Cindy Sander

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8+ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8+ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1+ NY-ESO-1–specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1–specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

CD8+ T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

TIGIT and PD-1 impair tumor antigen–specific CD8 + T cells in melanoma patients

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.

Immune Monitoring of the Circulation and the Tumor Microenvironment in Patients with Regionally Advanced Melanoma Receiving Neoadjuvant Ipilimumab

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2–19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3+/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

PD-1 Is a Regulator of NY-ESO-1-Specific CD8+ T Cell Expansion in Melanoma Patients

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8+ T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8+ T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8+ T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8+ T cells, NY-ESO-1-specific CD8+ T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8+ T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1+ APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8+ T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8+ T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8+ T cell numbers and functions, increasing the likelihood of tumor regression.

Immunogenicity and Antitumor Effects of Vaccination with Peptide Vaccine +/− Granulocyte-Monocyte Colony-Stimulating Factor and/or IFN-α2b in Advanced Metastatic Melanoma: Eastern Cooperative Oncology Group Phase II Trial E1696

Purpose: No therapy has ever shown prolongation of survival in stage IV metastatic melanoma. The association of cytokine-induced autoimmunity with improved prognosis led us to investigate the effect of multi-epitope melanoma vaccines alone and in combination with cytokines in this Eastern Cooperative Oncology Group multicenter phase II trial. Experimental Design: Eligible patients were required to have failed prior therapies and to be HLA-A2 positive. Three HLA class I-restricted lineage antigen epitopes were administered in a factorial 2 × 2 design. Peptide vaccine alone (arm A), or combined with granulocyte-monocyte colony-stimulating factor (GM-CSF; Immunex) 250 μg/d subcutaneously for 14 of 28 days each month (arm B), or combined with IFN-α2b (Intron A; Schering-Plough) 10 million units/m2 three times a week (arm C), or combined with both IFN-α2b and GM-CSF (arm D). The primary endpoint was immune response measured by enzyme-linked immunospot assay; secondary endpoints were clinical antitumor response, disease-free survival, and overall survival. Results: One hundred twenty patients enrolled and 115 patients were analyzed. Immune responses to at least one melanoma antigen were observed in 26 of 75 (35%) patients with serial samples. Neither IFN-α2b nor GM-CSF significantly improved immune responses. Six objective clinical responses were documented. At a median follow-up of 25.4 months, the median overall survival of patients with vaccine immune response was significantly longer than that of patients with no immune response (21.3 versus 13.4 months; P = 0.046). Conclusion: Immune response to vaccination correlates with prolonged survival in patients with metastatic melanoma and is not enhanced by immunomodulatory cytokines as tested in this trial.

PD-1 and Tim-3 Regulate the Expansion of Tumor Antigen–Specific CD8+ T Cells Induced by Melanoma Vaccines

Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freunds adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma.

Safety and Efficacy of Combination Immunotherapy With Interferon Alfa-2b and Tremelimumab in Patients With Stage IV Melanoma

PURPOSE We tested the hypothesis that the combination of tremelimumab and interferon alfa-2b acting via different and possibly synergistic mechanisms would overcome tumor immune tolerance and lead to significant and durable clinical responses. PATIENTS AND METHODS We conducted a phase II study in which patients were administered tremelimumab 15 mg/kg/course (three cycles [one cycle = 4 weeks]) intravenously every 12 weeks. High-dose interferon alfa-2b (HDI) was administered concurrently, including intravenous induction at 20 MU/m2/d for 5 d/wk for 4 weeks followed by maintenance at 10 MU/m2/d subcutaneously three times a week for 8 weeks per course. From course 2 onward, HDI maintenance was administered subcutaneously. RESULTS Thirty-seven patients with American Joint Committee on Cancer stage IV (9M1a, 6M1b, and 22M1c) were enrolled. Two patients had previously treated brain metastases. Grades 3 and 4 toxicities included neutropenia (six patients; 17%), diarrhea/colitis (four patients; 11%), liver enzyme increase (four patients; 11%), rash (four patients; 11%), fatigue (15 patients; 40%), and anxiety/depression (five patients; 14%). Response data were available for 35 patients. The best objective response rate (RR; Response Evaluation Criteria in Solid Tumors) by intention to treat was 24% (90% CI, 13% to 36%; four complete responses [CRs] and five partial responses [PRs] that lasted 6, 6, > 12, > 14, > 18, 20, > 28, 30, and > 37 months, respectively). Fourteen patients (38%) had stable disease (SD) that lasted 1.5 to 21 months. The median progression-free survival was 6.4 months (95% CI, 3.3 to 12.1 months). The median overall survival (OS) was 21 months (95% CI, 9.5 to not reached). There was a weak association between therapy-induced autoimmunity and clinical benefits (CR/PR/SD; P = .0059), baseline C-reactive protein (CRP) less than or equal to 2.7× the upper limit of normal and clinical benefits (P = .0494) and improved probability of survival (P = .0032), and baseline lymphocyte count of at least 1,000/μL and response (CR/PR; P = .0183) and clinical benefits (CR/PR/SD; P = .0255). Biomarker associations were not significant after adjustment for multiple comparisons. CONCLUSION HDI can be administered combined with tremelimumab with acceptable toxicity and promising durable antitumor efficacy that warrant further testing in a randomized trial.

Prognostic Significance of Serum S100B Protein in High-Risk Surgically Resected Melanoma Patients Participating in Intergroup Trial ECOG 1694

PURPOSE We evaluated adjuvant trial E1694 to more precisely define the prognostic significance of serum S100B in patients with high-risk resected melanoma. PATIENTS AND METHODS Sera from 670 E1694 patients banked at baseline and three additional time points were tested for S100B protein using chemiluminescence. RESULTS S100B testing results showed that the higher the S100B level is, the higher the risk of relapse and death, regardless of the cutoff value. Univariate analysis showed that baseline S100B > or = 0.15 microg/L is significantly correlated with overall survival (OS; P = .01). Multivariate analysis was performed adjusting for significant prognostic factors (ulceration and lymph node status) and treatment. Baseline S100B was a significant prognostic factor for survival (hazard ratio = 1.39; 95% CI, 1.01 to 1.92; P = .043). S100B values measured at later time points over 1 year were also demonstrated to be significant prognostic factors for relapse-free survival (RFS) and OS. Lower S100B values at baseline and during follow-up were associated with longer survival. A changing S100B from low at baseline to high on follow-up seemed to be associated with the worst RFS and OS. CONCLUSION For patients with high-risk surgically resected melanoma, a high baseline or increasing serum S100B is an independent prognostic marker of risk for mortality that may allow us to refine the application of adjuvant therapy in the future.

Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients.

Analog peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific cytotoxic T lymphocytes. Here, we report the results of a pilot trial designed to study the immunogenicity of the analog peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1–expressing melanoma. Eight patients were immunized either with Montanide and CpG (arm 1, 3 patients); Montanide and peptide NY-ESO-1 157-165V (arm 2, 2 patients); or with Montanide, CpG, and peptide NY-ESO-1 157-165V (arm 3, 3 patients). Only the 3 patients immunized with Montanide, CpG, and peptide NY-ESO-1 157-165V in arm 3 developed a rapid increase of effector-memory NY-ESO-1–specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28−). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1–specific CD8+ T cells in 2 patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1–specific T cells in 1 patient with no spontaneously tumor-induced CD8+ T-cell responses to NY-ESO-1. Collectively, our data support the capability of the analog peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1–specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1–specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1–specific CD8+ T cells after peptide-based vaccine strategies.