Graphs depict the mean ± SEM for one of three independent experiments (n = 4 or 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

plus HER2 vaccination (Fig. 3). CCL3 and CCL4 are important for mune tolerance to the tumor. Indeed, recent studies suggest that recruiting dendritic cells and T cells and promoting T-cell homing to checkpoint blockade using PD-1 or CTLA-4 blockade is more ef- sites of infection or inflammation (45, 46). Moreover, CCL3, CCL4, fective in tumors with a higher somatic mutation load (48, 49). and CCL5 are preferentially expressed in tumors with T-cell in- However, the mutational burden in prostate cancer is low relative to filtration in melanoma patients (47). The boost in these other cancer types and is more often associated with chromosomal by combination aOX40/aCTLA-4 therapy with HER2 vaccination rearrangements or deletions (50–53). Our data support these may drive the recruitment of TIL. Further studies in our laboratory studies, because combination therapy in the TRAMP model of are aimed at determining whether these chemokines are necessary spontaneous prostate adenocarcinoma was effective only when for the recruitment of CD8 effector T cells into the tumor. administered with a vaccine against a tumor-associated antigen. Despite an increase in antitumor immunity, the effectiveness of Moreover, because the induction of Th2 cytokines by aOX40/ combination therapy alone was reduced in mice with established aCTLA-4 limited the effectiveness of this therapy, using a vaccine tumors (13). Moreover, overcoming tumor tolerance and T-cell strategy to target cross-presenting dendritic cells specifically should anergy remains a concern for creating more effective therapeutic preferentially skew the response away from a Th2 T-cell response modalities. Prior studies demonstrate that aOX40 mAb in the ab- and toward a Th1 CD4 T-cell response, which is more favorable for sence of exogenous antigen is insufficient to overcome tolerance; tumor regression and patient survival (54–57). The use of anti– overcoming T-cell tolerance requires the administration of both DEC-205 mAb conjugated to NY-ESO-1 in patients with solid tu- aOX40 mAb and antigen (37). We hypothesized that combination mors (NCT01522820) finished testing with promising results (19) therapy with vaccination would be sufficient to overcome tumor and currently is being tested clinically in patients with acute myeloid tolerance and induce the expansion of CD8 T cells recognizing a leukemia (NCT01834248). Preclinical and clinical studies support tumor-associated antigen. Our data demonstrate that combination our data on vaccination with anti–DEC-205 antibodies. It will be aOX40/aCTLA-4 mAb with peptide vaccination specifically induced important for future clinical studies investigating combined aOX40/ the robust expansion of tumor-specific Pmel CD8 T cells (Fig. 5A). aCTLA-4 therapy to address whether Th2-biased T cells are in- Using a model of T-cell anergy combined with a spontaneous duced in patients subsequent to treatment, the extent to which they prostate cancer model, we have demonstrated that treatment with limit the efficacy of immunotherapy, and whether the inclusion of a aOX40/aCTLA-4 and vaccination can overcome T-cell anergy to a dendritic cell–targeted vaccination with combined immunotherapy tumor-associated antigen to promote a potent expansion of effector can restore Th1 polarization to enhance the efficacy of treatment. cells and significantly augment the number of Th1 cytokine- producing CD8 T cells (Fig. 5). Based on these data, it seems likely Materials and Methods

that aOX40/aCTLA-4 therapy will be more effective for patients in Mice. Wild-type C57BL/6 or BALB/c mice were purchased from Jackson Lab- INFLAMMATION the presence of a vaccination strategy designed to overcome im- oratories. OT-I Thy1.1 TCR transgenic mice, OX40−/− OT-I TCR transgenic mice, IMMUNOLOGY AND

Linch et al. PNAS | Published online January 4, 2016 | E325 Downloaded by guest on September 29, 2021 − − Pmel TCR transgenic/Rag-1 / transgenic mice, TRAMP mice, POET-1 mice, Tumor Challenge and Antibody Administration. TUBO cells (5 × 105), an HER2- and humanized CTLA-4 knock-in mice were bred and maintained under expressing cell line derived from a spontaneous mammary gland tumor from specific pathogen-free conditions in the Providence Portland Medical Center a BALB-neuT mouse (59), were injected into the right flank of BALB/C mice. vivarium. Humanized CTLA-4 knock-in mice were a gift from Jim Allison Tumor growth (area) was assessed every 2 to 3 d with microcalipers, and (University of Texas, MD Anderson Cancer Center, Houston). Experimental mice were killed when tumors reached >200 mm2. B16 F10 melanoma cells procedures were performed according to the NIH Guide for the Care and (3.5 × 105) were injected in to the right flank of C57BL/6 mice. Animals were Use of Laboratory Animals (58) and were approved by the Institutional treated with 200 μg rat IgG (Sigma), 200 μgaOX40(cloneOX86;Bio- Animal Care and Use Committee at Providence Cancer Center. XCell), and/or 200 μg aCTLA-4 (clone 9D9; BioXCell) mAb. All mAbs were verified to be endotoxin free and were injected i.p. (aCTLA-4) or s.c. (IgG FACS Analysis. For FACS analysis, cells were incubated for 30 min at 4 °C with or aOX40) into recipient mice. Murine anti–DEC-205–OVA and anti–DEC- Thy1.1 PE-Cy7, Thy1.1 eFluor 450, CD45 APC, CD8 eFluor 605, CD8 BV785, 205/HER2 mAbs were provided by Celldex Therapeutics, Inc., and 5 μg KLRG-1 APC, FoxP3 eFluor 450, CD25 Alexa Fluor 700, CD25 PE, OX40 PE, were injected s.c. along with 50 μg anti-CD40 (clone FGK4.5; BioXCell) or Fixable Viability Dye eFluor 780 and eFluor506, CD62L PerCP Cy5.5, CD127 50 μg poly(I:C) (InvivoGen) as an adjuvant. For the TUBO mammary car- PE-Cy7, Vb13 APC, or CD4 V500. All antibodies were obtained from eBio- cinoma model, treatment with IgG, OX40, and vaccine/adjuvant was on science, BD Biosciences, BioLegend, or Life Technologies. For intracellular days 10 and 14 after tumor implantation; aCTLA-4 was administered on staining, cells were fixed and permeabilized with the Foxp3 Staining Buffer days 10, 12, and 14. For the Pmel/B16F10 model, only 50 μg of aOX40 per dose kit (eBioscience) according to the manufacturer’s instructions. Cells were was used and was administered to tumor-bearing mice on days 8 and 9 after incubated for 30 min at 4 °C with IL-2 PE, TNF-α PE-Cy7, IFN-γ APC, Ki-67 FITC, tumor implantation, along with gp100 peptide and anti-CD40 or control IgG; granzyme B PE, and Foxp3 eF450. Cells were collected and analyzed using aCTLA-4 was administered on days 8, 10, and 12. In the TRAMP/POET model, the LSR II flow cytometer using Diva (BD Biosciences) or FlowJo (Tree Star) mice were vaccinated with 5 μganti–DEC-205-OVA, 50 μg anti-CD40 (on day 28 software. For the multiplex cytokine/chemokine ELISA, CD4 or CD8 T cells after adoptive transfer) along with mono- or combination therapy (50 μg − were purified by cell sorting (B220/CD11b/MHC II ), and 2 × 106 cells per well aOX40 or control rat IgG on days 28 and 29 and 200 μg aCTLA-4 on days 28, 30, were stimulated with medium or plate-bound anti-CD3 (2 μg/mL) in 24-well and 32). plates. Supernatants were collected after 4 h, and cytokine expression was determined using FlowCytomix Th1/Th2/Th17 and chemokine kits according TIL Isolation. TILs were harvested on day 21 by dissection of tissue into small to the manufacturer’s instructions (eBioscience). fragments followed by digestion in 1 mg/mL collagenase and 5 mg/mL DNase in PBS. After filtration through nylon mesh, lymphocytes were stained as Adoptive Transfer and Purification of OT-I T Cells. Single-cell suspensions were described above and were analyzed by flow cytometry. For immunohisto- prepared from spleens of OT-I Thy1.1 TCR transgenic or Pmel TCR transgenic/ chemistry, tumors were fixed in a 10% (wt/vol) zinc solution for 24 h and then − − + + + Rag-1 / mice. Cell suspensions were depleted of CD4 , CD11b , CD45R , were embedded in paraffin blocks. Paraffin blocks were sectioned, placed + + DX5 , and Ter-119 cells using the Dynabeads CD8 T-cell negative isolation kit onto slides, and dried overnight. Slides were deparaffinized in xylene and (Life Technologies), and cells were purified by negative selection according to stained with H&E. For fluorescence microscopy, slides were blocked with the manufacturer’s instructions. On day 7, 3 × 106 wild-type OT-I T cells or 5 × polyclonal IgG and PeroxAbolish (Biocare Medical) before staining with 105 Pmel CD8 cells in PBS were i.v. injected into recipient C57BL/6 mice or rabbit anti-mouse CD3 (SP7) (Spring Bioscience) followed by the anti-rabbit B16F10 tumor-bearing C57BL/6 mice, respectively. For the anergy model, 5 × secondary SuperPicTure Polymer Detection Kit (Life Technologies). The sig- 105 wild-type OT-I T cells were i.v. injected into TRAMP/POET recipients. nal was amplified using TSA-Cyanine 5 (PerkinElmer). Slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories) and visual- Lymphocyte Isolation and Analysis. Spleens were harvested and processed to ized using the Vectra Microscopy imaging system (PerkinElmer). Representa- × obtain single-cell suspensions. ACK lysing buffer was used to lyse red blood tive regions were captured at 200 magnification using Vectra Software and cells. Cells were rinsed with Roswell Park Memorial Institute [RPMI] 1640 were analyzed using Inform Image Analysis software (PerkinElmer). medium containing 10% (vol/vol) FBS (10% cRPMI) supplemented with 1 M Hepes, nonessential amino acids, sodium pyruvate, and penicillin-strepto- Statistical Analysis. Statistical significance was determined by unpaired Stu- mycin glutamine. Murine peripheral blood lymphocytes from the superficial dent t test, one-way ANOVA, or Kaplan–Meier survival where appropriate temporal vein were collected into tubes containing 50 μL heparin. One using GraphPad Prism software (GraphPad). milliliter of flow cytometry wash buffer (0.5% FBS, 0.5 mM EDTA, and 0.02%

NaN3 in PBS) was added, and the cells were vortexed and layered on top of ACKNOWLEDGMENTS. We thank Zefora Alderman and the Earle A. Chiles 700 μL of Ficoll-Paque medium (GE Healthcare) before centrifugation. Research Institute Vivarium for excellent technical assistance; Tibor Keler (Cell- – Lymphocytes were collected from the interface and washed with wash dex Therapeutics) for providing the anti DEC-205 mAb vaccines; and Drs. Michael Gough, Andrew Weinberg, and Walter Urba for helpful discus- buffer before staining. To measure antigen-specific cytokine production, μ sions and critical reading of the manuscript. This work was supported by the lymphocytes were incubated in 10% cRPMI with 5 g/mL of the OVA Providence Portland Medical Foundation, the Safeway Foundation, Susan μ (SIINFEKL) peptide (Anaspec) and 2.5 L/mL of Golgi-Plug solution containing G. Komen Grant CCR15329664, NIH Pathway to Independence Award brefeldin A (BD Biosciences) for 5 h at 37 °C. After washing, cells were 5R00CA136678, and American Cancer Society 2014 Roaring Fork Valley stained and fixed as described above. Postdoctoral Fellowship PF-14-249-01-LIB (to S.N.L.).

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