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 protein 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 growth factor 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-cytokine 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