Brendan Horton

Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment.

BackgroundBlockade of immune inhibitory pathways is emerging as an important therapeutic modality for the treatment of cancer. Single agent treatments have partial anti-tumor activity in preclinical models and in human cancer patients. Inasmuch as the tumor microenvironment shows evidence of multiple immune inhibitory mechanisms present concurrently, it has been reasoned that combination therapies may be required for optimal therapeutic effect.MethodsTo test this notion, we utilized permutations of anti-CTLA-4 mAb, anti-PD-L1 mAb, and/or the IDO inhibitor INCB23843 in the murine B16.SIY melanoma model.ResultsAll three combinations showed markedly improved tumor control over single treatments, with many mice achieving complete tumor rejection. This effect was seen in the absence of vaccination or adoptive T cell therapy. The mechanism of synergy was investigated to examine the priming versus effector phase of the anti-tumor immune response. Only a minimal increase in priming of anti-tumor T cells was observed at early time points in the tumor-draining lymph nodes (TdLN). In contrast, as early as three days after therapy initiation, a marked increase in the capacity of tumor-infiltrating CD8+ T cells to produce IL-2 and to proliferate was found in all groups treated with the effective combinations. Treatment of mice with FTY720 to block new T cell trafficking from secondary lymphoid structures still enabled restoration of IL-2 production and proliferation by intratumoral T cells, and also retained most of the tumor growth control.ConclusionsOur data suggest that the therapeutic effect of these immunotherapies was mainly mediated through direct reactivation of T cells in situ. These three combinations are attractive to pursue clinically, and the ability of intratumoral CD8+ T cells to produce IL-2 and to proliferate could be an important biomarker to integrate into clinical studies.

The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8+ T cells in the tumor microenvironment.

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous antitumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms, but also a T cell–intrinsic dysfunctional state. A more detailed study has been hampered by a lack of cell surface markers defining tumor-specific dysfunctional TILs, and PD-1 alone is not sufficient. Recently, we identified the transcription factor Egr2 as a critical component in controlling the anergic state in vitro. In this study, we show that the Egr2-driven cell surface proteins LAG-3 and 4-1BB can identify dysfunctional tumor antigen–specific CD8+ TIL. Co-expression of 4-1BB and LAG-3 was seen on a majority of CD8+ TILs, but not in lymphoid organs. Functional analysis revealed defective IL-2 and TNF production yet retained expression of IFN-&ggr; and regulatory T cell–recruiting chemokines. Transcriptional and phenotypic characterization revealed coexpression of multiple additional co-stimulatory and co-inhibitory receptors. Administration of anti–LAG-3 plus anti–4-1BB mAbs was therapeutic against tumors in vivo, which correlated with phenotypic normalization. Our results indicate that coexpression of LAG-3 and 4-1BB characterize dysfunctional T cells within tumors, and that targeting these receptors has therapeutic utility.

Intratumoral CD8+ T-cell Apoptosis Is a Major Component of T-cell Dysfunction and Impedes Antitumor Immunity

The apoptosis of tumor-infiltrating CD8+ T cells was found to be a major factor inhibiting antitumor immune responses. Blocking CD8+ T-cell apoptosis improved tumor control and should be considered as a strategy to enhancing current immunotherapies. Subsets of human tumors are infiltrated with tumor antigen–specific CD8+ T cells [tumor-infiltrating lymphocytes (TILs)] despite tumor progression. These TILs are thought to be inactivated by the immunosuppressive tumor microenvironment, through the engagement of inhibitory receptors such as CTLA-4 and PD-1. However, antigen-specific CD8+ TILs are not functionally inert but are undergoing activation in situ. Here, we show that antigen-specific CD8+ TILs are actively proliferating, yet also undergo high rates of apoptosis, leading to a vicious cycle of activation and death that limits immune efficacy. Preventing CD8+ TIL apoptosis by Bcl-xL overexpression enabled accumulation and improved tumor control. Effective combination immunotherapy with an agonist 4-1BB mAb plus either CTLA-4 or PD-L1 neutralization led to a marked accumulation of specific CD8+ TILs through decreased apoptosis rather than increased T-cell entry or proliferation. Our data suggest that antigen-driven apoptosis of CD8+ TILs is a barrier to effective spontaneous antitumor immunity and should be considered as a critical factor in the development of cancer immunotherapies. Cancer Immunol Res; 6(1); 14–24. ©2017 AACR.

Cancer Immunotherapy Targets Based on Understanding the T Cell-Inflamed Versus Non-T Cell-Inflamed Tumor Microenvironment

Most cancers express tumor antigens that can be recognized by T cells of the host. The fact that cancers become clinically evident nonetheless implies that immune escape must occur. Two major subsets of human melanoma metastases have been identified based on gene expression profiling. One subgroup has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I IFN signature. In contrast, the other major subset lacks this phenotype and has been designated as non-T cell-inflamed. The mechanisms of immune escape are likely distinct in these two phenotypes, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, FoxP3+ Tregs, and evidence for T cell-intrinsic anergy. Therapeutic strategies to overcome these inhibitory mechanisms are being pursued, and anti-PD-1 mAbs have been FDA approved. The presence of multiple inhibitory mechanisms in the same tumor microenvironment argues that combination therapies may be advantageous, several of which are in clinical testing. A new paradigm may be needed to promote de novo inflammation in cases of the non-T cell-infiltrated tumor microenvironment. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and cross-priming of T cells via CD8α+ DCs. New strategies are being developed to engage this pathway therapeutically, such as through STING agonists. The molecular mechanisms that mediate the presence or absence of the T cell-inflamed tumor microenvironment are being elucidated using parallel genomics platforms. The first oncogene pathway identified that mediates immune exclusion is the Wnt/β-catenin pathway, suggesting that new pharmacologic strategies to target this pathway should be developed to restore immune access to the tumor microenvironment.

The non-T-cell-inflamed tumor microenvironment: contributing factors and therapeutic solutions

The recent successes of cancer immunotherapy, first and foremost checkpoint blockade therapy, illustrate the power of the immune system to control cancer. As the number of patients receiving this therapy is increasing, the number of patients being resistant or establishing resistance toward immunotherapy is also increasing. We, therefore, need to further understand the mechanisms mediate resistance in order to prevent or overcome those mechanisms. Increasing evidence is being reported that alterations in tumor cell-intrinsic signaling pathways, including the activation of the WNT/β-catenin pathway, are associated with blunted T-cell infiltration. Infiltration of tumor by CD8 T cells is one of the most predictive biomarkers for the response toward immunotherapy and therefore the notion that alterations of certain tumor cell-intrinsic signaling pathways might mediate resistance should be considered. Understanding the molecular and immunological mechanisms mediating resistance will ultimately facilitate the development of effective treatment strategies counteracting immune evasion.

LAG-3 and 4-1BB identify dysfunctional antigen-specific T cells in the tumor microenvironment and combinatorial LAG-3/4-1BB targeting gives synergistic tumor control

Meeting abstracts Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous anti-tumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. One possible negative regulatory pathway is T cell-intrinsic anergy. Recently, we have

Agonistic 4-1bb antibodies in combination with inhibitory antibodies against CTLA-4, PD-L1 or LAG-3 ACT on CD8+ T cells in the tumor microenvironment and synergize to promote regression of established tumors

Tumors can be placed into two categories: those that are T cell-inflamed and those that are not, based on specific chemokine and type I interferon gene expression signatures and CD8+ tumor infiltrating T cells (TIL). That these immune-inflamed tumors are not destroyed by the CD8+ TIL argues that mechanisms must be present to render the CD8+ TIL dysfunctional. Baseline infiltration of CD8+ T cells appears to be critical for tumor regression in response to neutralizing antibodies against CTLA-4 and PD-L1, indicating that these immunotherapies work on T cells in the tumor microenvironment. To better characterize the functional properties and molecular targets in dysfunctional TIL, we have applied knowledge from in vitro T cell anergy studies towards an analysis of T cells in the tumor microenvironment using several mouse tumor models. These experiments led to the finding that dysfunctional CD8+ TIL express the inhibitory receptors PD-1 and LAG-3, but also paradoxically the co-stimulatory molecules 4-1BB and OX-40. We therefore investigated whether delivering positive signals through costimulatory receptors, in combination with blockade of specific inhibitory receptors, could act on dysfunctional TIL to restore their function and induce tumor regression. We found that an agonistic anti 4-1BB antibody combined with inhibitory antibodies against either PD-L1, LAG-3 or CTLA-4 induce tumor regression of established B16.SIY tumors. To test whether these combinations affect CD8+ T cells in the tumor microenvironment or in the periphery we measured the frequency of SIY reactive CD8+ T cells in the tumor, the tumor-draining lymph nodes, and in the spleen. We found that frequencies of SIY-reactive CD8+ T cells increased in both the periphery and the tumor after these antibody combinations. To test if newly primed cells from the periphery were required for tumor regression, we used the S1P1 inhibitor FTY720 to block T cell egress from lymph nodes. FTY720 treatment given continuously beginning hours before these antibody combinations did not prevent tumor control, suggesting that the effects of combinations of agonist 4-1BB antibody with inhibitory antibodies to CTLA-4, PD-L1 or LAG-3 occur within the tumor microenvironment. Effective combinations restored IL-2 production by CD8+ T cells within the tumor site. Our data suggest that reversing T cell dysfunction within the tumor microenvironment may be a common mechanism of multiple immunotherapy combinations, and that positive costimulatory signals can synergize with blockade of inhibitory receptors to reverse TIL dysfunction and support tumor regression.

Back from the dead: TIL apoptosis in cancer immune evasion

Tumours from multiple cancer types can be infiltrated by CD8þ T cells (TILs) (Spranger et al, 2016). TILs are thought to be suppressed by multiple immune inhibitory molecules in the tumour microenvironment, and this suppression has been associated with tumour progression (Gajewski et al, 2013). Therefore, despite tumour infiltration, almost all tumours containing TILs will progress if not treated. While several immune inhibitory mechanisms have been identified, immune inhibitory receptors expressed on activated T cells, like CTLA-4 and PD-1, have received the most attention over recent years owing to the immense clinical success of PD-1 and CTLA-4 neutralising antibodies (Hodi et al, 2010; Topalian et al, 2012). The engagement of inhibitory receptors expressed by TILs is thought to render TILs dysfunctional. However, evidence from both human tumour samples and mouse models has suggested that, despite inhibitory receptor expression, TILs are not functionally inert and actually retain the ability to proliferate, produce IFN-g (Daud et al, 2016; Williams et al, 2017), and show ex vivo cytotoxicity (Mahnke et al, 2012). These observations raise the question of why activated TILs are not able to spontaneously control progressing tumours, and how tumours that contain TILs might sometimes be resistant to immunotherapies such as checkpoint blockade. Current immunotherapies can induce durable tumour regression; however, they benefit a minority of patients: finding new strategies to increase the response rate to immunotherapies is of great interest to both researchers and clinicians. Two new studies have reported that CD8þ TILs undergo apoptosis within the tumour microenvironment, indicating that their apoptosis is a contributing factor to the frequent failure of TILs to reject tumours. Studies from Zhu et al (Zhu et al, 2017) and from our own laboratory (Horton et al, 2017) found that antigenspecific CD8þ T cells undergo apoptosis in the tumour microenvironment, limiting their ability to control progressing tumours and respond to immunotherapy. Zhu et al utilised an inducible melanoma model expressing a tumour antigen, P1A, to test for responsiveness to various types of immunotherapy. They found that adoptive cell transfer (ACT) of activated CD8þ T cells into the murine model was able to regress transplanted melanomas, but not induced melanomas. From this finding, the authors investigated the differences between the transplanted and induced melanoma models. The authors found that in induced melanomas, where ACT was unsuccessful, both the transferred and endogenous CD8þ TILs underwent increased apoptosis compared with the transplanted tumours. In their model, TIL apoptosis was dependent on IFN-g, which increased at the tumour site after ACT. Increased IFN-g led to induction of the apoptosis initiator FasL in the tumour microenvironment, particularly on myeloid-derived suppressor cells (MDSCs) which were enriched in the induced tumours. Blockade of FasL-Fas interactions with an Fas-Fc decreased TIL apoptosis, as did depletion of MDSCs. Furthermore, blockade of FasL-Fas signalling and MDSC depletion synergised with checkpoint blockade to slow the growth of induced melanomas (Figure 1). The study by Horton and colleagues also found that apoptosis of CD8þ TILs limited endogenous anti-tumour immunity, in this case utilising the transplantable B16.SIY melanoma model, a B16 melanoma line that was engineered to express the CD8þ T-cell antigen SIY. They tracked antigen-specific TILs using either K/ SIY pentamers or surface expression of both LAG-3 and 4-1BB, a phenotype which was previously found to identify dysfunctional, tumour antigen-specific cells in the tumour microenvironment (Williams et al, 2017). The authors found that LAG-3þ 4-1BBþ CD8þ TILs constantly proliferated in situ; however, despite this constant proliferation there was no accumulation of antigenspecific CD8þ TILs as the tumours progressed. Investigating this phenomenon, Horton and colleagues found that antigen-specific CD8þ TILs underwent apoptosis in the steady state during tumour progression, indicating that a futile cycle of proliferation and apoptosis was linked to T-cell dysfunction in the tumour microenvironment. Utilising multiple transplantable cell lines as well as an autochthonous, inducible melanoma model, they determined that apoptosis of antigen-specific TILs occurred in progressing, but not spontaneously regressing, tumours. This reduction in TIL apoptosis in regressing tumours is also consistent with observations by Zhu and colleagues, who found reduced TIL apoptosis during tumour regression induced by ACT. In their own

Abstract B154: Immune surveillance is thwarted by tumor-cell intrinsic β-catenin signaling

Growing evidence has emerged that subgroups of cancer patients have a spontaneous T cell-centered immune response reflected by infiltration of antigen-specific CD8+ T cells into the tumor microenvironment. However, another major subgroup of patients completely lacks this phenotype. Importantly, the presence of tumor-infiltrating CD8+ T cells has been correlated with clinical response to anti-PD-1 mAb and other immunotherapies. Recent work from our laboratory has revealed that activation of the Wnt/β-catenin pathway within tumor cells can mediate exclusion of T cells from the tumor microenvironment. Using an autochthonous inducible mouse model for melanoma (BrafV600E/PTEN-/-; BP) combined with or without stabilized β-catenin (BrafV600E/PTEN-/-/CAT-STA; BPC) we were able to show that this exclusion of antigen specific T cells from the tumor microenvironment was due to failed recruitment of Batf3-lineage dendritic cells. The absence of these dendritic cells led to defective early T cell priming and absence of systemic immunity. However, whether tumor-intrinsic β-catenin signaling might mediate tumor resistance at the effector phase of the anti-tumor immune response or even after an anti-tumor T cell response was established is not known. To test this notion, we used the regressor tumor model MC57-SIY, which is spontaneously rejected and leads to immunologic memory that can mediate immune surveillance. These tumors were implanted into naive BP and BPC mice that had been crossed to a Rosa26-LSL-SIY inducible antigen mouse. Following complete regression of the primary (MC57.SIY) tumor, the genetically induced melanomas were induced by topical tamoxifen application and tumor growth kinetics as well as infiltration of antigen-specific T cells analyzed. Although the primary SIY-specific CD8+ T cell response and the induced memory response were comparable between both tumor models, tumor protection was observed against BP-SIY tumors but not BPC-SIY tumors. This increased tumor control in BP-SIY mice was accompanied by strong T cell infiltration and a boosted memory response. These results suggest that effector T cell migration into tumor sites also might be excluded via tumor-intrinsic β-catenin signaling. To test this notion directly, we activated SIY-reactive T cell receptor transgenic T cells (2C T cells) in vitro and adoptively transferred them into BP-SIY and BPC-SIY tumor-bearing hosts. In fact, primed 2C cells were only found in BP-SIY tumors but not BPC-SIY tumors. These results were confirmed using in vivo 2-photon microscopy of the tumor microenvironment. Taken together, these data provide strong evidence that up-regulation of β-catenin in tumor cells is a very potent mechanism of immune evasion even after a primary immune response has been induced. As such, mechanisms of immune surveillance and editing of tumors expand beyond selection at the level of antigens but additionally via specific oncogene pathways activated within the tumor cells. Moreover, tumor-intrinsic β-catenin activation likely mediates resistance not only to checkpoint blockade therapy but also to T cell adoptive transfer. Citation Format: Stefani Spranger, Brendan Horton, Thomas F. Gajewski. Immune surveillance is thwarted by tumor-cell intrinsic β-catenin signaling. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B154.

Agonist anti-4-1BB plus neutralizing anti-CTLA-4 or -PD-L1 synergize to promote tumor regression by rescuing dying dysfunctional CD8+ T cells within the tumor microenvironment

Tumors can be broadly placed into two categories: those that are T cell-inflamed and those that are not, with T cell-inflamed tumors having specific chemokine and type I interferon gene expression signatures as well as CD8+ tumor-infiltrating T cells (TIL). The fact that these T cell-inflamed tumors are not destroyed by the CD8+ TIL argues that mechanisms in the tumor microenvironment must render the CD8+ TIL dysfunctional. In agreement with this argument, baseline infiltration of CD8+ T cells appears to be necessary for tumor regression in response to neutralizing antibodies against CTLA-4 and PD-L1, indicating that these immunotherapies work predominantly on T cells already infiltrating the tumor microenvironment. We analyzed CD8+ TIL in the mouse B16.SIY model of melanoma to identify additional immunotherapy targets that might synergize with blockade of CTLA-4 and the PD-1/PD-L1 pathway. Using knowledge from in vitro T cell anergy gene expression profiling and phenotypic analysis, we found that dysfunctional CD8+ TIL unexpectedly express several co-stimulatory molecules, including 4-1BB. We therefore investigated whether delivering positive signals through co-stimulatory receptors, in combination with blockade of specific inhibitory receptors, could act on dysfunctional TIL to restore their function and induce tumor regression. We found that an agonist anti 4-1BB antibody synergized with inhibitory antibodies against either CTLA-4 or PD-L1 to induce tumor regression of established B16.SIY tumors. By using the S1P1 inhibitor FTY720 to block T cell egress from lymph nodes, we found that these combinations did not require newly primed cells from the periphery for tumor regression, arguing that they work directly in the tumor microenvironment. Deeper analysis of TIL revealed expansion of SIY-reactive CD8+ TIL and restored IL-2 production, as well as decreased expression of inhibitory receptors PD-1, LAG-3 and 2B4. Investigation of the mechanism of increased TIL number revealed that proliferation of the TIL was not enhanced with these combinations. Rather, a marked decrease in apoptosis of SIY-reactive TIL was observed, as measured by decreased active caspase-3 levels during tumor regression induced by 4-1BB combination immunotherapy. 4-1BB may therefore provide critical survival signals that rescue dying dysfunctional CD8+ cells in the tumor microenvironment, allowing them to be re-functionalized by CTLA-4 or PD-L1 blockade to mediate tumor regression.