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Background Metastatic DNA mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer has a poor prognosis after treatment with conventional chemotherapy and exhibits high levels of tumour neoantigens, tumour-infiltrating lymphocytes, and checkpoint regulators. All of these features are associated with the response to PD-1 blockade in other tumour types. Therefore, we aimed to study nivolumab, a PD-1 immune checkpoint inhibitor, in patients with dMMR/MSI-H metastatic colorectal cancer.Methods In this ongoing, multicentre, open-label, phase 2 trial, we enrolled adults (aged >= 18 years) with histologically confirmed recurrent or metastatic colorectal cancer locally assessed as dMMR/MSI-H from 31 sites (academic centres and hospitals) in eight countries (Australia, Belgium, Canada, France, Ireland, Italy, Spain, and the USA). Eligible patients had progressed on or after, or been intolerant of, at least one previous line of treatment, including a fluoropyrimidine and oxaliplatin or irinotecan. Patients were given 3 mg/kg nivolumab every 2 weeks until disease progression, death, unacceptable toxic effects, or withdrawal from study. The primary endpoint was investigator-assessed objective response as per Response Evaluation Criteria in Solid Tumors (version 1.1). All patients who received at least one dose of study drug were included in all analyses. This trial is registered with ClinicalTrials.gov, number NCT02060188.Findings Of the 74 patients who were enrolled between March 12, 2014, and March 16, 2016, 40 (54%) had received three or more previous treatments. At a median follow-up of 12.0 months (IQR 8.6-18.0), 23 (31.1%, 95% CI 20.8-42.9) of 74 patients achieved an investigator-assessed objective response and 51 (69%, 57-79) patients had disease control for 12 weeks or longer. Median duration of response was not yet reached; all responders were alive, and eight had responses lasting 12 months or longer (Kaplan-Meier 12-month estimate 86%, 95% CI 62-95). The most common grade 3 or 4 drug-related adverse events were increased concentrations of lipase (six [8%]) and amylase (two [3%]). 23 (31%) patients died during the study; none of these deaths were deemed to be treatment related by the investigator.Interpretation Nivolumab provided durable responses and disease control in pre-treated patients with dMMR/MSI-H metastatic colorectal cancer, and could be a new treatment option for these patients.

Globally, about 10% of new cancers each year are CRC and ∼15% of these are MSI-H. Nivolumab (N), a fully human anti-PD-1 mAb, and ipilimumab (I), a humanized anti-CTLA-4 mAb, are immune checkpoint inhibitors with favorable safety and efficacy profiles in multiple tumor types. This phase 2 study evaluates N ± I in MSI-H and non-MSI-H pts with mCRC. MSI-H pts received N 3 mg/kg q2 wk (N3) or N 3 mg/kg + I 1 mg/kg q3 wk (N3 + I1) x 4 doses followed by N3 until disease progression (PD) or other discontinuation. Initial evaluation of N + I was also completed in non-MSI-H pts. Primary endpoint was investigator-reported ORR by RECIST 1.1. Other endpoints included safety, OS, PFS, and clinical activity in biomarker-defined subpopulations (KRAS, BRAF status, and PD-L1). 70 (N3) and 30 (N3 + I1) MSI-H pts and 3 (N1 + I1), 10 (N1 + I3), and 10 (N3 + I1) non-MSI-H pts were enrolled. All non-MSI-H pts and 87% (N3) and 93% (N3 + I1) of MSI-H pts had ≥2 prior regimens. 47 (67%; N3) and 18 (60%; N3 + I1) MSI-H pts remain on tx. Efficacy data for MSI-H pts are shown in the Table. Responses were also seen in non-MSI-H pts. Median (95% CI) PFS across all non-MSI-H pts was 1.4 mo (1.2, 1.9). Responses were observed regardless of tumor PD-L1 expression. Treatment-related adverse events (TRAEs) occurred in 41 (59%; N3) and 25 (83%; N3 + I1) MSI-H pts; 10 (14%; N3) and 8 (27%; N3 + I1) pts had Grade 3–4 TRAEs. One pt on N3 had a Grade 5 TRAE (sudden death). Additional biomarker data including MSI assessment and influence of BRAF/KRAS mutations will be presented.Tabled 1MSI-HaBy local screen efficacyN3 (n = 70)N3 + I1 (n = 30)ORR, n (%)bReported for patients with 12 weeks of follow-up, N3 (n = 47) and N3 + I1 (n = 27)12 (25.5)9 (33.3)CR PR SD PD Not determined/not reported0 12 (25.5) 14 (29.8) 17 (36.2) 4 (8.5)0 9 (33.3) 14 (51.9) 3 (11.1) 1 (3.7)Median DOR (95% CI), moNR (4.2, NE)NR (NE, NE)PFS rates, % (95% CI) 6 mo 9 mo 12 mo45.9 (29.8, 60.7) 45.9 (29.8, 60.7) 45.9 (29.8, 60.7)66.6 (45.5, 81.1) NR NROS rates, % (95% CI) 6 mo 9 mo 12 mo75.0 (58.5, 85.7) 65.6 (48.0, 78.6) 65.6 (48.0, 78.6)85.1 (65.0, 94.2) 85.1 (65.0, 94.2) NRBaseline PD-L1 expression, n (%) ≥1% <1% Indeterminate/not evaluable/missing17 (24.3) 29 (41.4) 24 (34.3)7 (23.3) 20 (66.7) 3 (10.0)NR, not reached; NE, not estimablea By local screenb Reported for patients with 12 weeks of follow-up, N3 (n = 47) and N3 + I1 (n = 27) Open table in a new tab N ± I demonstrated promising clinical activity with a favorable overall safety profile in pts with mCRC, regardless of tumor PD-L1 expression. Additional biomarker analyses are ongoing.

Background The FDA recently approved an anti-CTLA-4 antibody (Iplimumab) for the treatment of metastatic melanoma. This decision was based on Phase III results, which demonstrate that blocking this immune checkpoint provides a survival advantage in patients with advanced disease. As a single agent, ipilimumab is also being clinically evaluated in advanced (metastatic, castrate-resistant) prostate cancer and two randomized, placebo-controlled Phase III studies have recently completed accrual. Methods We used a well-described genetically engineered mouse (GEM), autochronous prostate cancer model (Pro-TRAMP) to explore the relative sequencing and dosing of anti-CTLA-4 antibody when combined with a cell-based, GM-CSF-secreting vaccine (GVAX). Results Our results show that combined treatment results in a dramatic increase in effector CD8 T cells in the prostate gland, and enhanced tumor-antigen directed lytic function. These effects are maximized when CTLA-4 blockade is applied after, but not before, vaccination. Additional experiments, using models of metastatic disease, show that incorporation of low-dose cyclophosphamide into this combined treatment regimen results in an additional pre-clinical benefit. Conclusions Together these studies define a combination regimen using anti-CTLA-4/GVAX immunotherapy and low-dose chemotherapy for potential translation to a clinical trial setting.

Abstract Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4+ and CD8+ T cells in three distinct transplantable tumors. Dual anti–LAG-3/anti–PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3−/−Pdcd1−/− mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer. Cancer Res; 72(4); 917–27. ©2011 AACR.

Abstract Checkpoint molecules like CTLA-4 are important regulators of tumor-specific tolerance, and monoclonal antibodies that block this class of molecules have emerged as important therapeutic modalities in multiple cancer types. Recent data suggest that tumor infiltrating, non-functional ("exhausted") CD8 cells may express more than one checkpoint molecule, implying a more subtle control system for modulating CD8 T cell function in vivo. Based on their frequent co-expression on tumor-infiltrating lymphocytes, we investigated the relative roles of PD-1 (programmed death - 1) and LAG-3 (lymphocyte activation gene -3) in tumor tolerance. Single knockout (KO) mice lacking either of these genes were relatively unaffected, with PD-1 KO mice developing a late-onset strain-specific myocarditis. However, double knockout mice (DKO) lacking both PD-1 and LAG-3 developed early autoimmune pathology, with infiltrating lymphocytes present in multiple organs, and eventual lethality. More significantly, DKO mice spontaneously rejected the poorly immunogenic B16 melanoma, while rejection was not observed when either PD-1 or LAG-3 was knocked out individually. Moreover, established tumors in wild-type mice could be cured by combined treatment anti-PD-1 and anti-LAG-3 antibodies, while single antibody treatment was sufficient only to delay tumor outgrowth. Taken together, these data demonstrate a previously unappreciated synergy between these two checkpoint molecules in tumor-specific tolerance, and suggest a novel combinatorial strategy for cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-251. doi:10.1158/1538-7445.AM2011-LB-251

Relative upregulation of the Ikaros family transcription factor Helios in natural regulatory T cells (Tregs) has been reported by several groups. However, a role for Helios in regulatory T cells has not yet been described. Here, we show that Helios is upregulated in CD4+CD25+ regulatory T cells. Chromatin-immunoprecipitation (ChIP) experiments indicated that Helios binds to the FoxP3 promoter. These data were further corroborated by experiments showing that knocking-down Helios with siRNA oligonucleotides results in down-regulation of FoxP3. Functionally, we found that suppression of Helios message in CD4+CD25+ T cells significantly attenuates their suppressive function. Taken together, these data suggest that Helios may play an important role in regulatory T cell function and support the concept that Helios may be a novel target to manipulate Treg activity in a clinical setting.

Abstract Among the various molecules that regulate T cell function, lymphocyte activation gene 3 (LAG- 3) has garnered significant interest. LAG-3 is expressed on activated T cells, B cells, NK cells, tumor infiltrating lymphocytes (TILs), and plasmacytoid dendritic cells. We previously showed that LAG-3 was relatively over-expressed on HA-specific transgenic T cells rendered anergic in vivo by encounter with cognate self antigen. In this system, regulatory activity could be functionally blocked with a LAG-3 specific monoclonal antibody (Huang et al). Observations in our lab using LAG-3 knockout mice demonstrate that CD8 T cells undergo enhanced homeostatic proliferation in vivo if LAG-3 is absent. Currently, we are conducting studies to understand the role of LAG-3 in human cancer. Via microarray, we compared CD4+25+GITR+ (Treg) T cells from the prostate to CD4+25-45RA+ (naïve) T cells from the peripheral blood. We found LAG-3 and CTLA-4 to be relatively upregulated in prostate infiltrating T regulatory cells. Also, at the expression level, we observed variable levels of LAG-3 expression on patient CD8 tumor infiltrating T lymphocytes (TILs). At a functional level, we have shown that a human anti-human LAG-3 antibody enhances T cell proliferation and cytokine function in a mixed lymphocyte reaction. Further studies that are currently underway suggest that LAG-3 could be a promising candidate for enhancing anti-tumor immunotherapy in a clinical setting.

Abstract Although CD4+ T cells that secrete IL-17 (TH17) have been fairly well-studied, we and others have shown that CD8 T cells can be cultured under similar skewing conditions, resulting in CD8 cells with a robust ability to secrete IL-17. The physiological role such CD8 T cells (Tc17) is not well understood. In our previous data, we showed that Tc17 can functionally convert to IFN-g producing cells, expand, and mediate autoimmunity in a self-antigen murine model. Because autoimmunity and antitumor immunity both involve the breaking of peripheral T cell tolerance, we performed experiments to test whether Tc17 could mediate anti-tumor immunity in implanted tumor models. In a preventive model, small numbers (0.1M) of sorted IFN-g-producing Tc1, IL-17-producing or IFN-g-producing Tc17 TCR-transgenic HA-recognizing CD8 T cells were adoptively transferred to immunocompetent recipient (Balb/C) mice on day -1, and 0.5M CT-26 tumor cells expressing HA as antigen were implanted into the footpad on day 0. Adoptive transfer of Tc17 was significantly superior to Tc1 in mediating an antitumor response and a better survival rate. Additional mechanisms are being explored.

LAG-3 (CD223) is a cell surface molecule expressed on activated T cells (Huard et al. Immunogenetics 39:213-217, 1994), NK cells (Triebel et al. J Exp Med 171:1393-1405, 1990), B cells (Kisielow et al. Eur J Immunol 35:2081-2088, 2005), and plasmacytoid dendritic cells (Workman et al. J Immunol 182:1885-1891, 2009) that plays an important but incompletely understood role in the function of these lymphocyte subsets. In addition, the interaction between LAG-3 and its major ligand, Class II MHC, is thought to play a role in modulating dendritic cell function (Andreae et al. J Immunol 168:3874-3880, 2002). Recent preclinical studies have documented a role for LAG-3 in CD8 T cell exhaustion (Blackburn et al. Nat Immunol 10:29-37, 2009), and blockade of the LAG-3/Class II interaction using a LAG-3 Ig fusion protein is being evaluated in a number of clinical trials in cancer patients. In this review, we will first discuss the basic structural and functional biology of LAG-3, followed by a review of preclinical and clinical data pertinent to a role for LAG-3 in cancer immunotherapy.

Lymphocyte Activation Gene-3 (LAG-3) is a transmembrane protein that binds MHC class II, enhances regulatory T cell activity, and negatively regulates cellular proliferation, activation, and homeostasis of T cells. Programmed Death 1 (PD-1) also negatively regulates T cell function. LAG-3 and PD-1 are both transiently expressed on CD8 T cells that have been stimulated during acute activation. However, both LAG-3 and PD-1 remain on CD8 T cells at high levels after stimulation within tolerizing environments. Our previous data demonstrated that blockade of either LAG-3 or PD-1 using mAb therapy in combination with vaccination restores the function of tolerized Ag-specific CD8 T cells in models of self and tumor tolerance. It is unclear whether tolerized CD8 T cells coexpress PD-1 and LAG-3 or whether PD-1 and LAG-3 mark functionally distinct populations of CD8 T cells. In this study, we describe three populations of CD8 T cells activated under tolerizing conditions based on LAG-3 and PD-1 staining, each with distinct phenotypic and functional characteristics. From a mechanistic perspective, both Ag concentration and proinflammatory signals control the expression of LAG-3 and PD-1 phenotypes on CD8 T cells under activating and tolerizing conditions. These results imply that signaling through the PD-1 and LAG-3 pathways have distinct functional consequences to CD8 T cells under tolerizing conditions and manipulation of both Ag and cytokine signaling can influence CD8 tolerance through LAG-3 and PD-1.