Roberta Zappasodi

Purpose: TRX518 is a mAb engaging the glucocorticoid-induced TNF receptor−related protein (GITR). This open-label, phase I study (TRX518-003) evaluated the safety and efficacy of repeated dose TRX518 monotherapy and in combination with gemcitabine, pembrolizumab, or nivolumab in advanced solid tumors. Patients and Methods: TRX518 monotherapy was dose escalated (Part A) and expanded (Part B) up to 4 mg/kg loading, 1 mg/kg every 3 weeks. Parts C–E included dose-escalation (2 and 4 mg/kg loading followed by 1 mg/kg) and dose-expansion (4 mg/kg loading) phases with gemcitabine (Part C), pembrolizumab (Part D), or nivolumab (Part E). Primary endpoints included incidence of dose-limiting toxicities (DLT), serious adverse events (SAE), and pharmacokinetics. Secondary endpoints were efficacy and pharmacodynamics. Results: A total of 109 patients received TRX518: 43 (Parts A+B), 30 (Part C), 26 (Part D), and 10 (Part E), respectively. A total of 67% of patients in Parts D+E had received prio...

Microbial diversity is associated with improved outcome in patients receiving allogeneic hematopoietic cell transplantation (allo-HCT), however, the mechanism underlying this observation is unclear. Unconventional T cells recognize specific metabolites of bacterial biosynthesis and their role in the post-HCT immunity has not yet been fully clarified. Here we have performed an observational study (n = 174 patients) using 16S rRNA sequencing of early post-HCT patient stool samples (day 7-21 after HCT) paired with multiparameter flow cytometry (performed at day 30 and day 100 after HCT) to explore the relationship between the intestinal microbiome early after HCT and the unconventional T cell populations in circulation. Our data extend findings of other groups suggesting that mucosal-associated invariant T (MAIT) cells are dependent on a diverse microbiome and are also associated with favorable allo-HCT outcome. In addition, we report for the first time that the Vδ2 subset of γδ T cell...

BackgroundThe majority of JAK2V617F-negative myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients, but the underlying reasons for this phenomenon are unknown. We speculate that this is due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifests. As a consequence of this MHC-I allele restriction, we reasoned that CALRMUT MPN patients would not efficiently respond to cancer vaccines composed of the CALRMUT neoantigen, but could do so when immunized with a properly epitope-optimized CALRMUT heteroclitic peptide vaccine approach.MethodsWe examined MHC-I allele frequency in CALRMUT MPN patients from two independen...

BackgroundImmune checkpoint blockade (ICB) has revolutionized the treatment of many cancer types; however, many patients do not benefit from these therapies due to inherent or acquired resistance. Preferential engagement in glycolysis is a hallmark of cancer cells and contributes to the progression and metastasis of many tumor types, including melanoma and triple-negative breast cancer (TNBC). Tumor reliance on glycolysis is emerging as a mechanism of resistance to immunotherapy, due in part to lactate-mediated acidification and competition for glucose in the tumor microenvironment. We recently found that knocking down the glycolytic enzyme lactate dehydrogenase A (LDHA) in 4T1 TNBC results in improved and long-lasting anti-tumor responses to CTLA-4 blockade in mice. These LDHA-defective tumors consume less glucose than wild-type tumors, leaving more glucose available in the TME for effector T cells to exert their tumoricidal function directly and via lineage destabilization of regu...

Regulatory T cells (Treg) are one of the major impediments to effective antitumor immunity and successful immunotherapy. Elevated intratumoral Treg frequencies, observed in a variety of malignancies, have been associated with poor prognosis. In this issue of Cancer Research, two studies underscore the potential of harnessing the unique migratory profile of tumor-infiltrating Tregs to selectively eliminate these cells without compromising peripheral tolerance. Both studies identify surface migratory receptors, CCR8 by Campbell and colleagues and GPR15 by Adamczyk and colleagues, as selective markers of intratumoral Tregs in tumor-bearing mice and patients with cancer. Genetic deletion of GPR15 or antibody-mediated depletion of CCR8 was found to preferentially decrease tumor-infiltrating Tregs and substantially delayed tumor progression. Together, these two studies highlight the significance of migratory molecules in intratumoral Tregs and propose two potential selective targets for p...

A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.

SUMMARY Limiting the metabolic competition in the tumor microenvironment (TME) may increase the effectiveness of immunotherapy. Because of its critical role in glucose metabolism of activated T cells, CD28 signaling has been proposed as a T-cell metabolic biosensor1. Conversely, CTLA-4 engagement has been shown to down-regulate T-cell glycolysis1. Here, we investigated the impact of CTLA-4 blockade on the metabolic fitness of intra-tumor T cells in relationship to the tumor glycolytic capacity. We found that CTLA-4 blockade promotes immune cell infiltration and metabolic fitness especially in glycolysis-low tumors. Accordingly, anti-CTLA-4 achieved better therapeutic outcomes in mice bearing glycolysis-defective tumors. Intriguingly, tumor-specific CD8+ T-cell responses correlated with phenotypic and functional destabilization of tumor-infiltrating regulatory T cells (Tregs) toward IFN-γ- and TNF-α-producing cells in glycolysis-defective tumors. By mimicking the highly and poorly glycolytic TME in vitro, we show that the effect of CTLA-4 blockade to promote Treg destabilization is dependent on Treg glycolysis and CD28 signaling. These findings indicate that decreasing tumor competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumor glycolysis. Moreover, these results reveal a new mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose.

BackgroundThe majority of JAK2V617F-negative myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients, but the underlying reasons for this phenomenon are unknown.MethodsIn this study, we examine class-I major histocompatibility complex (MHC-I) allele frequency in CALRMUT MPN patients from two independent cohorts and observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are under-represented in CALRMUT MPN patients. We speculate that this is due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifests. As a consequence of this MHC-I allele restriction, we reasoned that CALRMUT MPN patients...

BackgroundCD4 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.3–5MethodsOur 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...

BackgroundMultiple suppressive mechanisms within the tumor microenvironment are capable of blunting anti-tumor T cell responses, including the engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8+ T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).1 2 While immune checkpoint blockade therapies aimed at reinvigorating T cell effector function have demonstrated their clinical effectiveness,3 4 not all patients demonstrate long-term disease control.5 The refractory nature of terminally differentiated, exhausted CD8+ T cells to be reinvigorated by PD-1 blockade is one potential cause.6–8 This limitation warrants the need to explore modulatory pathways that potentially program T cells toward exhaustion.MethodsSingle cell-RNA sequencing (scRNA-seq) data derived from the tumo...

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

Only a limited fraction of patients derives durable clinical benefit upon immune checkpoint blockade. Deepening our understanding of mechanisms of response and resistance to these therapies is thus needed to optimize their employment in rational combinations. Cellular energy metabolism reprogramming is a critical hallmark of cancer. High glucose consumption and lactate production by tumor cells restrict nutrient availability in the tumor microenvironment (TME) for effector T cells, which also rely on glycolysis to replicate and function. In addition, immune checkpoints and co-stimulatory molecules are emerging as important regulators of T cell metabolism. Exploiting the capacity of immune checkpoint blockade to perturb T cell metabolism in combination with inhibition of tumor glycolysis may thus be a rational and more effective anti-cancer approach. We investigated the link between tumor glycolysis and immune cell function using RNA sequencing data sets from patients treated with checkpoint blockade and in tumor:T cell co-culture systems. We then studied the effects of checkpoint blockade in syngeneic glycolysis-defective murine tumor models and explored mechanisms underlying anti-tumor activity. We found that expression of glycolysis-related genes is inversely correlated with infiltration of most immune cell types in melanomas from patients before CTLA-4 blockade. However, after treatment, immune-related and glycolysis-related genes were more often co-expressed, suggesting that anti-CTLA-4 may partially restore immune cell fitness in the glycolytic TME. To directly assess the effect of tumor metabolism on T cell function, we co-cultured activated T cells with the highly glycolytic murine mammary carcinoma 4T1 and observed that tumor cells, or similar non-toxic concentrations of exogenous lactate, significantly limited T cell activation and viability. We thus investigated whether inhibition of tumor glycolysis could potentiate the activity of CTLA-4 blockade. We found that neoadjuvant treatment with anti-CTLA-4 significantly prolonged survival in mice bearing glycolysis defective 4T1 tumors, where lactate dehydrogenase A (LDH-A) - the critical enzyme controlling lactate production in aerobic glycolysis - was knocked down (4T1-KD). Intriguingly, tumor protection was associated with intratumoral regulatory T cell (Treg) functional phenotypic destabilization towards IFN-gamma and TNF-alpha producing Tregs. By mimicking the LDH-A-KD and control 4T1 TME in vitro using higher and lower concentrations of glucose, respectively, we showed that CTLA-4 blockade promotes glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. These findings indicate that rebalancing glucose utilization in favor of immune cells in the TME may facilitate the activity of CTLA-4 blockade and reveal an additional mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose. Citation Format: Roberta Zappasodi, Inna Serganova, Ivan Cohen, Masatomo Maeda, Masahiro Shindo, Yasin Senbabaoglu, Avigdor Leftin, Rachana Maniyar, Svena Verma, Matthew Lubin, Myat Kyaw Ko, Mayuresh M. Mane, Arnab Ghosh, Ellen Ackerstaff, Jason A. Koutcher, Ping-Chih Ho, Greg M. Delgoffe, Ronald Blasberg, Jedd D. Wolchok, Taha Merghoub. CTLA-4 blockade drives loss of regulatory T cell functional stability in glycolysis defective tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3257.

BackgroundImmune checkpoint blockade (ICB) has revolutionized cancer treatment. However, long-term benefits are only achieved in a small fraction of patients. Understanding the mechanisms underlying ICB activity is key to improving the efficacy of immunotherapy. A major limitation to uncovering these mechanisms is the limited number of responders within each ICB trial. Integrating data from multiple studies of ICB would help overcome this issue and more reliably define the immune landscape of durable responses. Towards this goal, we formed the TimIOs consortium, comprising researchers from the Society for Immunotherapy of Cancer Sparkathon TimIOs Initiative, the Parker Institute of Cancer Immunotherapy, the University of North Carolina-Chapel Hill, and the Institute for Systems Biology. Together, we aim to improve the understanding of the molecular mechanisms associated with defined outcomes to ICB, by building on our joint and multifaceted expertise in the field of immuno-oncology....

3006 Background: To evaluate the safety and tolerability of vaccination with DCs loaded with killed tumor cells, 18 patients with measurable relapse/refractory follicular (12) and lymphoplasmocytoid (6) lymphoma have been enrolled in a phase I study. Methods: Each patient received at 2-weekly intervals 4 SC injections of 50x10e6 tumor-loaded DCs. Immature DCs were generated by 5-days culture of autologous monocytes in the presence of IL-4 and GM-CSF. Autologous CD19+ tumor cells, harvested from lymph nodes (12) and/or peripheral blood (6), were heat shocked and then irradiated by UVC. DCs were loaded for 48 hrs with killed tumor cells and then were cultured for 12 hrs in the presence of TNF-a. Median prior number of treatment regimens was 2 (range 1–5) comprising 4 patients treated with high-dose chemotherapy. The vaccination was started at least 6-months after the last chemotherapy treatment. Results: All patients were evaluable for toxicity and for efficacy with a median follow-up...

2729 Histone deacetylase inhibitors (HDACis) are among the most promising novel classes of anti-cancer compounds thanks to their ability of simultaneously targeting multiple pro-survival and anti-apoptotic pathways, while retaining a relatively good safety profile. Through the promotion of histone and non-histone protein acetylation, they can modulate not only expression and activation of key signaling proteins but also non-coding small RNAs (microRNAs) that post-transcriptionally control gene expression. Increasing evidence is pointing to an altered pattern of microRNA expression at the basis of the genesis and the maintenance of lymphoid malignancies and in particular…

1562 Our previous findings have made it clear that the significant clinical efficacy attained by dendritic cell-based vaccination in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients is firmly associated with multifaceted immunologic responses, including the development of anti-heat shock protein (HSP)105 humoral immunity (Di Nicola et al., Blood 2009 113:18–27; Zappasodi et al., Cancer Res. 2010 70:9062–9072; Zappasodi et al., Blood 2011 118:4421–4430). Human HSP105 is a high-molecular-weight chaperone constitutively expressed at low levels within the cytoplasm, and can also be induced in the nucleus by various forms of stress. It is overexpressed in several solid tumors, including melanoma, breast, thyroid and gastroenteric cancers. We have recently shown that this is also true for B-NHLs, in which HSP105 levels increase in function of their aggressiveness and proliferation index (Zappasodi et al., Blood 2011 118:4421–4430). Accordingly, in normal lymph nodes HSP105 expression...

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.

The clinical successes of checkpoint blockade have demonstrated that proper modulation of T cell co-inhibitory/co-stimulatory pathways can achieve substantial anti-tumor activity. However, many patients are or become refractory to checkpoint blockade. Additional forms of immunotherapy targeting alternative immune pathways are thus needed. Antibodies (Abs) engaging the TNF receptor GITR can enhance T cell functions and counteract regulatory T cell (Treg) suppression and have shown potent anti-tumor activity in animal models. Based on this evidence, we initiated the first in-human phase-I trial with the humanized aglycosylated anti-GITR Ab TRX518 (NCT01239134). Here, we report the immune effects of a single ascending dose of TRX518 monotherapy in 37 advanced cancer patients in this phase-I trial and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade. We found that TRX518 frequently reduces circulating Tregs. In 8 patients for whom pre- and post-treatment tumor biopsies were available, reductions in intra-tumor and circulating Tregs after TRX518 were positively correlated. Yet, these patients did not experience substantial clinical responses. To explain this outcome, we modeled sensitivity and refractoriness to anti-GITR by treating B16F10-melanoma-bearing mice with the Ab DTA-1 on day 4 (curative regimen) or day 7 (refractory regimen) after tumor implantation respectively. We found that Tregs were significantly reduced and CD8+:Treg and Teff:Treg ratios increased in both responding and refractory tumors. Interestingly, CD8+ T cells from refractory tumors overexpressed T cell exhaustion markers and did not up-regulate memory and functional markers. We thus tested whether counteracting exhaustion could overcome resistance of advanced tumors to anti-GITR. PD-1 blockade in combination with anti-GITR starting on day 7 after tumor implantation controlled tumor growth similar to the curative anti-GITR monotherapy regimen (day 4 treatment) and achieved 50% complete response rate associated with long-lasting anti-tumor immunological memory. This was associated with more activated and less exhausted profiles of intra-tumor CD8+ T cells, which displayed enhanced tumor-lytic capacity compared to CD8+ T cells from non-responding tumors treated with each agent alone. These results indicate that Treg reduction can serve as a pharmacodynamic biomarker of anti-GITR in patients. However, Treg elimination from advanced tumors may not be sufficient to activate cytotoxic CD8+ T cell responses unless the T cell exhaustion process is concurrently blocked. This provides the rationale to combine immunotherapies targeting Tregs with strategies able to counteract exhaustion, such as anti-PD-1, to regress advanced tumors. Based on these observations, we have started to investigate TRX518 in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574). Citation Format: Roberta Zappasodi, Cynthia Sirard, Yanyun Li, Sadna Budhu, Moshen Abu-Akeel, Cailian Liu, Xia Yang, Hong Zhong, Walter Newman, Jingjing Qi, Philip Wong, David Schaer, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Jedd Wolchok, Taha Merghoub. Rational combination of GITR agonism with PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2711.

Introduction: At 52.5% identity in their extracellular domains (ECDs), mouse and human GITR (CD357) sequences are only modestly conserved. Thus transitioning promising murine anti-tumor activity data with the anti-GITR DTA-1 and 2F8 Mabs to clinical trials with the non-crossreactive anti-human GITR Mab TRX518 poses a considerable limitation. Here we investigated three elements of the GITR mouse and human antibodies to address this limitation: 1) the functional epitopes targeted by anti-human GITR (TRX-518) and anti-murine GITR (DTA-1 and 2F8) antibodies; 2) their biological activities on primary T cells; and 3) the role of anti-GITR Fc receptor engagement. Results and Conclusions: Alanine scanning, amino acid substitutions and flow cytometry experiments were performed to define critical residues important for the binding of all three Mabs to their respective GITR targets. Swaps of 5-8 of these critical residues showed both the loss of murine and the gain of human directed Mab binding when residues critical for TRX18 binding were substituted into murine GITR. Likewise, the substitution of residues critical for DTA-1/2F8 binding into human GITR resulted in loss of TRX518 binding and the gain of 2F8/DTA-1 binding. We then mapped these critical residues to 3D structural models of the murine and human GITR ECDs built by contact-map guided fragment assembly simulations. Regions defined by these critical residues showed a high degree of overlap, indicating structural conservation even without amino acid identity. Thus the epitopes defined by both DTA-1 and 2F8 are homologous to those defined on human GITR by TRX518. In in vitro standard proliferation/suppression assays with effector T cells (Teff) and regulatory T cells (Tregs) isolated from human healthy donors and naive mice respectively, TRX518, similarly to DTA-1, significantly increased proliferation, activation and pro-inflammatory cytokine production of human and murine Teff cultured alone or in the presence of Tregs. These observations indicate that Mabs directed to similar mouse and human GITR ECD epitopes result in similar biological activities. Lastly, we questioned the importance of a functional Fc domain in anti-GITR Mabs in order to translate these in vitro effects into anti-tumor activity in vivo. To this end, we compared the anti-tumor activity of DTA-1 and its aglycosyl version in the aggressive and poorly immunogenic B16 melanoma model. Both Fc and non-Fc binding DTA-1 were able to control tumor growth. This suggests that the aglycosyl-anti-GITR Mab TRX518 has the potential to be effective in the clinic despite the lack of Fc receptor engagement. These results align with our preliminary results in patients treated with TRX518 monotherapy showing reductions in Treg frequencies both in tumor biopsies and in the periphery. TRX518 would therefore appear to be well-poised for use in the treatment of solid tumor malignancies. Note: This abstract was not presented at the meeting. Citation Format: Roberta Zappasodi, Heidi Heath, Yang Zhang, Michael Haas, Min Yang, Christopher Mirabelli, Jedd D. Wolchok, Cyndi Sirard, Walter Newman, Taha Merghoub. GITR cancer immunotherapy: Epitope swapping of anti-GITR TRX518 to inform functional translatability from mouse to human [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2402.

Modulating T cell homeostatic mechanisms with checkpoint blockade can efficiently promote endogenous anti-tumor T cell responses1–11. However, many patients still do not benefit from checkpoint blockade12, highlighting the need for targeting of alternative immune pathways13. Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is an attractive target for immunotherapy, owing to its capacity to promote effector T cell (Teff) functions14,15 and hamper regulatory T cell (Treg) suppression16–20. On the basis of the potent preclinical anti-tumor activity of agonist anti-GITR antibodies, reported by us and others16,21,22, we initiated the first in-human phase 1 trial of GITR agonism with the anti-GITR antibody TRX518 (NCT01239134). Here, we report the safety profile and immune effects of TRX518 monotherapy in patients with advanced cancer and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. We demonstrate that TRX518 reduces circulating and intratumoral Treg cells to similar extents, providing an easily assessable biomarker of anti-GITR activity. Despite Treg reductions and increased Teff:Treg ratios, substantial clinical responses were not seen. Similarly, in mice with advanced tumors, GITR agonism was not sufficient to activate cytolytic T cells due to persistent exhaustion. We demonstrate that T cell reinvigoration with PD-1 blockade can overcome resistance of advanced tumors to anti-GITR monotherapy. These findings led us to start investigating TRX518 with PD-1 pathway blockade in patients with advanced refractory tumors (NCT02628574).GITR agonism can be exploited in combination with checkpoint blockade to overcome immune refractoriness 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 CD4Foxp3 T cells expressing PD-1 (4PD1) and observed that 4PD1 accumulate intratumorally as a function of tumor burden. Interestingly, CTLA-4 blockade promotes intratumoral and peripheral 4PD1 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 reduction after anti-PD-1 correlates with poor prognosis. Mechanistically, we provide evidence that mouse and human circulating and intra-tumor 4PD1 inhibit T cell functions in a PD-1/PD-L1 dependent fashion and resemble follicular helper T cell (T)-like cells. Accordingly, anti-CTLA-4 activity is improved in T deficient mice.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring g...

Background: The tumor necrosis factor receptor GITR is expressed at high levels on regulatory T cells (Tregs) and up-regulated on activated T cells. Evidence from preclinical studies has shown the dual activity of GITR stimulation to counteract Tregs functionally and quantitatively and enhance T-cell effector functions, thus providing the rationale to develop GITR agonist antibodies for cancer immunotherapy. These agents have demonstrated potent therapeutic effects associated with both Treg reduction and modulation in tumor-bearing mice. Here we investigate Treg modulation as a biomarker of activity of the fully humanized agonist anti-GITR antibody TRX-518 in advanced solid cancer patients and the potential mechanisms underlying this effect. Methods: Patients were accrued to 9 cohorts (up to 6 patients/cohort) to receive a single dose of TRX518 (dose range: 0.0001-8 mg/kg). Pharmacodynamic analyses included flow cytometric evaluation of frequency and phenotype of circulating T cells at different time points up to 12 weeks after treatment. Relevant changes observed with these analyses were monitored in pre- and post-treatment tumor biopsies by immunofluorescence staining. Effects of GITR stimulation with TRX-518 on human Tregs were studied in in vitro cultures with CD4+ T cells from healthy donors. Results: Analysis of peripheral blood mononuclear cells from 37 advanced refractory solid cancer patients treated with >=0.005 mg/kg TRX-518 (cohorts 3-9) revealed frequent reduction in circulating Tregs after treatment with TRX-518. Proliferation potential of Tregs was not altered by TRX-518, as indicated by their persistent Ki67 expression. We then asked whether a subset of Tregs was preferentially affected and found that GITR+ Tregs and CD45RA-Foxp3hi effector Tregs (eTregs) were specifically targeted by TRX-518 in a dose-dependent manner. Accordingly, we observed that eTregs express GITR at higher levels than naive CD45RA+Foxp3+ Tregs. In a subset of patients for whom pre- and post-treatment tumor biopsies were available (n=8; 3 melanoma, 2 lung, 2 colorectal and 1 bladder cancer patients), we tested whether intra-tumor Tregs, which are typically enriched in eTregs, were also affected. Noteworthy, we observed that changes in intra-tumor and circulating Tregs after TRX-518 were positively correlated. As a potential mechanism responsible for this effect, our initial in vitro analyses indicate that GITR stimulation with TRX-518 destabilizes Treg phenotype by down-regulating Foxp3 and enhancing expression of the prototype Th1-lineage transcription factor T-bet. This suggests that TRX-518 may favor Treg to effector-T-cell conversion, thus explaining the effect of Treg loss observed in patients. Conclusions: Circulating Treg reduction is a potential pharmacodynamic biomarker of TRX518 biological activity. This parameter may allow predictive correlation with changes in intratumoral Treg infiltration. We plan to further investigate this effect and its relevance for the association with clinical responses in our recently opened TRX518 multi-dose study. Clinical trial information: NCT01239134. Citation Format: Roberta Zappasodi, Yanyun Li, Mohsen Abu-Akeel, Jingjing Qi, Philip Wong, Cynthia Sirard, Michael Postow, David A. Schaer, Walter Newman, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Jedd D. Wolchok, Taha Merghoub. Intratumor and peripheral Treg modulation as a pharmacodynamic biomarker of the GITR agonist antibody TRX-518 in the first in-human trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT018. doi:10.1158/1538-7445.AM2017-CT018

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, tumor cells and tumor endothelium. PS has been shown to promote immunosuppressive signals in the tumor microenvironment. Antibodies that target PS have been shown to reactivate anti-tumor immunity by polarizing tumor associated macrophages into a pro-inflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. In a mouse B16 melanoma model, targeting PS in combination with immune checkpoint blockade promoted greater anti-tumor activity than either agent alone. This combination was shown to enhance CD4+ and CD8+ T cell infiltration and activation in the tumors of treated animals. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. The abscopal effect, a phenomenon in which tumor regression occurs outside the site of RT, has been observed in both preclinical and clinical trials when RT is combined with immunotherapy. In this study, we show that irradiation treatment of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of local immune responses in the tumors of treated animals revealed an increase in tumor-associated macrophages with a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the systemic immune responses in the spleen and tumor draining lymph nodes revealed an increase in CD8 T cell activation, effector cytokine production and differentiation into effector memory cells in the triple combination. This finding highlights the potential of combining these three agents to improve outcome in patients with advanced-stage melanoma and other cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade. Citation Format: Sadna Budhu, Olivier De Henau, Roberta Zappasodi, Rachel Giese, Luis F. Campesato, Christopher Barker, Bruce Freimark, Jeff Hutchins, Jedd D. Wolchok, Taha Merghoub. Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2017-574

e18513 Background: We reported that dendritic cell-based vaccination in relapsed indolent NHL patients achieved clinical benefits strongly associated with B and T cell responses. Hence, we exploited the antibody (Ab) repertoires of responders (R) and non-R (NR) to investigate whether the recognized ags might represent novel NHL targets for therapy. Methods: Vaccine-induced Ab cross-reactivity with allogeneic paraffin-embedded specimens and live tumor cells was tested by immunohistochemistry (IHC) and flow cytometry (FC), using biotinylated Abs purified from pre- and post-vaccine sera. Ag discovery was performed by a modified serological proteomics- based approach. Proteins differentially revealed by post-vaccine Abs of R were analyzed by mass spectrometry (MS). Abs-mediated antitumor activity of was tested in a standard proliferation assay. Statistical analysis was performed by Student t-test. Results: Post-vaccine Abs from R displayed increased allo-reactivity compared to matched pre-vaccine or NR sample...

Background: Breast cancer (BC) has historically been considered immunologically silent; however, several observations indicate that potentiation of immune functions can benefit BC patients. Intratumor T-cell infiltration has prognostic significance in patients with BC across different molecular and histological categories. In addition, PD-L1 can be overexpressed in BC, in particular in the highly aggressive triple negative BC (TNBC) subtype. This is associated with poor prognosis specifically in patients with luminal B and basal-like phenotypes, thus making these subtypes rational targets of PD-1/PD-L1 axis blockade treatments. However, initial results from early-phase clinical trials show a modest activity of immune checkpoint blockade monotherapy against BC, with 19% TNBC and 3-12% hormone receptor-positive patients achieving a clinical response to anti-PD-1/PD-L1 therapies. Lack of inflammation and T-cell infiltration at the tumor site are characteristic features of tumors that do not respond to checkpoint blockade. Since BC is typically poorly infiltrated by T cells, having strategies that reverse this immune exclusion as well as non-invasive modalities to predict this effect are crucial to improve the clinical efficacy of immune checkpoint blockade. Experimental Design: Our working model is that tumor glycolysis (lactate) and T-cell infiltration are mechanistically interdependent, since we reasoned that a highly glycolytic tumor microenvironment (due to lactate production) could hamper survival, expansion and effector functions of T cells in BC lesions, thus explaining T-cell exclusion. Therefore, we tested whether genetic and/or pharmacologic inhibition of lactate production/consumption could restore intratumor T-cell infiltration and immune function in BC models, thus favoring tumor responsiveness to checkpoint blockade. Results: By interrogating a compendium of 4 BC patient gene expression datasets, we found that patients harboring tumors with high expression of lactate dehydrogenase A (LDH-A) or the lactate transporters MCT-1/4 have a significantly higher risk to develop metastases (p −16 ), whereas those having tumors with increased expression of CD3 and CD8 transcripts experience a better prognosis (p −16 ). Gene expression data from MSKCC9s cBio Portal showed an inverse correlation between glycolysis- and immune-related gene expression signatures in BC patients, which was in agreement with our hypothesis. To mechanistically demonstrate the impact of lactate metabolism on intratumor T-cell infiltration in BC, we generated an LDH-A-knocked-down variant (LDH-A KD) of the metastatic TNBC murine model 4T1, and studied its growth and immune infiltrate in vivo in comparison with the control 4T1 tumor. Animals implanted with LDH-A KD 4T1 showed a 5-fold increase in tumor-infiltrating CD3 + T cells and a 4-fold reduction in tumor-associated macrophages, and experienced a significantly prolonged survival. LDH-A KD tumors could be completely eradicated in immunocompetent but not in immunodeficient mice, further supporting the immunologic basis of the antitumor effects of LDH-A inhibition. In vitro analyses showed that proliferation, activation and pro-inflammatory cytokine release of activated CD8 T cells were significantly hampered when they were co-cultured with 4T1 cells. Of note, blocking lactate transport with two new MCT-1/4 small-molecule inhibitors promoted immune functions of CD8 T cells cultured with 4T1, without showing significant toxicity in cultures of T cells alone. Citation Format: Roberta Zappasodi, Arnab Ghosh, Inna Serganova, Ivan Cohen, Yasin Senbabaoglu, Masahiro Shindo, Mayuresh M. Mane, Avigdor Leftin, Ellen Ackerstaff, Jason A. Koutcher, Jedd D. Wolchok, Ronald G. Blasberg, Taha Merghoub. Overcoming intratumor T-cell exclusion by modulation of lactate metabolism to improve immune checkpoint therapies in aggressive breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR06.

The recent clinical successes of CTLA4 and PD-1 blockade have clearly demonstrated the therapeutic efficacy to enhance anticancer immune responses. Novel targets of immunomodulation are now under investigation to increase the number of immunotherapeutic options available for relapsing/refractory patients. Activation of co-stimulatory molecules with agonist monoclonal antibody (mAbs) represents the most rationale alternative option. Based on the impressive preclinical activity of stimulating the glucocorticoid-induced TNF receptor (GITR) with the mAb DTA-1, an agonist anti-human GITR mAb has very recently entered the clinical evaluation. Although DTA-1, as a monotherapy, proved effective in curing established immunogenic tumors, it failed against advanced poorly immunogenic models, such as B16 melanoma. As anti-GITR therapy is translated into clinic, defining the basis of its activity and failure is key to identify prognostic markers and more effective combination therapies. We thus studied how GITR stimulation with the agonist antibody DTA-1 affects T cells in B16 melanoma-bearing mice treated in curative (early) or refractory (advanced) condition, and verified their functional changes in vitro. In vivo, the comparison of advanced versus early B16 microenvironment before treatment revealed a significant increase in Treg frequency, which caused the Teff/Treg ratio to decrease, and a dysfunctional Teff phenotype. Moreover, B16 cells, in the setting of established tumors, expressed lower levels of MHC-I and MHC-II molecules. DTA-1 consistently reduced intratumor Treg frequency and expression of suppression markers in both curative and refractory conditions. However, Teff activation and tumor-associated MHCs were induced only when DTA-1 was administered in curative conditions. In vitro, pre-incubation of Tregs with DTA-1 completely abolished their ability to inhibit proliferation of target Teff, without affecting Treg expression of the lineage markers Foxp3 and Helios. In addition, treatment with DTA-1, in comparison with the matched IgG isotype control, boosted the release of IFN-gamma, TNF-alpha, IL-17 and IL-6 cytokines in cultures of Tregs, pointing to the promotion of a Th1/Th17 differentiation profile following GITR engagement. Interestingly, when B16 cells were added in culture with Tregs and incubated with DTA-1, they up-regulated MHC-I and MHC-II molecules in a Treg and/or DTA-1 dependent manner. The presence of tumor cells during exposure of Tregs to anti-GITR stimulation did not further alter their function and phenotype, indicating a major direct effect of DTA-1 on Tregs. To confirm the functional modulation of GITR stimulation in human Tregs, we tested their activity in vitro upon pre-incubation with a recombinant human GITR ligand (GITRL). We consistently found that proliferation of activated Teff was no longer suppressed if Tregs were previously treated with GITRL. These results validate GITR as a target to revert immune tolerance in the human setting. Our data indicate that anti-GITR therapy counteracts Treg-mediated immunosuppression independently from the stage of the disease, whereas T-cell activation and tumor immune recognition are favored selectively in the presence of low tumor burden. These findings suggest that, in the setting of advanced and poorly immunogenic tumors, anti-GITR therapy should be exploited in combination with treatments promoting Teff functions and/or tumor killing. Citation Format: Roberta Zappasodi, Sadna Budhu, Cailian Liu, Kyle Draleau, David Schaer, Hong Zhong, Xia Yang, Taha Merghoub, Jedd D. Wolchok. Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment. [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 B183.

Immunotherapy for the treatment of cancer has made significant progresses over the last 20 years. Multiple efforts have been attempted to restore immune-mediated tumor elimination, leading to the development of several targeted immunotherapies. Data from recent clinical trials suggest that these agents might improve the prognosis of patients with advanced genito-urinary (GU) malignancies. Nivolumab has been the first immune checkpoint-inhibitor approved for pre-treated patients with metastatic renal cell carcinoma. Pembrolizumab and atezolizumab have shown promising results in both phase I and II trials in urothelial carcinoma. Brentuximab vedotin has demonstrated early signals of clinical activity and immunomodulatory effects in highly pre-treated patients with testicular germ cell tumors. In this review, we have summarized the major clinical achievements of immunotherapy in GU cancers, focusing on immune checkpoint blockade as well as the new immunomodulatory monoclonal antibodies (mAbs) under clinical evaluation for these malignancies.

Background - Treatment of ovarian cancer remains very challenging, with 80-85% of the cases still dying after relapse to standard chemotherapy, and alternative treatments are urgently needed. Expansion of regulatory T cells (Tregs) is considered the major factor limiting spontaneous immune responses to ovarian cancer. Agonist antibodies against the co-stimulatory receptor OX40 have recently demonstrated to abrogate Treg functions and are under clinical evaluation. We thus studied whether OX40 constituted a valid target of ovarian cancer-associated Tregs. Methods -Treg immunophenotypic analyses were performed by flow cytometry in ascites and ovarian cancer specimens and studied in association with patients9 outcome. Results - CD4+CD25+Foxp3+ Treg% and OX40 expression were measured in 50 diagnostic ovarian cancer cases (36, high grade serous carcinomas; 14, other histotypes). Treg% was significantly higher in ovary (47 evaluable cases) relative to ascites (39 evaluable cases) (p Conclusions - Our study underscores a negative prognostic impact of OX40 expression on ovarian cancer-infiltrating Tregs and indicates that OX40 may constitute a rational target to counteract effector Treg functions and unleash potentially effective immune responses against ovarian cancer. Citation Format: Massimo Di Nicola, Roberta Zappasodi, Alessia Burocci, Giusi Ruggiero, Fabio Martinelli, Claudio Tripodo, Domenica Lorusso, Filippo De Braud, Francesco Raspagliesi, Mario P. Colombo. OX40 expression in tumor-associated Tregs as a potential prognostic biomarker and immunotherapeutic target in ovarian cancer. [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 B157.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We reported that the splice variant of human HER2 lacking exon 16 (delta16HER2) represents a highly penetrating HER2 oncogenic alteration identified in human primary breast tumor specimens and is able to influence the response to Trastuzumab. This HER2 variant forms covalent cysteine bonds that generate constitutively active homodimers, thereby activating multiple oncogenic downstream signaling pathways that we recently found to be mediated through activated Src kinase. To examine the ability of delta16HER2 to transform mammary epithelium in vivo and to monitor delta16HER2-driven tumorigenesis in live mice, we generated a FVB transgenic mouse model for the human delta16HER2 isoform. Transgenic female mice developed multifocal mammary tumors with a rapid onset starting at about 12 weeks of age and progressively thereafter, clearly pointing to the candidacy of the delta16HER2 isoform as the transforming form of the human HER2 oncoprotein. Histological and immunohistochemical analysis (IHC) of primary mammary nodules revealed a population of polygonal cells with classical epithelia-like aspects distinctly expressing HER2 and also a population of smaller spindle-shaped cells arranged in fascicles with lower levels of HER2 expression, suggesting the onset of the epithelial-to-mesenchymal transition (EMT). Consistent with these findings, FACS analysis of delta16HER2-positive tumor cells immunomagnetically purified from disaggregated transgenic primary tumors indicated the increased mean fluorescence intensity of HER2 staining with increasing tumor cell size. IHC analysis of the lung metastases that had formed in the majority of female mice revealed monomorphic and classical epithelial tumor cells homogeneously expressing high levels of delta16HER2. FACS and IHC analyses confirmed the lower binding efficacy of Trastuzumab to delta16HER2-overexpressing primary tumor cells cultured both under bidimensional (2D) and tridimensional (3D) conditions as compared to monoclonal reagents directed to different HER2 extracellular domain epitopes. Experiments in both primary and metastatic in vitro and in vivo delta16HER2-positive models are in progress to determine whether delta16HER2-driven tumor aggressiveness and Trastuzumab susceptibility depend not only on genetic changes intrinsic to the tumor cell, i.e., the EMT process, but also on extrinsic tumor surrounding microenvironment-related factors such as an imbalance between extracellular and intracellular pH, redox state and hypoxia. Preliminary FACS and IHC analyses indicate that delta16HER2-positive primary tumor cells are reactive for known epithelial markers as EpCAM, E-cadherin- and ck-18 and, a small subset of these mammary tumor cells, also stain positive for the mesenchymal differentiation markers vimentin, N-cadherin and ck14 significantly indicating an active EMT program. Supported by AIRC and Ministry of Health Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916. doi:1538-7445.AM2012-916

We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete re...

The rationale to treat lymphomas with immunotherapy comes from long-standing evidence on their distinctive immune responsiveness. Indolent B-cell non-Hodgkin lymphomas, in particular, establish key interactions with the immune microenvironment to ensure prosurvival signals and prevent antitumor immune activation. However, reports of spontaneous regressions indicate that, under certain circumstances, patients develop therapeutic antitumor immunity. Several immunotherapeutic approaches have been thus developed to boost these effects in all patients. To date, targeting CD20 on malignant B cells with the antibody rituximab has been the most clinically effective strategy. However, relapse and resistance prevent to cure approximately half of B-NHL patients, underscoring the need of more effective therapies. The recognition of B-cell receptor variable regions as B-NHL unique antigens promoted the development of specific vaccines to immunize patients against their own tumor. Despite initial promising results, this strategy has not yet demonstrated a sufficient clinical benefit to reach the regulatory approval. Several novel agents are now available to stimulate immune effector functions or counteract immunosuppressive mechanisms, such as engineered antitumor T cells, co-stimulatory receptor agonist, and immune checkpoint-blocking antibodies. Thus, multiple elements can now be exploited in more effective combinations to break the barriers for the induction of anti-lymphoma immunity.