Combine and Conquer: Double CTLA-4 and PD-1 Blockade Combined with Whole Tumor Antigen Vaccine Cooperate to Eradicate Tumors
Authors: Krisztian Homicsko, Jaikumar Duraiswamy, Marie-Agnès Doucey, George Coukos
Journal: Cancer Research (2016)

Visit the Cancer Research 75th Anniversary timeline.See related article by Duraiswamy et al., Cancer Res 2013;73:3591–603.Cancers hijack the normal regulatory pathways of normal inflammatory reactions. Dysregulation of fundamental immune checkpoints is present virtually in all tumors with underlying immune recognition and is crucial in the development and maintenance of cancer immune tolerance. The dissection of immune checkpoints showed that cancers can corrupt not one but multiple immune control mechanisms. Prior research showed that disinhibiting malfunctioning immune checkpoints (1) could help in mounting an antitumor immunity. In light of the multiple potential checkpoint targets, the article by Duraiswamy and colleagues (2) from our laboratory set out to dissect the potential of cotargeting two key components of T-cell dysregulation in tumors, cytotoxic T lymphocyte–associated protein 4 (CTLA-4), and programmed cell death 1 (PD-1) to potentially synergize their efficacies in tumor eradication. Furthermore, we aimed at enhancing checkpoint blockades with a whole tumor antigen vaccination strategy. Our overall aim was to provide preclinical evidence of a combination immune therapy that could be translated to human clinical trials.In Duraiswamy and colleagues' work, we made use of two immunocompetent transplantation murine tumor models, a colon cancer model, CT26, and the model of ovarian cancer ID8-VEGF (2). These models are faithful representations of human cancer development in an immunocompetent host. First, we could show that both of these tumors have a baseline T-cell infiltrate, but one that is severely impaired functionally. CTLA-4 and PD-1 were coexpressed in a subset of tumor-infiltrating lymphocytes (TIL), which showed the greatest level of inhibition of T-cell function, including antigen recognition and cytokine release. In both tumor models, one can identify and track tumor-specific CD8+ T cells directed against known antigens of CT26 or ID8-VEGF tumors, namely the gp70423–431 peptide of AH-1 and the FR161–169 of the folate receptor, respectively. Importantly, PD-1+CTLA-4− and PD-1+CTLA-4+ TILs were sorted from untreated tumors and tested for response ex vivo to the above cognate peptide in each model, which was presented in these experiments by pulsed splenocytes. Reproducible results were found in both tumor models, whereby the frequency of IFNγ+, TNFα+, IL2+, or CD107a/b+ responder cells was approximately double among the single PD-1+ (PD-1+CTLA-4−) TILs relative to the double-positive (PD-1+CTLA-4+) TILs. In addition, the single-positive TILs were the only population to proliferate in response to peptide, whereas the double-positive population failed to proliferate. The double-positive population also expressed higher levels of additional inhibitory receptors, such as 2B4, LAG-3, and TIM-3, and exhibited higher levels of CD44 and lower levels of CD62L and CD127, suggesting that the double-positive TILs represent an antigen-experienced, functionally exhausted effector cell phenotype. Importantly, PD-1 blockade alone enhanced the proliferation of single PD-1 positive and weakly that of double-positive TILs in response to peptide. CTLA-4 blockade alone was able to enhance the proliferation only of PD-1+CTLA-4+ CD8+ TILs and had no effect on CTLA-4− cells. However, dual blockade potently enhanced the proliferation of PD-1+CTLA-4+ CD8+ TILs in response to peptide presented by antigen-presenting cells. Thus, our work clearly demonstrated that double PD-1/CTLA-4 blockade can rescue double-positive PD-1+CTLA-4+ TILs from their severely dysfunctional status and partly restore their response to tumor antigen.Therapeutic blockade of either PD-1 or CTLA-4 in vivo enhanced the frequency of effector T cells infiltrating tumors. However, simultaneous therapeutic blockade further enhanced the frequency of TILs and had significant and additive effects in activating CD8+ and CD4+ effector TILs as revealed by proliferation and effector function (expression of granzyme B and tumor antigen–specific polyfunctional cytokine secretion). Furthermore, the combination uniquely reduced the frequency of T regulatory cells (Treg) in tumors. These pharmacodynamics effects were in agreement with an important effect on response and cure rate of the animals treated with double blockade.We compared anti-αPD-1 and αPD-L1 antibodies in these double blockade experiments. Although the clinical efficacy of the single PD-L1 blockade was found to be slightly higher than that of PD-1 blockade, the efficacy of double PD-1/CTLA-4 and PD-L1/CTLA-4 blockade was similar. PD-1 and PD-L1 induced similar effects on TILs with respect to activation of effector function, although at a signaling level, we found some differences. 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 blockade was associated with a marked increase in pT-bet expression, which is expressed preferentially by type I effector T cells. PD-L1 blockade combined with CTLA-4 blockade further increased pEomes, whereas PD-1 blockade combined with CTLA-4 blockade further increased pS6K. PD-L1/CTLA-4 blockade also induced higher numbers of TILs and a higher ratio of CD8+ to Treg cells. Clinical evidence to date has not revealed major differences in the efficacy of individual PD-1 versus PD-L1–blocking antibodies, and further results are awaited with the PD-L1/CTLA-4 blockade.At the time of publication, clinical data was not available to substantiate these preclinical findings. Since then, we have seen that the dual blockade of CTLA-4 and PD-1 can outperform monotherapies of either agent (3). As shown in metastatic melanoma, in a phase III clinical trial, upfront CTLA-4 and PD-1 blockade results in responses in up to 60% of patients. These responses are mostly durable, but definitive overall survival data are eagerly awaited. Similar efficacy was seen with dual therapy in mismatch repair–deficient metastatic colorectal cancer (4) as well as non–small cell lung cancer (5). The combination is currently evaluated in many cancer types in 83 clinical trials.Although the response rates in metastatic melanoma and other patients are encouraging, clearly not all patients respond, and patient selection will be a key both to find patients who benefit from the combination therapy and to propose alternative options to those who are unlikely to respond. Emerging data indicate that the absence of PD-L1 expression alone may be a weak predictor of lack of benefit from dual therapy and may not be helpful in clinical practice.Both in mice (6) and in human patients (7), tumors showing deregulation of T-cell infiltration are unlikely to respond to PD-1 blockade, and a recent publication (8) confirms our preclinical finding in that dual CTLA-4 and PD-1–positive T cells are key predictors of responsiveness to immunotherapy. In fact, benefit from PD-1–inhibitory therapy was strongly correlated with higher frequency of double-positive PD-1hi/CTLA-4hi TILs. Thus, PD-1 monotherapy is not expected to be effective in patients with nonimmunogenic tumors lacking TILs, but whether the double CTLA-4 and PD-1/PD-L1 inhibition could be effective in TIL-negative tumors remains to be tested. CTLA-4 blockade can effectively immobilize immunity against nonimmunogenic tumors, but this is usually observed in mouse models when tumor antigen is simultaneously released through combinations, for example radiation. Thus, CTLA-4 and PD-1 inhibition may still lead to marginal benefit in patients with TIL-negative tumors in the absence of additional combinations.Important efforts must be made in the clinic to understand how to effectively mobilize immunity against tumors that are void of TILs. Some important leads have already emerged from preclinical models in the recent years. For example, some tumors exclude T cells, which accumulate in the surrounding stroma or at the invasive margin. We previously reported that endothelial Fas ligand (FasL) expression might be responsible for preventing successful extravasation and engraftment of effector T cells in numerous solid tumor types, including melanoma and many adenocarcinomas, both in man and in mouse. Given the direct role of VEGF and prostaglandin E2 in inducing endothelial FasL expression, targeting the tumor vascular barrier with antiangiogenesis regimens may prove beneficial in enhancing T-cell homing to tumors (9). Combinations of VEGF blockade individually with CTLA-4 or PD-L1 blockade have already been tested in the clinic, with strong positive effects reported on T-cell infiltration, confirming the relevance of these pathway interactions (10).Tumors eliciting minimal immune recognition may pose yet different challenges. In our mouse models, GVAX significantly increased the complete response rate in the two mouse models when added to the checkpoint blockade combination. Vaccines expand the pool of tumor-specific effector and memory T cells and allow for exploiting the beneficial effects of CTLA-4 blockade on T-cell priming. In our models, the vaccine increased the frequency of tumor-reactive CD8+ TILs, and dual PD-1 and CTLA-4 inhibition combined with vaccination led to enhanced infiltration of tumor-specific CD8+ T cells, producing IFNγ and TNFα in tumors. Thus, effective vaccination against tumor antigens could in fact provide an important partner to CTLA-4 and PD-1/PD-L1 blockade. Corroborating evidence comes from human and mouse melanoma, where the administration of effective CD103+ dendritic cells could restore the sensitivity of tumors and overcome intrinsic resistance to combined CTLA-4/PD-L1 blockade, which in this case was driven by constitutive tumor activation of β-catenin signaling (11).Parallel with CTLA-4 and PD-1, many other immune checkpoints are in place in cancers. The definition of these regulatory molecules in cancer of individual patients will be essential to define the optimal therapy. Hence, personalization of immunotherapy could be achieved by careful identification of the unique neoepitope repertoire shared by many cancer cells of an individual to prepare cancer vaccines and by selectively targeting immune checkpoints most abundant in that tumor.The field of cancer immunotherapy is advancing rapidly as clinical data are emerging, and preclinical models provide better understanding of mechanisms underlying resistance to therapy, which would allow developing biomarkers for patient selection and targeted therapeutic combinations to overcome therapeutic resistance. Although intrinsic resistance accounts today for many early therapeutic failures, evidence of adaptive resistance is provided by patients who progress after an initial response. The determination of the dysregulated cancer immune networks is not only important prior to initiation of immune therapies but also during response and at adaptation/progression. Adaptive resistance mechanisms have mainly been described for small-molecule kinase inhibitors. However, recent studies show that tumor cells as well as the immune microenvironment can evolve under immune pressure, leading to escape. For example, in response to PD-1 blockade, tumor cells can lose IFN response genes and escape cell death (12).In conclusion, our article showed that combination immune therapies should be applied to eradicate tumors. Further research should define how to best select optimal combinations for personalized care. In addition, as we have shown, immunocompetent mouse models are essential for the mechanistic understanding of treatment efficacy and failure. These mouse models should be optimized to carefully reproduce the immune phenotypes of human cancers, which will enhance our understanding of gene- and cell-regulatory circuitries underlying response and allow developing optimal therapies.K. Homicsko reports receiving a commercial research grant from Roche and is a consultant/advisory board member for BMS, Merck Serono, Roche, and AMGEN. G. Coukos reports receiving commercial research grants from Boehringer-Ingelheim and Celgene, has received speakers bureau honoraria from Genentech and Roche, and is a consultant/advisory board member for Genentech, Novartis, Roche, and Sanofi-Aventis. No potential conflicts of interest were disclosed by the other authors.Krisztian Homicsko was supported by the Leenaards Foundation, Nuovo-Soldati Foundation, and by a research fellowship from the University of Lausanne. George Coukos was supported by NIH transformative grant R01CA156695, ERC advanced grant 1400206AdG-322875, Leenaards Foundation, Marcus Foundation, and Research Fellowship by University of Lausanne.