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Induction of potent immune responses to self-antigens remains a major challenge in tumor immunology. We have shown that a vaccine based on alphavirus replicon particles (VRP) activates strong cellular and humoral immunity to tyrosinase-related protein-2 (TRP2) melanoma antigen, providing prophylactic and therapeutic effects in stringent mouse models. Here, we report that the immunogenicity and efficacy of this vaccine is increased in combination with either antagonist anti-CTL antigen-4 (CTLA-4) or agonist anti-glucocorticoid-induced TNF family–related gene (GITR) immunomodulatory monoclonal antibodies (mAb). In the challenging therapeutic setting, VRP–TRP2 plus anti-GITR or anti–CTLA-4 mAb induced complete tumor regression in 90% and 50% of mice, respectively. These mAbs had similar adjuvant effects in priming an adaptive immune response against the vaccine-encoded antigen, augmenting, respectively, approximately 4- and 2-fold the TRP2-specific CD8+ T-cell response and circulating Abs, compared with the vaccine alone. Furthermore, while both mAbs increased the frequency of tumor-infiltrating CD8+ T cells, anti–CTLA-4 mAb also increased the quantity of intratumor CD4+Foxp3− T cells expressing the negative costimulatory molecule programmed death-1 (PD-1). Concurrent GITR expression on these cells suggests that they might be controlled by anti-GITR mAbs, thus potentially explaining their differential accumulation under the two treatment conditions. These findings indicate that combining immunomodulatory mAbs with alphavirus-based anticancer vaccines can provide therapeutic antitumor immune responses in a stringent mouse model, suggesting potential utility in clinical trials. They also indicate that tumor-infiltrating CD4+Foxp3−PD-1+ T cells may affect the outcome of immunomodulatory treatments. Cancer Immunol Res; 2(5); 448–58. ©2014 AACR.

PDF file - 428K, Supplementary Figure S1. Depigmentation after combination therapy. Supplementary Figure S2. Tumor immune infiltrate after immunomodulation. Supplementary Figure S3. PD-1 expression on T cell subsets. Supplementary Figure S4. Effect of anti-CTLA4 on TILs.

PDF file - 144K, Supplemental Figure 1 NY-ESO-1 antigen-specific CD4+ T cell lines were CD8 negative.

PDF file - 106K, Supplemental Table 1 Summary of NY-ESO-1 Immune responses.

CD4+ T cells provide help to enhance and sustain cytotoxic CD8+ T-cell responses. A direct lytic role for this cell population in mouse models further supports the use of tumor-reactive CD4+ T cells for cancer immunotherapy. CTLA-4 blockade has been shown to expand antigen-specific cytotoxic CD4+ T cells in mouse models. We took advantage of spontaneous immunity to the NY-ESO-1 cancer-testis antigen to investigate quantitative and qualitative changes in antigen-specific CD4+ T-cell responses after ipilimumab (anti-CTLA-4 monoclonal antibody) treatment in patients with advanced melanoma. Four patients with NY-ESO-1 seropositive melanoma were chosen upon the availability of suitable blood specimens for characterizing the functions of NY-ESO-1 antigen-specific CD4+ T-cell response by enzyme-linked immunospot (ELISPOT), intracellular cytokine staining (ICS), and cytotoxicity assays. Multiple NY-ESO-1 antigen-specific CD4+ T-cell responses with TH1 dominance were induced or enhanced after ipilimumab treatment in peripheral blood in all four patients. NY-ESO-1 antigen–specific CD4+ T-cell lines established from all four patients after ipilimumab treatment recognized naturally processed NY-ESO-1 protein in antigen-presenting cells, expressed master transcription factor Eomesodermin (Eomes), and secreted perforin and Granzyme B. Finally, we showed that these NY-ESO-1 antigen-specific CD4+ T-cell lines directly lysed autologous melanoma cell lines expressing NY-ESO-1 in an MHC class II restricted manner. Our results show that antigen-specific cytotoxic CD4+ T-cell responses are induced after ipilimumab therapy in human cancer patients. Ipilimumab may induce the expression of lytic granules on antigen-specific cytotoxic CD4+ T cells via Eomes, revealing a novel consequence of immunologic checkpoint blockade. Cancer Immunol Res; 1(4); 235–44. ©2013 AACR.

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The repertoire of tumor-infiltrating lymphocytes (TILs) can be vast, and many of these TILs are not endowed with tumor reactivity. While a number of reports have shown that tumor-reactive TILs express CD39, few reports have demonstrated that conversely, CD39 can be leveraged to serve as a proxy of tumor-reactive CD8 T cells. Using single-cell CITE/RNA/TCRseq, we show that CD39+ CD8 T cells in human lung cancers demonstrate transcriptional and proteomic features of exhaustion, tumor reactivity, and clonal expansion. Moreover, TCR cloning revealed that CD39 enriched for tumor-reactive CD8 T cell clones. Flow cytometry of 440 lung cancer specimens revealed that CD39 level on CD8 T cells is only weakly correlated with tumoral features that currently guide lung cancer therapy, such as histology, driver mutation, PD-L1 and tumor mutation burden. PD-1 axis blockade, but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8 T cells. CD39 correlated with PD-1 expression on CD8 T cells and high pre-treatment/early-on-treatment levels were associated with improved clinical outcomes, but not immune-related adverse events, from immune checkpoint blockade therapy. This comprehensive profiling of the clinical, pathological and molecular features highlights the utility of CD39 as a proxy for tumor-reactive CD8 T cells in human lung cancer.

Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

Transcription factors ThPOK and Runx3 regulate the differentiation of “helper” CD4+ and “cytotoxic” CD8+ T cell lineages respectively, inducing single positive (SP) T cells that enter the periphery with the expression of either the CD4 or CD8 co-receptor. Despite the expectation that these cell fates are mutually exclusive and that mature CD4+CD8+ double positive (DP) T cells are present in healthy individuals and augmented in the context of disease, yet their molecular features and pathophysiologic role are disputed. Here, we show DP T cells in murine and human tumors as a heterogenous population originating from SP T cells which re-express the opposite co-receptor and acquire features of the opposite cell type’s phenotype and function following TCR stimulation. We identified distinct clonally expanded DP T cells in human melanoma and lung cancer by scRNA sequencing and demonstrated their tumor reactivity in cytotoxicity assays. Our findings indicate that antigen stimulation induces SP T cells to differentiate into DP T cell subsets gaining in polyfunctional characteristics.

Irreversible T cell exhaustion limits the efficacy of programmed cell death 1 (PD-1) blockade. We observed that dual CD40-TLR4 stimulation within a single tumor restored PD-1 sensitivity and that this regimen triggered a systemic tumor-specific CD8(+) T cell response. This approach effectively treated established tumors in diverse syngeneic cancer models, and the systemic effect was dependent on the injected tumor, indicating that treated tumors were converted into necessary components of this therapy. Strikingly, this approach was associated with the absence of exhausted PD-1(hi) T cells in treated and distant tumors, while sparing the intervening draining lymph node and spleen. Furthermore, patients with transcription changes like those induced by this therapy experienced improved progression-free survival with anti–PD-1 treatment. Dual CD40-TLR4 activation within a single tumor is thus an approach for overcoming resistance to PD-1 blockade that is unique in its ability to cause the loss of exhausted T cells within tumors while sparing nonmalignant tissues.

Preclinical and clinical studies have shown that intratumoral oncolytic viruses (OVs) can potentiate host anti-tumor immunity and overcome resistance to immune checkpoint blockade, although clinical responses to OVs have been modest to date. While T cell infiltration of tumors is frequently cited as a measure of OV immunogenicity, this measure is non-specific as OVs elicit a strong virus-directed T cell response, and it remains unknown what proportion of T cells expanded by OV therapy are virus-specific vs. tumor-specific or how these T cells distribute across virus-treated and distant tumors. We employed oncolytic Newcastle disease virus (NDV) in a bilateral flank melanoma mouse model to identify and phenotypically characterize anti-viral and anti-tumor T cells using single cell (sc) RNA and T cell receptor (TCR) sequencing. Intratumoral NDV therapy to a single flank tumor resulted in increased infiltration of CD4+ and CD8+ T cells in the injected (enestic) and distant (non-enestic) tumors and increased the breadth of the TCR repertoire at both sites with preferential expansion of several dominant clonotypes. There was substantial expansion of the proportion of T cell clonotypes shared between enestic and nonenestic tumors as well as the spleen, indicative of inter-tumor TCR repertoire normalization. In both treated and distant tumors, we observed a significant increase in the frequency of convergent TCR clonotypes, i.e. TCRs encoded by different nucleotide sequences that converge on the same amino acid sequence, implying that the presence of these TCRs in tumors is non-random. Using scRNA and paired TCR sequencing, we demonstrate that NDV therapy leads to expansion of unique clusters of terminally differentiated and activated CD4+ and CD8+ T cells associated with distinct TCR-based clonotypes. Notably, the predominant phenotypic clusters were distinct between the enestic and non-enestic tumors. Enestic tumors were dominated by CD8+ T cells exhibiting a signature associated with terminal dysfunction (PDCD1, LAG3, TOX), while the predominant expanded CD8+ T cells in non-enestic tumors exhibited an activation signature associated with high expression of cytolytic markers. While phenotypic states were conserved for the dominant TCR clones shared across the enestic and non-enestic tumors, TCRs unique to the non-enestic tumor were predominantly associated with an activated T cell state. Taken together, these studies highlight that T cells expanded by OV therapy exhibit unique functional states and TCRs in treated and distant tumors and imply that virus- and tumor-specific T cells may be identified by distinct TCR repertoires and phenotypes. Understanding the balance between virus- and tumor-directed T cells elicited by OV therapy will be key to engineering OVs and designing combination strategies that drive stronger tumor-directed T cell response. Citation Format: Bharat Burman, Nicholas Ceglia, Daniel Hirschhorn, Sadna Budhu, Levi Mangarin, Anton Oseledchyk, Yonina Bykov, Andrew McPherson, Sohrab Shah, Jedd Wolchok, Taha Merghoub, Dmitriy Zamarin. Defining the balance of anti-viral and anti-tumor T cell responses to oncolytic virus therapy using single cell approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5217.

Immune Checkpoint Blockade (ICB) with anti- programmed cell death protein (PD)-1 and anti-cytotoxic T lymphocyte associated protein (CTLA)-4 have had immense success in cancer treatment. However, primary and acquired resistance to these therapies limits their clinical benefit. Accumulating evidence indicates that chemotherapeutic agents such as Cyclophosphamide (CTX) that can induce anticancer immunity stand out as particularly promising partners for use in combination with ICBs. A combination approach to reorchestrate the anti-tumor immune response could magnify the clinical benefit of ICBs. CTX is an alkylating chemotherapeutic which is directly tumoricidal that can also modulate immune cells. CTX is known to preferentially deplete certain T cell subsets (such as T regulatory cells, T regs) and lead to homeostatic proliferation of antigen-specific T cells in addition to the modulation of myeloid cells. Here, we hypothesized that the addition of ICBs will augment the immunomodulatory changes induced by CTX including but not limited to the homeostatic proliferation of T cells which will reset the T cell receptor (TCR) repertoire to favor tumor antigen-specific T cells. The murine melanoma tumor model, B16-F10, is known to be refractory to treatment with ICBs, particularly anti-PD-1, as these only modestly slow the growth of small tumors. Here, we show that a single dose of CTX one day prior to starting an ICB (anti-PD-1 and anti-CTLA-4) regimen slows tumor progression of B16-F10 compared to CTX or ICB alone. Similarly, the triple combination (CTX+ anti- PD-1 + anti-CTLA-4) also significantly prolongs the survival of tumor bearing mice. Further, the combination of CTX with anti- PD-1 and anti-CTLA-4 increased the number of activated and proliferating CD8+ tumor infiltrating lymphocytes (TILs). This increase in cytotoxic T cells was accompanied by a decrease in Tregs, which further augments the tumor control. Additionally, we also observed a significant increase in a highly cytolytic population of CD4+ CD8+ double positive TILs. The depletion of CD8+ cells but not CD4+ cells abrogates the therapeutic effect of the triple combination suggesting that the anti-tumor effect is CD8+ T cell dependent. Overall, our results suggest that the combination of CTX with the ICBs, anti- PD-1 and anti-CTLA-4, is a potent combinatorial approach that can prime an anti-tumor response and promote robust control of tumor growth. Thus, these findings form the basis for further investigations in understanding the mechanisms of combinatorial cancer therapies in tumor models that are refractory to ICB therapies and can inform the design of future therapeutic interventions that combine ICB with chemotherapy. Note: M.M.G, A.B.W, and L.H contributed equally to this work Citation Format: Mariam M. George, Allison Betof Warner, Linda Hamadene, Daniel Hirschhorn, Alan N. Houghton, Jedd D. Wolchok, Taha Merghoub. Combination of cyclophosphamide with immune checkpoint blockade inhibition elicits potent tumor control in a preclinical melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4167.

Background CD4 and CD8 T cells are genetically and functionally distinct cell subsets of the adaptive immune system that play pivotal roles in immune surveillance and disease control. During development in the thymus, transcription factors ThPOK and Runx3 regulate the differentiation and maturation of these two lineages into single positive T cells that enter the periphery with mutually exclusive expression of either the CD4 or CD8 co-receptor.1–2 Despite our expectation that these two cell fates are fixed, mature CD4+CD8+ double positive (DP) T cells have been described in the context of numerous immunological responses, including cancer, but their molecular and functional properties and therapeutic relevance remain controversial and largely unknown.3–5 Methods Our lab has identified and characterized a heterogenous DP T cell population in murine and human melanoma tumors comprised of CD4 and CD8 T cells re-expressing the opposite co-receptor and a parallel uptake in the opposite cell type’s phenotype and function. Using CD4 (Trp1) and CD8 (Pmel) transgenic TCR T cells specific to B16 melanoma antigens gp75 and gp100 respectively, we demonstrate the re-expression of the opposite co-receptor following adoptive T cell transfer in B16 melanoma tumor bearing mice. Results Specifically, up to 50% of transferred CD4 Trp1 T cells will re-express CD8 to become a DP T cell in the tumor microenvironment. Further, these CD4 derived DP T cells upregulate CD8 lineage regulator Runx3 and cytolytic genes Gzmb, Gzmk, and Prf1 to become potent cytotoxic T cells. Alternatively, a subset of CD8 Pmel T cells differentiate into DP T cells characterized by the increased expression of CD4, ThPOK, and regulatory marker FoxP3 (figure 1). In addition, we utilized 10x single cell and ATAC sequencing to further characterize these divergent DP T cell populations among open repertoire T cells isolated from murine and human melanoma tumors. Conclusions Our findings highlight the capability of single positive T cells to differentiate in response to antigen and local stimuli into novel T cell subsets with polyfunctional characteristics. The resulting cell subsets will potentially affect the tumor microenvironment in distinct ways. Our studies may inform therapeutic approaches to identify antigen specific T cells as well as innovative signaling pathways to target when genetically engineering T cells to optimize cytotoxic function in the setting of adoptive cell therapy. Ethics Approval The human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #06-107 References Ellmeier W, Haust L & Tschismarov R. Transcriptional control of CD4 and CD8 coreceptor expression during T cell development. Cell Mol Life Sci 2013;70:4537–4553. Luckey MA, et al. The transcription factor ThPOK suppresses Runx3 and imposes CD4+ lineage fate by inducing the SOCS suppressors of cytokine signaling. Nature Immunology 2014; 15, 638–645. Bohner P, et al. Double positive CD4(+)CD8(+) T Cells are enriched in urological cancers and favor T Helper-2 polarization. Front Immunol 2019; 10, 622. Nascimbeni M, Shin E-C, Chiriboga L, Kleiner DE & Rehermann B. Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood 2004;104:478–486. Nishida K, et al. Clinical importance of the expression of CD4+CD8+ T cells in renal cell carcinoma. Int Immunol 2020;32:347–357.

Targeted immune therapies such as adoptive T cell transfer (ACT) are often ineffective because tumors evolve over time under selective pressure to display antigen loss variant. A classic example is de-differentiation and loss of expression of antigenic proteins. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here we show that melanoma-specific CD4+ ACT therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas that contain clonal escape variants. As expected, early on-target recognition of melanoma antigens by adoptively transferred tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was partially dependent on neutrophils. Supporting these findings, extensive neutrophil extracellular traps were found in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Our findings uncover a novel interplay between T cells mediating the initial tumor- and tissue-specific immune response, and neutrophils mediating tumor destruction of antigen loss variants.

BackgroundTargeted immune-based therapies such as adoptive T cell transfer (ACT) are often ineffective because tumors evolve over time and under selective pressure display antigen loss variant clones. A classic example in melanoma is de-differentiation and loss of expression of antigenic proteins. Therapies that activate multiple branches of the immune system may eliminate such escape variantsMethodsHere we show that melanoma-specific CD4+ ACT therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate large melanoma tumors with clonal escape variants.ResultsEarly on-target recognition of melanoma antigens by adoptively transferred tumor-specific CD4+ T cells was required. Surprisingly, however, complete tumor eradication was partially dependent on neutrophils. Supporting these findings, extensive neutrophil activation and neutrophil extracellular traps were found in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade.ConclusionsOur findings uncover a novel interplay between T cells mediating the initial tumor- and tissue-specific immune response, and neutrophils mediating tumor destruction of antigen loss variants.Ethics ApprovalAll tissues were collected at MSKCC following study protocol approval by the MSKCC Institutional Review Board. All mouse procedures were performed in accordance with institutional protocol guidelines at Memorial Sloan-Kettering Cancer Center (MSKCC) under an approved protocol.

Immune checkpoint blockade (ICB) with anti-CTLA-4 and anti-PD-1 is increasingly used to treat a variety of malignancies, but primary and acquired resistance limit the clinical benefit. Mechanism-guided approaches to overcome resistance and re-orchestrate an anti-tumor immune response could expand the clinical impact of ICBs. Cyclophosphamide (CTX) is an alkylating chemotherapeutic which is directly tumoricidal and also modulates immune response. CTX preferentially depletes certain T cell subsets (such as Tregs) and leads to homeostatic proliferation of antigen-specific T cells. We hypothesized that a dose of CTX prior to initiating ICB would counteract several known patterns of resistance and enhance anti-tumor response to ICB. The B16 murine melanoma model is largely refractory to inhibition of PD-1, CTLA-4, or combination CTLA-4+PD-1, all of which only modestly slow the growth of small tumors. One dose of CTX one day prior to each of these treatments significantly slows tumor growth and prolongs survival compared to CTX or ICB alone. This effect is most pronounced for the triple combination of CTX+ anti-CTLA-4+ anti-PD-1, where tumor regression was observed in 70% of mice and tumors were completely eradicated in 20%. In addition to standard ICB, we hypothesized that CTX would enhance response to immune modulatory molecules in clinical development. Glucocorticoid-induced TNFR family related gene (GITR) is a costimulatory molecule expressed primarily on the surface of T cell subsets. GITR engagement enhances activation, proliferation, and clonal expansion of effector T cells while hampering the suppressive function of Tregs. However, agonist anti-GITR antibodies have neither effectively controlled tumor growth when given as a monotherapy in preclinical models nor showed efficacy in clinical trials. In the B16 melanoma model, CTX + GITR engagement with the agonist monoclonal antibody DTA-1 slows tumor growth, elicits some tumor regression, and prolongs survival, whereas neither therapy alone controls tumor growth. Similar efficacy is observed in other tumor models (MCP-11 myeloma and CT26 colon carcinoma). Combination therapy with CTX + DTA-1 increases the ratio of CD8+ effector T cells to Tregs, at least in part by inducing Treg-specific activation-induced cell death. Single cell sequencing and flow cytometry demonstrated a marked increase in pro-inflammatory cytokines, cytolytic granules, and activation markers accompanied by reduced exhaustion markers in intratumoral CD8+ T cells. Overall, we demonstrate that a single dose of CTX added to standard and novel immune modulators is a potent combinatorial approach that primes an anti-tumor response, enhances T cell fitness, and overcomes primary resistance to ICB. The data presented here serve as the foundation for mechanistically-driven clinical trials in development. Citation Format: Allison Betof Warner, Daniel Hirschhorn, Levi M. Mangarin, Linda Hamadene, Adam D. Cohen, Gabrielle A. Rizzuto, Jedd D. Wolchok, Taha Merghoub. Combination of cyclophosphamide chemotherapy with immune modulation overcomes resistance to checkpoint blockade in a pre-clinical melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1795.

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells and has been shown to promote immunosuppressive signals in the tumor microenvironment (TME). Antibodies that block PS interaction with its receptors have been shown to repolarize tumor associated macrophages (TAMs) into a pro-inflammatory (M1) state, reduce the number of myeloid derived suppressor cells (MDSCs) in tumors and promote maturation of dendritic cells (DCs). Radiation therapy (RT) is an effective focal treatment of isolated solid tumors but is less effective in controlling metastatic cancers as a monotherapy. Tumor-directed RT is known to induce immunogenic cell death and enhance tumor-specific T cell infiltration into treated tumors. We found that a single dose of 15 Gy of tumor-directed RT on mouse B16 melanoma causes an increase in expression of PS on the surface of viable immune infiltrates. We hypothesize that expression of PS on immune cells may act similar to immune checkpoint molecules and provide a negative feedback mechanism to immune cells. Therefore, we posit that blocking PS may enhance the efficacy of tumor-directed RT. Indeed, we found that treatment with an antibody that targets PS (mch1N11) enhanced the anti-tumor efficacy of tumor-directed RT and improved overall survival. This combination led to an increase in M1 TAMs and a corresponding decrease in M2 TAMs which translated to a more pro-inflammatory TME. The addition of anti-PD-1 to RT and mch1N11 led to even greater anti-tumor efficacy and overall survival. Anti-PD-1 exerted its effects primarily by enhancing CD8+ T cell infiltration, activation, and effector function in the triple combination. Consistent with the mouse studies, we found increased PS expression on several immune subsets in the blood in patients with metastatic melanoma after receiving tumor-directed RT. These findings highlight the potential of combining PS targeting with RT and PD-1 pathway blockade to improve outcomes in patients with advanced-stage cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade.

SUMMARY Phosphatidylserine (PS) is exposed on the surface of apoptotic cells and is known to promote immunosuppressive signals in the tumor microenvironment (TME). Antibodies that block PS interaction with its receptors have been shown to repolarize the TME into a proinflammatory state. Radiation therapy (RT) is an effective focal treatment of isolated solid tumors but is less effective at controlling metastatic cancers. We found that tumor-directed RT caused an increase in expression of PS on the surface of viable immune infiltrates in mouse B16 melanoma. We hypothesize that PS expression on immune cells may provide negative feedback to immune cells in the TME. Treatment with an antibody that targets PS (mch1N11) enhanced the anti-tumor efficacy of tumor-directed RT and improved overall survival. This combination led to an increase in proinflammatory tumor-associated macrophages. The addition of anti-PD-1 to RT and mch1N11 led to even greater anti-tumor efficacy and overall survival. We found increased PS expression on several immune subsets in the blood of patients with metastatic melanoma after receiving tumor-directed RT. These findings highlight the potential of combining PS targeting with RT and PD-1 pathway blockade to improve outcomes in patients with advanced-stage cancers., In Brief Budhu et al. show that tumor-directed irradiation of murine B16 melanoma causes an increase in PS on the surface of infiltrating immune cells. Blocking PS and RT improves the anti-tumor efficacy and overall survival, which can be further improved with the addition of anti-PD-1. Melanoma patients exhibit increased PS on their PBMCs after RT., Graphical Abstract

Adoptive cell therapy has emerged as a viable strategy to treat cancer. T cells that recognize tumor antigens can be reinvigorated ex-vivo or autologous T cells can be genetically modified to express anti-tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs). However, once re-infused into patients, these tumor specific T cells are subjected to immunosuppressive signals within the tumor. A critical immune checkpoint within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells and tumor cells. Innate cells exposed to PS secrete suppressive cytokines that can significantly impair the function of tumor specific T cells. Antibodies that target PS can reactivate anti-tumor immunity by reducing the number of MDSCs in tumors and promoting the maturation of functional APCs. Our lab has shown that the mouse chimeric version of PS Targeting monoclonal antibody Bavituximab (1N11), in combination with transgenic CD4+ T cells that recognize melanoma antigen Trp1, can regress advanced melanoma tumors in mice. Here, we demonstrate a 2nd generation CAR T cell, that binds Trp1 on the surface of B16 melanoma, in combination with 1N11 can improve anti-tumor activity and survival in B16 tumor bearing mice. Flow cytometry analysis of immune responses in the tumor of mice treated with tumor specific T cells and 1N11 shows a decrease in M2 macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with PS targeting can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS Targeting antibodies with CAR T cell therapy in solid tumors.

A significant proportion of cancer patients do not respond to immune checkpoint blockade. To better understand the molecular mechanisms underlying these treatments, we explored the role of CD4(+)Foxp3(−) T cells expressing PD-1 (4PD1(hi)) and observed that 4PD1(hi) accumulate intratumorally as a function of tumor burden. Interestingly, CTLA-4 blockade promotes intratumoral and peripheral 4PD1(hi) increases in a dose-dependent manner, while combination with PD-1 blockade mitigates this effect and improves anti-tumor activity. We found that lack of effective 4PD1(hi) reduction after anti-PD-1 correlates with poor prognosis. Mechanistically, we provide evidence that mouse and human circulating and intra-tumor 4PD1(hi) inhibit T cell functions in a PD-1/PD-L1 dependent fashion and resemble follicular-helper-T-cell(T(FH))-like cells. Accordingly, anti-CTLA-4 activity is improved in T(FH) deficient mice.

Far too many employees are unhappy at work. For many, it starts with an inadequate and unfair salary. Today, one of the major problems resulting from weak, selfish,and callous management and leadership is the great number of workers -- including white-collar ones -- who are grossly underemployed, underpaid, and maltreated by management.

Why have war, crime, and other forms of vile wrongdoing essentially been ongoing from the beginning of time? Why do so many people routinely engage in all sorts of lesser mean-spirited, overly-aggressive actions leading to the detriment and victimization of others? Far too many of us are unhappy today because of the actions of terrible, immoral people. Too often, we become pained by the routine immorality, nastiness, unfairness, deceit, cheating, manipulation, carelessness, greed, hostility, and even physical violence coming from so many of the people all around us — leaving us emotionally and physically injured in their wake. In essence, part of human nature is that most people are at least somewhat routinely sadistic. The capacity to do evil deeds is within us from birth, a manifestation of our evolution starting with our early ancestry; we emerged from the most highly aggressive and intelligent primate the earth has ever known, the "killer ape."

CD4+ T cells provide help to enhance and sustain cytotoxic CD8+ T-cell responses. A direct lytic role for this cell population in mouse models further supports the use of tumor-reactive CD4+ T cells for cancer immunotherapy. CTLA-4 blockade has been shown to expand antigen-specific cytotoxic CD4+ T cells in mouse models. We took advantage of spontaneous immunity to the NY-ESO-1 cancer-testis antigen to investigate quantitative and qualitative changes in antigen-specific CD4+ T-cell responses after ipilimumab (anti-CTLA-4 monoclonal antibody) treatment in patients with advanced melanoma. Four patients with NY-ESO-1 seropositive melanoma were chosen upon the availability of suitable blood specimens for characterizing the functions of NY-ESO-1 antigen-specific CD4+ T-cell response by enzyme-linked immunospot (ELISPOT), intracellular cytokine staining (ICS), and cytotoxicity assays. Multiple NY-ESO-1 antigen-specific CD4+ T-cell responses with TH1 dominance were induced or enhanced after ipilimumab treatment in peripheral blood in all four patients. NY-ESO-1 antigen–specific CD4+ T-cell lines established from all four patients after ipilimumab treatment recognized naturally processed NY-ESO-1 protein in antigen-presenting cells, expressed master transcription factor Eomesodermin (Eomes), and secreted perforin and Granzyme B. Finally, we showed that these NY-ESO-1 antigen-specific CD4+ T-cell lines directly lysed autologous melanoma cell lines expressing NY-ESO-1 in an MHC class II restricted manner. Our results show that antigen-specific cytotoxic CD4+ T-cell responses are induced after ipilimumab therapy in human cancer patients. Ipilimumab may induce the expression of lytic granules on antigen-specific cytotoxic CD4+ T cells via Eomes, revealing a novel consequence of immunologic checkpoint blockade. Cancer Immunol Res; 1(4); 235–44. ©2013 AACR.

Therapy for cancer can be achieved by artificially stimulating antitumor T and natural killer (NK) lymphocytes with agonist monoclonal antibodies (mAb). T and NK cells express several members of the TNF receptor (TNFR) family specialized in delivering a costimulatory signal on their surface. Engagement of these receptors is typically associated with proliferation, elevated effector functions, resistance to apoptosis, and differentiation into memory cells. These receptors lack any intrinsic enzymatic activity and their signal transduction relies on associations with TNFR-associated factor (TRAF) adaptor proteins. Stimulation of CD137 (4-1BB), CD134 (OX40), and glucocorticoid-induced TNFR (GITR; CD357) promotes impressive tumor-rejecting immunity in a variety of murine tumor models. The mechanisms of action depend on a complex interplay of CTL, T-helper cells, regulatory T cells, dendritic cells, and vascular endothelium in tumors. Agonist mAbs specific for CD137 have shown signs of objective clinical activity in patients with metastatic melanoma, whereas anti-OX40 and anti-GITR mAbs have entered clinical trials. Preclinical evidence suggests that engaging TNFR members would be particularly active with conventional cancer therapies and additional immunotherapeutic approaches. Indeed, T-cell responses elicited to tumor antigens by means of immunogenic tumor cell death are amplified by these immunostimulatory agonist mAbs. Furthermore, anti-CD137 mAbs have been shown to enhance NK-mediated cytotoxicity elicited by rituximab and trastuzumab. Combinations with other immunomodulatory mAb that block T-cell checkpoint blockade receptors such as CTLA-4 and PD-1 are also promising. Clin Cancer Res; 19(5); 1044–53. ©2013 AACR.

Targeting tumour-infiltrating suppressive myeloid cells with a selective PI3Kγ inhibitor overcomes resistance to checkpoint blockade therapy in various mouse myeloid-rich tumour models. Therapeutic blockade of immune checkpoints with antibodies against CTLA-4 and PD-1 has proved effective against some cancer types, but clinical benefit has been limited to a subset of patients. Here Olivier De Henau et al. show that resistance to checkpoint blockade is associated with a high level of infiltration by suppressive myeloid cells in various mouse tumour models. In addition, targeting the myeloid-derived suppressor cells with a selective inhibitor of the γ isoform of phosphoinositide 3-kinase (PI3Kγ) increases sensitivity to checkpoint blockade therapy in a melanoma mouse model. Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers1. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity2,3,4. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance5,6. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

OX40 engagement induces a cytotoxic CD4+ T cell subpopulation to eradicate advance melanomas, Harnessing the adaptive immune response to treat malignancy is now a clinical reality. Several strategies are used to treat melanoma; however, very few result in a complete response. CD4+ T cells are important and potent mediators of anti-tumor immunity and adoptive transfer of specific CD4+ T cells can promote tumor regression in mice and patients. OX40, a costimulatory molecule expressed primarily on activated CD4+ T cells, promotes and enhances anti-tumor immunity with limited success on large tumors in mice. We show that OX40 engagement, in the context of chemotherapy-induced lymphopenia, induces a novel CD4+ T cell population characterized by the expression of the master regulator eomesodermin that leads to both terminal differentiation and central memory phenotype, with concomitant secretion of Th1 and Th2 cytokines. This subpopulation of CD4+ T cells eradicates very advanced melanomas in mice, and an analogous population of human tumor-specific CD4+ T cells can kill melanoma in an in vitro system. The potency of the therapy extends to support a bystander killing effect of antigen loss variants. Our results show that these uniquely programmed effector CD4+ T cells have a distinctive phenotype with increased tumoricidal capability and support the use of immune modulation in reprogramming the phenotype of CD4+ T cells.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor formation, facilitate immune escape, and enable tumor progression. MDSCs are important contributors to the development of an immunosuppressive tumor microenvironment that blocks the action of cytotoxic antitumor T effector cells. Heterogeneity in these cells poses a significant barrier to studying the in vivo contributions of individual MDSC subtypes. Herein, we show that granulocyte-macrophage colony stimulating factor, a cytokine critical for the numeric and functional development of MDSC populations, promotes expansion of a monocyte-derived MDSC population characterized by expression of CD11b and the chemokine receptor CCR2. Using a toxin-mediated ablation strategy to target CCR2-expressing cells, we show that these monocytic MDSCs regulate entry of activated CD8 T cells into the tumor site, thereby limiting the efficacy of immunotherapy. Our results argue that therapeutic targeting of monocytic MDSCs would enhance outcomes in immunotherapy. Cancer Res; 72(4); 876–86. ©2011 AACR.

A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express antitumor T cell receptors or chimeric antigen receptors (CARs). Although the potency and specificity of tumor-specific T cells can be manipulated ex vivo, once reinfused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells, tumor cells and tumor endothelium. Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of antitumor T cells. Antibodies that target PS have been shown to reactivate antitumor immunity by polarizing tumor-associated macrophages into a proinflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. Our lab has previously shown that a PS targeting monoclonal antibody (mch1N11), in combination with transgenic CD4+ T cells that recognize the melanoma antigen Trp1, can regress very advanced melanomas in all treated mice. Here, we further those studies with data showing that a 2nd-generation CAR T cell that binds Trp1 on the surface of B16 melanoma, in combination with mch1N11 can improve antitumor activity and overall survival in B16 tumor-bearing mice. Additionally, in vitro killing assays with antigen-specific T cells sorted from the tumor reveal that mch1N11 enhances the cytolytic function of these T cells against B16 melanoma. Flow cytometry analysis of local immune responses in the tumors of animals treated with tumor-specific T cells and mch1N11 showed a decrease in anti-inflammatory (M2) macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with mch1N11 can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS targeting antibodies with CAR T cell therapy in solid tumors. Citation Format: Sara Schad, Daniel Hirschhorn-Cymerman, Sadna Budhu, Hong Zhong, Xia Yang, Joseph Shan, Steven King, Taha Merghoub, Jedd Wolchok. Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3568.

Stimulation of glucocorticoid-induced tumor necrosis factor receptor (GITR) has been shown to enhance antitumor immunity by stimulating effector CD4+ and CD8+ T cells and attenuating suppression and depleting by CD4+Foxp3+ regulatory T cells (Treg). However, GITR monotherapy does not effectively control tumor growth. We hypothesize that cyclophosphamide (CTX), a cytotoxic chemotherapeutic agent with key immunomodulatory properties, can enhance the potency of GITR engagement anti-tumor effects. Further, we hypothesize that CTX can induce neoantigen formation by increasing tumor mutation burden. In this study, we administered high-dose CTX (250 mg/kg) followed the next day by an agonist anti-GITR antibody (DTA-1) to C56BL/6 mice bearing poorly immunogenic B16 melanoma tumors. We found that the combination therapy can cause regression of established tumors whereas the single agents did not. Mechanistically, we found that CTX administration results in tumor antigen release, which enhances the antitumor T cell response after treatment with DTA-1. This effect is lost when lower doses of CTX are given or CTX is administered prior to tumor inoculation, suggesting that tumor cell death and cross-presentation of tumor antigens to T cells is necessary. Furthermore, Treg cells are an important target of combination therapy. Intratumoral Treg levels were substantially reduced in treated mice, which greatly increased the ratio of T effector/Treg in the tumor. In conclusion, CTX and agonist anti-GITR antibody slows tumor progression and can induce regression of palpable, poorly immunogenic B16 melanoma tumors, likely by enhancing cross-priming and increasing the T effector/Treg ratio in the tumor. Thus, this combination warrants further investigation as a novel immunotherapy approach and provides rational for exploring combination of GITR agonist antibodies with chemotherapeutic treatments in clinical settings. Citation Format: Allison Betof Warner, Daniel Hirschhorn, Adam Cohen, Cailian Liu, Walter Newman, Cyndi Sirard, Jedd Wolchok, Taha Merghoub. Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1699.

Cyclophosphamide (CTX), a commonly used chemotherapeutic agent can enhance immune responses. The ability of CTX to promote the proliferation of effector T cells and abrogate the function of regulatory T cells (Tregs) has been described. In this study, we examined the effects of CTX treatment on dendritic cell (DC) subsets and the subsequent outcome on the effector and suppressive arms of adaptive immunity. In secondary lymphoid tissues, tissue-derived migratory DCs (migratory DCs), lymphoid tissue–resident DCs (resident DCs), and plasmacytoid DCs (pDCs) are well described. CTX has profound and selective cytotoxic effects on CD8+ resident DCs, but not skin-derived migratory DCs or pDCs in lymph nodes (LNs) and spleen, causing an imbalance among these DC subsets. CTX treatment increases the potency of DCs in antigen presentation and cytokine secretion, and partially inhibits the suppressor activity of Tregs. Adoptive transfer of CD8+ DCs can reconstitute this population in regional draining LNs and abrogate the immune-enhancing effects of CTX in vivo. These findings demonstrate that CTX may improve immune responses by preferentially depleting CD8+ lymphoid-resident DCs, which leads to diminished Treg suppression and enhanced effector T-cell function in vivo.

Successful immune-based therapies that treat tumors are often accompanied by immune-related adverse events (irAE). Immunotherapy-treated patients who experience favorable anti-tumor responses typically manifest irAEs. The primary immunotherapies currently in clinic include agents that activate T cell responses such as checkpoint blockade of inhibitory pathways and infusion of ex-vivo tumor-derived or T cell receptor (TCR)-transgenic or chimeric antigen receptor (CAR)-modified T cells. While the beneficial and toxic effects of T cell-based immunotherapies in the clinic are being extensively explored, the precise mechanisms of tumor elimination and irAE remain the subject of intense investigation. In the present study, we treated established tumors with melanoma-specific adoptive CD4+ T cell transfer and costimulation via anti-OX40 or anti-CTLA-4. We found that, in spite of co-opting the adaptive immune response, acute local inflammation plays a fundamental role in tumor elimination and related toxicities in a model irAE. While stimulated T cells are necessary for initiating a therapeutic response, activation of endogenous neutrophils constitute an important and necessary effector mechanism of tumor destruction and irAE. Extensive neutrophils extracellular traps (NETs) were associated with irAE. Melanoma patients treated with checkpoint blockade, suffering from skin rashes equivalent to irEA found in mice, showed increased survival and NETois in biopsies from rashes and tumors. Our results bring forward a novel paradigm where T cells prime an anti-tumor immune response followed by an inflammatory effector mechanism, provided by the innate immune system with curative as well as morbid effects.

A significant proportion of cancer patients do not respond to immune checkpoint blockade therapy. To deepen our understanding of the mechanisms of resistance to immunotherapy, we studied a population of CD4+Foxp3− T cells expressing PD-1 (4PD1hi), which we found to be up-regulated in B16-melanoma bearing mice after CTLA-4 blockade in association with limited response to treatment. We observed that 4PD1hi accumulate intratumorally as a function of tumor burden in untreated tumor-bearing hosts. Interestingly, CTLA-4 blockade promotes intratumoral and peripheral 4PD1hiincreases in a dose-dependent manner, while combination with PD-1 blockade mitigates this effect in both B16-bearing mice and melanoma patients and significantly improves anti-tumor activity. In addition, we found that persistence of high levels of 4PD1hi after PD-1 blockade correlates with poor prognosis in melanoma patients. Mechanistically, we show that mouse and human circulating and intra-tumor 4PD1hi inhibit T-cell functions in a PD-1/PD-L1 dependent fashion. In addition, we found that mouse and human 4PD1hi resemble follicular-helper-T-cell(TFH)-like cells and that CTLA-4 blockade activity is improved in TFH deficient mice. These findings broaden our understanding of the mechanisms that limit anti-tumor immunity, providing an additional explanation for the incremental activity of combined CTLA-4 and PD-1 blockade. Our study also defines 4PD1hi as a new prognostic and pharmacodynamic biomarker for the design of optimal checkpoint blockade combination schedules and dosage.

We report that OX40 stimulation drives all lineages of CD4 T cell development, including regulatory T cells (Tregs), and the plasticity of the response is dependant on local cytokines. In TGF-β1-treated cultures, an OX40 agonist increased IFN-γ and IL-4 production and diverted T cells from the Treg lineage. However, cytokine blockade in the context of OX40 stimulation promoted enhanced Treg accumulation. This observation was evident in naive mice, as OX40 engagement enhanced Treg proliferation and accumulation in vivo. Lastly, OX40 agonist administration influenced experimental autoimmune encephalomyelitis disease severity in opposing directions, depending on the timing of administration. Given during Ag priming, the OX40 agonist drove Treg expansion and inhibited disease, whereas given later it enhanced T cell effector cytokine production in the CNS and exacerbated disease. Hence, OX40 signaling can augment the accumulation of all CD4 T cell lineages; however, its accentuation of immune responses may have vastly different biologic outcomes depending upon the local cytokine milieu.

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

Using the immune system to control cancer has been investigated for over a century. Yet it is only over the last several years that therapeutic agents acting directly on the immune system have demonstrated improved overall survival for cancer patients in phase III clinical trials. Furthermore, it appears that some patients treated with such agents have been cured of metastatic cancer. This has led to increased interest and acceleration in the rate of progress in cancer immunotherapy. Most of the current immunotherapeutic success in cancer treatment is based on the use of immune-modulating antibodies targeting critical checkpoints (CTLA-4 and PD-1/PD-L1). Several other immune-modulating molecules targeting inhibitory or stimulatory pathways are being developed. The combined use of these medicines is the subject of intense investigation and holds important promise. Combination regimens include those that incorporate targeted therapies that act on growth signaling pathways, as well as standard chemotherapy and radiation therapy. In fact, these standard therapies have intrinsic immune-modulating properties that can support antitumor immunity. In the years ahead, adoptive T-cell therapy will also be an important part of treatment for some cancer patients. Other areas which are regaining interest are the use of oncolytic viruses that immunize patients against their own tumors and the use of vaccines against tumor antigens. Immunotherapy has demonstrated unprecedented durability in controlling multiple types of cancer and we expect its use to continue expanding rapidly.