Derek Ng Tang

Preoperative CTLA-4 Blockade: Tolerability and Immune Monitoring in the Setting of a Presurgical Clinical Trial

Purpose: Cytotoxic T lymphocyte associated antigen (CTLA-4) blockade is being explored in numerous clinical trials as an immune-based therapy for different malignancies. Our group conducted the first preoperative clinical trial with the anti–CTLA-4 antibody ipilimumab in 12 patients with localized urothelial carcinoma of the bladder. Experimental Design: Six patients were treated with 3 mg/kg/dose of anti–CTLA-4 and six patients were treated with 10 mg/kg/dose of antibody. Primary end points of the study were safety and immune monitoring. Results: Most drug-related adverse events consisted of grade 1/2 toxicities. All patients had measurable immunologic pharmacodynamic effects, consisting of an increased frequency of CD4+ICOShi T cells in tumor tissues and the systemic circulation. To determine if CD4+ICOShi T cells could be a correlative marker for clinical outcome after treatment with anti–CTLA-4, a cohort of metastatic melanoma patients was studied retrospectively for frequency of CD4+ICOShi T cells and survival. Data from this small cohort of patients indicated that an increased frequency of CD4+ICOShi T cells, sustained over a period of 12 weeks of therapy, correlates with increased likelihood of clinical benefit consisting of overall survival. Conclusions: Our trial shows that anti–CTLA-4 therapy has a tolerable safety profile in the presurgical setting and that a preoperative model can be used to obtain biological data on human immune responses, which can efficiently guide the monitoring of patients treated in the metastatic disease setting. Clin Cancer Res; 16(10); 2861–71. ©2010 AACR.

Anti-CTLA-4 therapy results in higher CD4+ICOShi T cell frequency and IFN-γ levels in both nonmalignant and malignant prostate tissues

Cytotoxic lymphocyte antigen-4 (CTLA-4) blockade is an active immunotherapeutic strategy that is currently in clinical trials in cancer. There are several ongoing trials of anti-CTLA-4 in the metastatic setting of prostate cancer patients with reported clinical responses consisting of decreases in the prostate specific antigen (PSA) tumor marker for some patients. Immunologic markers that correlate with these clinical responses are necessary to guide further development of anti-CTLA-4 therapy in the treatment of cancer patients. We recently reported that CD4+ inducible co-stimulator (ICOS)hi T cells that produce interferon-γ (IFN-γ) are increased in the peripheral blood and tumor tissues of bladder cancer patients treated with anti-CTLA-4 antibody. Here we present data from the same clinical trial in bladder cancer patients demonstrating a higher frequency of CD4+ICOShi T cells and IFN-γ mRNA levels in nonmalignant prostate tissues and incidental prostate tumor tissues removed at the time of radical cystoprostatectomy. Our data suggest immunologic markers that can be used to monitor prostate cancer patients who receive anti-CTLA-4 therapy and indicate that the immunologic impact of anti-CTLA-4 antibody can occur in both tumor and nonmalignant tissues. These data should be taken into consideration for evaluation of efficacy as well as immune-related adverse events associated with anti-CTLA-4 therapy.

Identification of Human Regulatory T Cells in the Setting of T-Cell Activation and Anti–CTLA-4 Immunotherapy on the Basis of Expression of Latency-Associated Peptide

UNLABELLED Effector and regulatory T cells (Treg) share multiple markers that make it difficult to discern differences in these populations in humans. The transcription factor FoxP3 has been shown to identify Tregs. However, the detection of FoxP3 requires cell permeabilization, thereby preventing isolation of viable Tregs. Subsequently, the extracellular marker CD127 was established for the identification of Tregs. However, these studies were not conducted in the setting of immunotherapy. Here, we conducted studies to analyze CD127 and FoxP3 expression on T cells before and after in vitro activation as well as in the setting of patients treated with antibodies directed against cytotoxic T-lymphocyte antigen-4 (CTLA-4). We show that latency-associated peptide (LAP), as opposed to CD127, was capable of identifying Tregs after in vitro activation as well as after treatment with anti-CTLA-4. Therefore, we propose that LAP should be used as a marker of Tregs for immune monitoring studies in patients treated with active immunotherapy such as anti-CTLA-4. SIGNIFICANCE Tregs play an important role in human diseases, including cancer and autoimmunity; however, it has been difficult to study these cells because of a lack of an appropriate marker. Here, we propose LAP as a marker that can be used to identify Tregs in patients treated with immunotherapy, thereby permitting isolation of these cells for functional studies and for ex vivo expansion.

A study of genes and microRNAs that may predict clinical responses to anti-CTLA-4 therapy.

285 Background: Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, Ipilimumab (BMS), has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of Ipilimumab for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4 therapy. In order to identify genes, microRNAs, and signaling pathways that are modulated by anti-CTLA-4, which may be used for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4+ T cells from patients treated with anti-CTLA-4 for changes in gene and microRNA expression profiles. METHODS On an IRB-approved Phase Ia presurgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of Ipilimumab at 10 mg/kg at weeks 1 and 4. Pre-therapy and post-therapy blood samples were collected for CD4+ T cell enrichment by using the T cell isolation kit from Miltenyi Biotec (Auburn, CA). RNA and microRNA were isolated from purified CD4+ T cells using Qiagen RNA isolation kits for Affymetrix microarray and micoRNA array analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. RESULTS Ipilimumab treatment resulted in modulation of differentially expressed genes (DEGs). After two doses of treatment, Ipilimumab significantly changed expression of a total of 289 DEGs. Further pathway analyses indicated that Ipilimumab induced a variety of pathways involved in cell proliferation and immune modulation, including PI3K/AKT, MAP/ERK, and IFN/JAK-STAT pathways. We have also identified 9 microRNAs that potentially regulate the expression of genes changed by anti-CTLA-4 therapy. CONCLUSIONS Ipilimumab treatment results in modulation of multiple genes, microRNAs, and pathways, which likely play important roles in anti-tumor immune responses. We are currently testing a number of these identified genes and microRNAs as potential predictive biomarkers for anti-CTLA-4 therapy in a small cohort of patients who had complete response vs. progression of disease after anti-CTLA-4 therapy.

Abstract 1265: Sustained PI3-kinase signaling is mediated by ICOS, not CD28, for anti-tumor T-cell responses elicited by anti-CTLA-4.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The generation of anti-tumor T-cell responses requires at least two distinct signals consisting of T-cell receptor signaling and CD28-costimulation to mediate PI3-kinase signaling. In previous studies, we reported that cancer patients treated with anti-CTLA-4 had a significant increase in T-cells expressing inducible costimulator (ICOS). Here, we observed that PI3-kinase signaling was significantly increased in ICOShi as compared to ICOSlow CD28+ T-cells from anti-CTLA-4 treated patients. Our data also indicate that after T-cell activation, ICOS-mediated PI3-kinase signaling plays an important role in downstream T-cell responses such as expression of the T-bet transcription factor and anti-tumor Th1 cytokine production. An ICOS-specific siRNA transfected into activated human T-cells leads to diminished PI3K-signaling and T-bet expression, which controls production of anti-tumor Th1 cytokines. Furthermore, T-cells from ICOS-deficient mice have impaired PI3-kinase signaling after in vitro activation. Our data also demonstrate that T-cells from ICOS-deficient mice have impaired PI3-kinase signaling in vivo, which correlates with impaired tumor rejection after treatment with anti-CTLA-4. These data offer strong evidence to support that CD28-mediated PI3-kinase signaling is limited and ICOS provides a critical third signal to enable sustained PI3-kinase signaling for optimal anti-tumor T-cell responses in the setting of anti-CTLA-4 therapy. Citation Format: Hong Chen, Tihui Fu, Dimitra Tsavachidou, Sijin Wen, Derek Ng Tang, Qiuming He, Jing Jing Sun, Padmanee Sharma. Sustained PI3-kinase signaling is mediated by ICOS, not CD28, for anti-tumor T-cell responses elicited by anti-CTLA-4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1265. doi:10.1158/1538-7445.AM2013-1265

Abstract 5368: A phase II study of ipilumimab plus androgen deprivation therapy in castration-sensitive prostate carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Anti-CTLA-4 therapy (Ipilimumab, Yervoy™) was recently FDA-approved for the treatment of metastatic melanoma based on improved median overall survival observed in Phase III clinical trials. Since anti-CTLA-4 targets a T cell specific molecule, as opposed to a tumor specific molecule, it is being investigated as treatment for multiple malignancies, including prostate cancer. Given that anti-CTLA-4 mediates anti-tumor responses by enhancing T cell responses, it has been postulated that standard agents that enable tumor cell death may allow for priming of a T cell immune response that can be augmented by combination with CTLA-4 blockade. Therefore, we hypothesized that adding ipilimumab to standard androgen ablation (AA) therapy in men with metastatic prostate cancer (mPC) would improve immune and clinical responses. We designed a clinical trial whereby men with castrate-sensitive mPC, ECOG performance status ≤ 2 and normal organ and marrow function are registered ≤1 month of initiation of an LHRH agonist or antagonist (LHRHa). Men with autoimmune diseases, known brain metastases, small cell carcinoma morphology, known HIV, Hepatitis B or C infection, untreated symptomatic spinal cord compressions or chronic immunosuppressive therapies are excluded. Starting on day 29, patients receive 4 monthly doses of 10mg/kg ipilimumab. The primary endpoint of the trial is to estimate the rate of PSA ≤ 0.2ng/mL at 7 months, shown to be a powerful predictor of survival (Hussain et al. J Clin Oncol. 2006; 24:3984). Secondary endpoints include: immune responses, time to testosterone recovery (>50ng/mL), time to disease progression off AA, overall survival and safety of the combination.Accrual is ongoing. Data from 8 patients accrued to date show a significant increase in the frequency of T cells expressing the inducible costimulator (ICOS), as we have previously shown with ipilimumab monotherapy (Liakou et al. Proc Natl Acad Sci U S A. 2008;105:14987; Carthon et al. Clin Cancer Res. 2010;16:2861), and significant increases in memory T cell markers. ICOS+ T cells play an important role in anti-tumor responses mediated by anti-CTLA-4 therapy (Fu et al. Cancer Res. 2011;71:5445). Significant changes in dendritic, natural killer and macrophage cell populations have not been observed. To date, one patient developed an asymptomatic grade 3 transaminitis after 2 doses of ipilimumab that resolved with high dose steroids. Assessment of immune responses induced by the combination of ipilimumab and AA, and evaluation of clinical endpoints are ongoing. Updated data will be presented. 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 5368. doi:1538-7445.AM2012-5368

Abstract 4388: Combination therapy with anti-CTLA-4 plus leuprolide acetate in the pre-surgical setting of patients with regional, high-risk prostate cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Anti-CTLA-4 is a monoclonal antibody (Ipilimumab or Yervoy, Bristol-Myers Squibb) that was recently FDA-approved for the treatment of patients with metastatic melanoma. Since anti-CTLA-4 targets a T cell specific molecule, as opposed to a tumor-specific molecule, it is being tested for therapeutic efficacy in multiple malignancies, including prostate cancer. In addition, since anti-CTLA-4 enhances anti-tumor T cell responses by blocking an inhibitory signal on activated T cells, it has been postulated that traditional therapies that lead to tumor cell death, thereby releasing tumor antigens to prime and potentially activate T cell responses, can be used in combination with CTLA-4 blockade to sustain activated T cell responses and enhance anti-tumor immune responses. Pre-clinical mouse models have shown this to be true. To determine the impact of combination therapy on human immune responses, we designed a pre-surgical clinical trial with a hormonal agent consisting of leuprolide acetate, a standard agent that has therapeutic efficacy against prostate cancer, plus anti-CTLA-4 (ipilimumab), in the setting of patients with regional, high-risk prostate cancer. As of October 2011, we have treated 4 out of a planned 20 patients on trial. Patients receive one injection of leuprolide acetate (Lupron, Tap Pharmaceuticals) at a dose of 22.5 mg on week 0 and ipilimumab at 10 mg/kg/dose on weeks 1 and 4. Patients then undergo surgery at week 8. Tumor tissues are collected prior to any therapy and then at the time of surgery. Blood is collected throughout the study prior to each therapeutic intervention and after surgery. Immunologic analyses are ongoing on all collected patients’ samples. These data will be compared to those obtained from previously completed trials consisting of a trial whereby patients were treated with ipilimumab monotherapy and a trial whereby patients were treated with hormonal therapy prior to surgery. Our preliminary data indicate that combination therapy impacts T cell responses including a measurable increase in the frequency of T cells expressing the inducible costimulator (ICOS) molecule. In addition, four patients have completed the trial, including surgery, without any serious adverse events related to the combination therapy. 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 4388. doi:1538-7445.AM2012-4388