CR 75th Anniversary Commentary Special Lecture Combine and Conquer: Double CTLA-4 and PD-1 Blockade Combined with Whole Tumor Antigen Vaccine Cooperate to Eradicate Tumors Krisztian Homicsko1,2,3, Jaikumar Duraiswamy4, Marie-Agnes Doucey1, and George Coukos1,2 þ See related article by Duraiswamy et al., Cancer Res 2013;73: cells was approximately double among the single PD-1 (PD- þ À þ þ 3591–603. 1 CTLA-4 ) TILs relative to the double-positive (PD-1 CTLA-4 ) Cancers hijack the normal regulatory pathways of normal TILs. In addition, the single-positive TILs were the only popula- inflammatory reactions. Dysregulation of fundamental immune tion to proliferate in response to peptide, whereas the double- checkpoints is present virtually in all tumors with underlying positive population failed to proliferate. The double-positive immune recognition and is crucial in the development and main- population also expressed higher levels of additional inhibitory tenance of cancer immune tolerance. The dissection of immune receptors, such as 2B4, LAG-3, and TIM-3, and exhibited higher checkpoints showed that cancers can corrupt not one but multiple levels of CD44 and lower levels of CD62L and CD127, suggesting immune control mechanisms. Prior research showed that disin- that the double-positive TILs represent an antigen-experienced, hibiting malfunctioning immune checkpoints (1) could help in functionally exhausted effector cell phenotype. Importantly, PD-1 mounting an antitumor immunity. In light of the multiple poten- blockade alone enhanced the proliferation of single PD-1 positive tial checkpoint targets, the article by Duraiswamy and colleagues and weakly that of double-positive TILs in response to peptide. (2) from our laboratory set out to dissect the potential of cotarget- CTLA-4 blockade alone was able to enhance the proliferation only þ þ þ À ing two key components of T-cell dysregulation in tumors, cytotoxic of PD-1 CTLA-4 CD8 TILs and had no effect on CTLA-4 cells. T lymphocyte–associated protein 4 (CTLA-4), and programmed However, dual blockade potently enhanced the proliferation of þ þ þ cell death 1 (PD-1) to potentially synergize their efficacies in tumor PD-1 CTLA-4 CD8 TILs in response to peptide presented by eradication. Furthermore, we aimed at enhancing checkpoint antigen-presenting cells. Thus, our work clearly demonstrated blockades with a whole tumor antigen vaccination strategy. Our that double PD-1/CTLA-4 blockade can rescue double-positive þ þ overall aim was to provide preclinical evidence of a combination PD-1 CTLA-4 TILs from their severely dysfunctional status and immune therapy that could be translated to human clinical trials. partly restore their response to tumor antigen. In Duraiswamy and colleagues' work, we made use of two Therapeutic blockade of either PD-1 or CTLA-4 in vivo enhanced immunocompetent transplantation murine tumor models, a colon the frequency of effector T cells infiltrating tumors. However, cancer model, CT26, and the model of ovarian cancer ID8-VEGF simultaneous therapeutic blockade further enhanced the frequen- (2). These models are faithful representations of human cancer cy of TILs and had significant and additive effects in activating þ þ development in an immunocompetent host. First, we could show CD8 and CD4 effector TILs as revealed by proliferation and that both of these tumors have a baseline T-cell infiltrate, but one effector function (expression of granzyme B and tumor antigen– that is severely impaired functionally. CTLA-4 and PD-1 were specific polyfunctional cytokine secretion). Furthermore, the coexpressed in a subset of tumor-infiltrating lymphocytes (TIL), combination uniquely reduced the frequency of T regulatory cells which showed the greatest level of inhibition of T-cell function, (Treg) in tumors. These pharmacodynamics effects were in agree- including antigen recognition and cytokine release. In both tumor ment with an important effect on response and cure rate of the þ models, one can identify and track tumor-specificCD8 Tcells animals treated with double blockade. directed against known antigens of CT26 or ID8-VEGF tumors, We compared anti-aPD-1 and aPD-L1 antibodies in these namely the gp70423–431 peptide of AH-1 and the FR161–169 of the double blockade experiments. Although the clinical efficacy of þ À folate receptor, respectively. Importantly, PD-1 CTLA-4 and PD- the single PD-L1 blockade was found to be slightly higher than þ þ 1 CTLA-4 TILs were sorted from untreated tumors and tested for that of PD-1 blockade, the efficacy of double PD-1/CTLA-4 and response ex vivo to the above cognate peptide in each model, PD-L1/CTLA-4 blockade was similar. PD-1 and PD-L1 induced which was presented in these experiments by pulsed splenocytes. similar effects on TILs with respect to activation of effector Reproducible results were found in both tumor models, whereby function, although at a signaling level, we found some differences. þ þ þ þ the frequency of IFNg , TNFa , IL2 , or CD107a/b responder Although both antibodies increased phosphorylated (p)Eomes, which is expressed in memory T cells, and pS6K, which acts to activate cell proliferation, protein translation, and survival, PD-L1 1Ludwig Institute for Cancer Research, Lausanne Branch, Lausanne, Switzerland. blockade was associated with a marked increase in pT-bet expres- 2Department of Oncology, University Hospital of Lausanne, Lausanne, Switzer- sion, which is expressed preferentially by type I effector T cells. land. 3Swiss Institute for Experimental Cancer Research at EPFL, Lausanne, PD-L1 blockade combined with CTLA-4 blockade further increased 4 Switzerland. Department of Pathology, University of Chicago, Chicago, Illinois. pEomes, whereas PD-1 blockade combined with CTLA-4 blockade Corresponding Author: George Coukos, Department of Oncology and Ludwig further increased pS6K. PD-L1/CTLA-4 blockade also induced þ Branch, CHUV, University of Lausanne, Rue du Bugnon 46, Lausanne BH09-701, higher numbers of TILs and a higher ratio of CD8 to Treg cells. Switzerland. E-mail: [email protected] Clinical evidence to date has not revealed major differences in the doi: 10.1158/0008-5472.CAN-16-2868 efficacy of individual PD-1 versus PD-L1–blocking antibodies, and Ó2016 American Association for Cancer Research. further results are awaited with the PD-L1/CTLA-4 blockade. www.aacrjournals.org 6765 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. CR 75th Anniversary Commentary At the time of publication, clinical data was not available to combined with vaccination led to enhanced infiltration of tumor- þ substantiate these preclinical findings. Since then, we have seen specificCD8 T cells, producing IFNg and TNFa in tumors. Thus, that the dual blockade of CTLA-4 and PD-1 can outperform effective vaccination against tumor antigens could in fact provide monotherapies of either agent (3). As shown in metastatic mel- an important partner to CTLA-4 and PD-1/PD-L1 blockade. Cor- anoma, in a phase III clinical trial, upfront CTLA-4 and PD-1 roborating evidence comes from human and mouse melanoma, þ blockade results in responses in up to 60% of patients. These where the administration of effective CD103 dendritic cells could responses are mostly durable, but definitive overall survival data restore the sensitivity of tumors and overcome intrinsic resistance to are eagerly awaited. Similar efficacy was seen with dual therapy in combined CTLA-4/PD-L1 blockade, which in this case was driven mismatch repair–deficient metastatic colorectal cancer (4) as well by constitutive tumor activation of b-catenin signaling (11). as non–small cell lung cancer (5). The combination is currently Parallel with CTLA-4 and PD-1, many other immune check- evaluated in many cancer types in 83 clinical trials. points are in place in cancers. The definition of these regulatory Although the response rates in metastatic melanoma and other molecules in cancer of individual patients will be essential to patients are encouraging, clearly not all patients respond, and define the optimal therapy. Hence, personalization of immuno- patient selection will be a key both to find patients who benefit therapy could be achieved by careful identification of the unique from the combination therapy and to propose alternative options neoepitope repertoire shared by many cancer cells of an individ- to those who are unlikely to respond. Emerging data indicate that ual to prepare cancer vaccines and by selectively targeting immune the absence of PD-L1 expression alone may be a weak predictor of checkpoints most abundant in that tumor. lack of benefit from dual therapy and may not be helpful in The field of cancer immunotherapy is advancing rapidly as clinical practice. clinical data are emerging, and preclinical models provide better Both in mice (6) and in human patients (7), tumors showing understanding of mechanisms underlying resistance to therapy, deregulation of T-cell infiltration are unlikely to respond to PD-1 which would allow developing biomarkers for patient selection blockade, and a recent publication (8) confirms our preclinical and targeted therapeutic combinations to overcome therapeutic finding in that dual CTLA-4 and PD-1–positive T cells are key resistance. Although intrinsic resistance accounts today for many predictors of responsiveness to immunotherapy. In fact, benefit early therapeutic failures, evidence of adaptive resistance is pro- from PD-1–inhibitory therapy was strongly correlated with higher vided by patients who progress after an initial response. The frequency of double-positive PD-1hi/CTLA-4hi TILs. Thus, PD-1 determination of the dysregulated cancer immune networks is monotherapy is not expected to be effective in patients with not only important prior to initiation of immune therapies but nonimmunogenic tumors lacking TILs, but whether the double also during response and at adaptation/progression. Adaptive CTLA-4 and PD-1/PD-L1 inhibition could be effective in TIL- resistance mechanisms have mainly been described for small- negative tumors remains to be tested.