Diane J. Poole

Ipilimumab and a poxviral vaccine targeting prostate-specific antigen in metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial

BACKGROUND Therapeutic cancer vaccines have shown activity in metastatic castration-resistant prostate cancer (mCRPC), and methods are being assessed to enhance their efficacy. Ipilimumab is an antagonistic monoclonal antibody that binds cytotoxic T-lymphocyte-associated protein 4, an immunomodulatory molecule expressed by activated T cells, and to CD80 on antigen-presenting cells. We aimed to assess the safety and tolerability of ipilimumab in combination with a poxviral-based vaccine targeting prostate-specific antigen (PSA) and containing transgenes for T-cell co-stimulatory molecule expression, including CD80. METHODS We did a phase 1 dose-escalation trial, with a subsequent expansion phase, to assess the safety and tolerability of escalating doses of ipilimumab in combination with a fixed dose of the PSA-Tricom vaccine. Patients with mCRPC received 2×10(8) plaque-forming units of recombinant vaccinia PSA-Tricom subcutaneously on day 1 of cycle 1, with subsequent monthly boosts of 1×10(9) plaque-forming units, starting on day 15. Intravenous ipilimumab was given monthly starting at day 15, in doses of 1, 3, 5, and 10 mg/kg. Our primary goal was to assess the safety of the combination. This study is registered with ClinicalTrials.gov, number NCT00113984. FINDINGS We completed enrolment with 30 patients (24 of whom had not been previously treated with chemotherapy) and we did not identify any dose-limiting toxic effects. Grade 1 and 2 vaccination-site reactions were the most common toxic effects: three of 30 patients had grade 1 reactions and 26 had grade 2 reactions. 21 patients had grade 2 or greater immune-related adverse events. Grade 3 or 4 immune-related adverse events included diarrhoea or colitis in four patients and grade 3 rash (two patients), grade 3 raised aminotransferases (two patients), grade 3 endocrine immune-related adverse events (two patients), and grade 4 neutropenia (one patient). Only one of the six patients previously treated with chemotherapy had a PSA decline from baseline. Of the 24 patients who were chemotherapy-naive, 14 (58%) had PSA declines from baseline, of which six were greater than 50%. INTERPRETATION The use of a vaccine targeting PSA that also enhances co-stimulation of the immune system did not seem to exacerbate the immune-related adverse events associated with ipilimumab. Randomised trials are needed to further assess clinical outcomes of the combination of ipilimumab and vaccine in mCRPC. FUNDING US National Institutes of Health.

Pilot Study of Vaccination with Recombinant CEA-MUC-1-TRICOM Poxviral-Based Vaccines in Patients with Metastatic Carcinoma

Purpose: Poxviral vectors have a proven safety record and can be used to incorporate multiple transgenes. Prior clinical trials with poxviral vaccines have shown that immunologic tolerance to self-antigens can be broken. Carcinoembryonic antigen (CEA) and MUC-1 are overexpressed in a substantial proportion of common solid carcinomas. The primary end point of this study was vaccine safety, with immunologic and clinical responses as secondary end points. Experimental Design: We report here a pilot study of 25 patients treated with a poxviral vaccine regimen consisting of the genes for CEA and MUC-1, along with a triad of costimulatory molecules (TRICOM; composed of B7.1, intercellular adhesion molecule 1, and lymphocyte function–associated antigen 3) engineered into vaccinia (PANVAC-V) as a prime vaccination and into fowlpox (PANVAC-F) as a booster vaccination. Results: The vaccine was well tolerated. Apart from injection-site reaction, no grade ≥2 toxicity was seen in more than 2% of the cycles. Immune responses to MUC-1 and/or CEA were seen following vaccination in 9 of 16 patients tested. A patient with clear cell ovarian cancer and symptomatic ascites had a durable (18-month) clinical response radiographically and biochemically, and one breast cancer patient had a confirmed decrease of >20% in the size of large liver metastasis. Conclusions: This vaccine strategy seems to be safe, is associated with both CD8 and CD4 immune responses, and has shown evidence of clinical activity. Further trials with this agent, either alone or in combination with immunopotentiating and other therapeutic agents, are warranted.

A Pilot Study of MUC-1/CEA/TRICOM Poxviral-Based Vaccine in Patients with Metastatic Breast and Ovarian Cancer

Purpose: PANVAC is a recombinant poxviral vaccine that contains transgenes for MUC-1, CEA, and 3 T-cell costimulatory molecules. This study was conducted to obtain preliminary evidence of clinical response in metastatic breast and ovarian cancer patients. Experimental design: Twenty-six patients were enrolled and given monthly vaccinations. Clinical and immune outcomes were evaluated. Results: These patients were heavily pretreated, with 21 of 26 patients having 3 or more prior chemotherapy regimens. Side effects were largely limited to mild injection-site reactions. For the 12 breast cancer patients enrolled, median time to progression was 2.5 months (1–37+) and median overall survival was 13.7 months. Four patients had stable disease. One patient had a complete response by RECIST and remained on study for 37 months or more, with a significant drop in serum interleukin (IL)-6 and IL-8 by day 71. Another patient with metastatic disease confined to the mediastinum had a 17% reduction in mediastinal mass and was on study for 10 months. Patients with stable or responding disease had fewer prior therapies and lower tumor marker levels than patients with no evidence of response. For the ovarian cancer patients (n = 14), the median time to progression was 2 months (1–6) and median overall survival was 15.0 months. Updated data are presented here for one patient treated with this vaccine in a previous trial, with a time to progression of 38 months. Conclusions: Some patients who had limited tumor burden with minimal prior chemotherapy seemed to benefit from the vaccine. Further studies to confirm these results are warranted. Clin Cancer Res; 17(22); 7164–73. ©2011 AACR.

The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines.

Abstract An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon γ production has been used to analyze specific T cell responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.

Evaluation of human carcinoembryonic-antigen (CEA)-transduced and non-transduced murine tumors as potential targets for anti-CEA therapies

The MC-38 C57BL/6 mouse colon adenocarcinoma cell line has been transduced with a retroviral construct containing cDNA encoding the human carcinoembryonic antigen (CEA) gene [Robbins PF, Kantor JA, Salgaller M, Horan Hand P, Fernsten PD, Schlom J (1991) Cancer Res 51: 3657]. Two clones, MC-38-ceal and MC-38-cea2, expressed high levels of CEA on their cell surface. A third CEA-expressing cell line, MCA-102-cea3, was similarly derived by transduction of the MCA-102 C57BL/6 mouse fibrosarcoma cell line and is described here. In this study, the three CEA-transduced murine tumor cell lines (MC-38-cea1, MC-38-cea2, MCA-102-cea3) were evaluated for their tumorigenic potential, as well as their ability to serve as in vivo model systems for active and passive immunotherapy studies. Parameters that were investigated include tumor growth rate, the antibody response of the host to CEA, and the CEA content of the tumors. The MC-38-cea2 model appeared to be the most appropriate for immunotherapy studies. Biodistribution studies, using an125I-labeled anti-CEA mAb, demonstrated efficient tumor targeting of MC-38-cea2 tumors in C57BL/6 and athymic mice.

New gene expressed in prostate: a potential target for T cell-mediated prostate cancer immunotherapy

New gene expressed in prostate (NGEP) is a prostate-specific gene encoding either a small cytoplasmic protein (NGEP-S) or a larger polytopic membrane protein (NGEP-L). NGEP-L expression is detectable only in prostate cancer, benign prostatic hyperplasia and normal prostate. We have identified an HLA-A2 binding NGEP epitope (designated P703) which was used to generate T cell lines from several patients with localized and metastatic prostate cancer. These T cell lines were able to specifically lyse HLA-A2 and NGEP-expressing human tumor cells. NGEP-P703 tetramer binding assays demonstrated that metastatic prostate cancer patients had a higher frequency of NGEP-specific T cells when compared with healthy donors. Moreover, an increased frequency of NGEP-specific T cells was detected in the peripheral blood mononuclear cells of prostate cancer patients post-vaccination with a PSA-based vaccine, further indicating the immunogenicity of NGEP. These studies thus identify NGEP as a potential target for T cell-mediated immunotherapy of prostate cancer.

Effect of Talactoferrin Alfa on the Immune System in Adults With Non-Small Cell Lung Cancer

BACKGROUND Talactoferrin alfa (talactoferrin), an agent with immune-stimulating properties, has demonstrated safety and preliminary efficacy in clinical trials. METHODS Ten patients (five males and five females) with stage IV non-small cell lung cancer (NSCLC) in a single-arm pilot study received orally administered talactoferrin (1.5 g, b.i.d.) for up to 24 weeks. Radiographic and immunologic studies were performed at baseline and at weeks 6 and 12. Circulating immune cells (natural killer cells [NKCs], CD4+, CD8+, and regulatory T cells) and systemic cytokine levels were measured to assess immune response. RESULTS Patients enrolled in the study had received a median of four prior chemotherapy regimens, and all patients were symptomatic. Talactoferrin was well tolerated, with no grade 3 or 4 toxicities. Median time to progression (TTP) and overall survival were 6 weeks and 14.5 weeks, respectively. The four patients with ≥9 weeks TTP had evidence of immunologic activity (three with increased NKC activity). CONCLUSIONS The median of four previous chemotherapy regimens, with elevated levels of interleukin (IL) 6 and tumor necrosis factor-alfa in most patients, suggests these patients were poor candidates for immunotherapy.

Abstract 2546: Analysis of immune cell subsets in a multidrug therapeutic regimen for patients with metastatic castration-resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: To investigate the effects of docetaxel-based combination therapy with bevacizumab and dexamethasone premedication on the immune response in patients with metastatic castration-resistant prostate cancer (mCRPC). Experimental Design: We studied immune responses in 13 patients enrolled in a phase II trial at the National Cancer Institute (NCI). The study was designed as a 13 patient expansion of a previously reported phase II study to evaluate the immunologic response after 2 cycles of treatment with a docetaxel-based chemotherapy regimen including docetaxel (75 mg/m2 every 3 weeks) and bevacizumab (15 mg/kg every 3 weeks). Dexamethasone pre-medication (4 mg) was given 12 h and 1 h prior to chemotherapy, and again after 12 h. Patients were evaluated before treatment and on day 40, 3 weeks after the second cycle. We compared PBMC and serum samples collected at baseline and after 40 days of treatment. We investigated CD4+ and CD8+ T-cells and regulatory T-cells (CD4+ CD25hi CD127- FoxP3+) by flow cytometry. T-cell proliferation, as well as NK-cell functional activity, was evaluated. Serum samples were analyzed for levels of cytokines, chemokines, sCD27, sCD40L and vascular endothelial growth factor (VEGF). Results: The baseline characteristics were: median age 64 years, Gleason score 9, PSA 100 ng/ml, and Halabi Predicted Survival 10.6 months. Patients had a median PSA decline of 66% after 2 cycles. The median TTP was 14.1 months, and OS 18.7 months. At 3 weeks after the second cycle we found no significant changes in absolute lymphocyte count, CD4+ and CD8+ T-cell proliferation and NK-cell function. The number of CD4+ T-cells decreased. CTLA4+ regulatory T-cells did not change. There was no change in the serum levels of IL-6, IL-8, IL-10 and TNFα. As expected, the serum levels of VEGF decreased substantially after therapy. The serum levels of sCD40L did not change after treatment. Interestingly, an increase in the serum level of sCD27 correlated with longer OS (P= 0.037, R= 0.58). Conclusions: Treatment of mCRPC patients with docetaxel-based combination therapy with bevacizumab and dexamethasone premedication for 40 days did not alter the immune response in a way that would decrease the likelihood of successful immunotherapy, either before or after this treatment. Citation Format: Caroline Jochems, Benjamin Boyerinas, Ravi A. Madan, Diane J. Poole, Yang-Min Ning, William D. Figg, David J. Liewehr, Seth M. Steinberg, James L. Gulley, Kwong-Yok Tsang, Jeffrey Schlom. Analysis of immune cell subsets in a multidrug therapeutic regimen for patients with metastatic castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2546. doi:10.1158/1538-7445.AM2014-2546

Generation of human T cells directed against an agonist epitope of Brachyury, a transcription factor involved in human tumor cell epithelial to mesenchymal transition (EMT)

Purpose The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to metastasis. An HLA-A2 epitope of Brachyury has been shown to expand human T cells that are capable of lysing Brachyury-expressing tumor cells in an HLA-dependent manner. A phase I clinical trial is ongoing at the NCI using a recombinant yeast Brachyury vaccine. We have previously demonstrated that agonist epitopes of tumorassociated antigens are more effective than native epitopes at activating antigen-specific T cell responses. The current study sought to identify an agonist of the Brachyury HLA-A2 epitope in order to increase T cell activation and tumor lysis.

Abstract 1260: Generation of human T cells directed against an agonist epitope of a transcription factor involved in epithelial to mesenchymal transition (EMT).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to metastasis. An HLA-A2 epitope of Brachyury has been shown to expand human T cells that are capable of lysing Brachyury-expressing tumor cells in an HLA-dependent manner. This study sought to define an agonist of this epitope in order to increase T cell activation and tumor lysis. Experimental Design: A novel agonist epitope of Brachyury was generated by residue substitution of the native epitope. Characterization of this epitope as an agonist included; comparison of HLA binding affinity and stability, interferon γ production by epitope-specific T cell lines, as well as Brachyury-and HLA-specific lysis of tumor cells. The presence of Brachyury-specific T cells that would recognize the agonist peptide, within the circulating PBMC of cancer patients, was determined by ELISPOT. Results: The agonist epitope was shown to bind HLA-A2 with higher affinity and stability than the native. T cell lines generated from both the native and agonist epitopes produced higher levels of interferon γ in response to stimulation with the agonist epitope. The agonist-specific T cell line was able to lyse a variety of Brachyury-expressing tumor cells more efficiently than the T cell line generated with the native epitope, and specificity of lysis was confirmed by cold-target inhibition and HLA-A2 blocking. Tetramer staining revealed Brachyury agonist-specific T cells in the PBMC of a prostate cancer patient after in vitro stimulation with the agonist peptide. PBMC from colon and ovarian cancer patients reacted to the agonist peptide in an interferon γ ELISPOT assay. Conclusions: An agonist epitope for Brachyury has been identified, which increased T cell activation and function as compared to the native epitope. T cells that react to the agonist peptide were detected in patients with carcinomas known to express high levels of Brachyury. This study supports the use of Brachyury as a cancer vaccine strategy, including the Brachyury agonist epitope. Citation Format: Jo A. Tucker, Diane J. Poole, Claudia Palena, Caroline Jochems, Jeffrey Schlom, Kwong Y. Tsang. Generation of human T cells directed against an agonist epitope of a transcription factor involved in epithelial to mesenchymal transition (EMT). [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 1260. doi:10.1158/1538-7445.AM2013-1260