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Abstracts J Immunother Volume 33, Number 8, October 2010 knockdown of the genes dramatically decreased the tumorigenic capacity, indicating that the genes could be associated with tumorinitiating capacity of CSCs. We could induce cytotoxic T cells (CTLs) from peripheral blood lymphocytes of cancer patients after in vitro stimulation with SOX2-derived peptides. SP cells were susceptible to cytotoxicity of CTLs, whereas they were resistant to chemotherapeutic drugs. In addition, DNA vaccine encoding the CSC gene showed higher tumor suppressive capacity in vivo in a mouse tumor model. Our studies indicate that cancer vaccine targeting CSCs might serve as potent immunotherapy for cancer. Evaluation of FOXP3 Microsatellite Polymorphisms in High-Risk Melanoma Patients Receiving Adjuvant Interferon Ena Wang*, Helen Gogasw, Alessandro Monaco*, Lorenzo Uccellini*, Charalampos S. Floudasw, Yanis Metaxasw, Urania Dafniw, George Fountzilasw, Maria Spyropoulou-Vlachouw, Francesco M. Marincola*. *Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD; w Hellenic Cooperative Oncology Group, Athens, Greece. Background: Attempts to identify patients who benefit from adjuvant treatment with interferon alfa-2b (IFN) have been disappointing. The development of autoimmunity during adjuvant therapy with IFN cannot assist selection of patients for therapy at baseline. The FOXP3 is located at Xp11.23 within an area of autoimmune disease linkage and therefore is an excellent positional candidate gene for autoimmunity at this locus. Consequently we performed a microsatellite analysis of FOXP3 gene in the exon 0 region in high-risk melanoma patients enrolled in a study of two regimens of high-dose IFN. Methods: A fragment analysis was performed in the DNA of 259 stage IIb, IIc and III melanoma patients. After amplification and electrophoresis run, data from each sample were analyzed using Genemapper software (Applied Biosystems) which objectively calls the fragments size. Results: Thirteen alleles were detected (size: 284, 286, 288, 290, 292, 298, 302, 304, 306, 310, 312, 314, 316). The tabular distribution of these alleles in melanoma patients is presented below (Table 1). At a median follow up of 71 months (range 7.1 to 138.7 mo), 144 patients have recurred and 95 have died. There were no statistically significant differences in the incidence of FOXP3 alleles between melanoma patients who recurred and those with no evidence of recurrence. RFS did not differ significantly between patients with recurrence and those without for each of the alleles detected. Conclusions: No allele defined by the microsatellite analysis of the FOXP3 gene in exon 0 was correlated with improved RFS in this high-risk group of melanoma patients. Reshaping CD4 and CD8 Memory T Cell Proliferation by Treating Cancer Patients with an OX40 Agonist: Immunologic Assessment of a Phase I Clinical Trial Magdalena Kovacsovics-Bankowski, Edwin Walker, Lana Chisholm, Kevin Floyd, Walter Urba, Brendan Curti, Andrew Weinberg. Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, OR. OX40, a member of the TNF superfamily, is a potent costimulatory molecule expressed upon activation at the surface of CD4 and CD8 T cells. Its engagement improves T cell effector function and survival. Preclinical studies have shown that OX40 agonists injected into tumor-bearing mice increase anti-tumor immunity leading tumor-free survival in several mouse models. These results prompted the initiation of phase I clinical trial using a mouse anti-human OX40 agonist antibody in patients diagnosed with a variety of solid malignancies. In this dose-escalation study, the agonist OX40 antibody was administered on Day 1, 3 and 5 at 0.1, 0.4 and 2 mg/kg, respectively. Immune monitoring analysis, over a 2 month period, was performed on PBL by flow cytometry directly ex vivo, using a 10-color antibody panel detecting CD3, CD4, CD8, CD95, CD28, CD25, CD127, CCR7, FoxP3 and Ki-67. On Day 8-15, we have observed a 2-3-fold increase in the proliferating CD4+ CD95+ T cells as detected by increased Ki-67 staining, mostly in the FoxP3- population with no significant increase in CD4+ FoxP3+ T cells proliferation (Treg). The proportion of cycling CD8+ CD95+ T cells peaked later 15-29 days after the administration of the antibody with a 2-4.5-fold increase of cycling cells compared to a group of nine controls. Interestingly, the middle dose (0.4 mg/kg) showed the greatest sustained increase in proliferation for both the CD4 (non-Treg) and CD8 memory T cell population. Administration of anti-OX40 antibody, not only triggered T cell proliferation, but also increased the activation status of the CD8+ T cells as measured by the co-expression of CD38 and HLA-DR on the cycling cells. Using these two surface markers, cycling CD8+ T cells were sorted and a gene array analysis showed increases in mRNA for CD86, CTLA- 4, and 4-1BB when compared to the non-cycling CD8+ T cells. Flow cytometry data confirmed that Ki-67+ CD8+ T cells were enriched for these markers. Finally, in 2 out of 3 patients, where autologous tumor was obtained the infusion of anti-OX40 antibody appeared to increase the proportion of tumor specific T cells. PBMC collected pre and post anti-OX40 administration were co-cultured with melanoma cell lines. CD8 T cells, from PBMC obtained after anti-OX40 administration showed an increase in INF-g secretion toward autologous or HLA-matched melanoma cell lines. These results suggest that administration of an anti-OX40 antibody in cancer patients increases tumor-specific CD4+ and CD8+ T cells by enhancing their proliferation and the production of type I cytokines. TABLE 1. Genes No Relapse, N = 115 (%) Relapse, N = 144 (%) Allele_284 2 (1.74) 2 (1.39) 1 Allele_286 2 (1.74) 1 (0.69) 0.586 Allele_288 79 (68.70) 90 (62.50) 0.358 Allele_290 4 (3.48) 2 (1.39) 0.411 Allele_292 0 (0.00) 1 (0.69) 1 Allele_298 1 (0.87) 5 (3.47) 0.231 Allele_302 1 (0.87) 3 (2.08) 0.632 Allele_304 1 (0.87) 2 (1.39) 1 Allele_306 6 (5.22) 11 (7.64) 0.463 Allele_310 16 (13.91) 18 (12.50) 0.853 Allele_312 1 (0.87) 5 (3.47) 0.231 Allele_314 2 (1.74) 2 (1.39) 1 Allele_316 0 (0.00) 2 (1.39) 0.504 P Characterization of Antigen Specific T Cell Activation and Cytokine Expression Induced by Sipuleucel-T Johnna D. Wesley, Eric Chadwick, Ling-Yu Kuan, Corazon dela Rosa, Mark Frohlich, David Urdal, Nadeem Sheikh, Nicole M. Provost. Dendreon, Seattle, WA. Sipuleucel-T (PROVENGE s ) is an autologous cellular immunotherapy designed to stimulate an immune response to prostate cancer. PA2024 [a recombinant human antigen consisting of prostatic acid phosphatase (PAP) and granulocyte macrophagecolony stimulating factor (GM-CSF)] is cultured ex vivo for 2 days with peripheral blood mononuclear cells (PBMCs) isolated from a standard leukapheresis procedure. Sipuleucel-T obtained at Wks 0, 2, and 4, after which cells are infused back into subjects. This study examined the T cell activation profile and cytokine production in sipuleucel-T from men with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer enrolled in a Phase 3 study (D9902B, IMPACT). PBMCs from each leukapheresis were incubated with either PA2024 or GM-CSF (sargramostim), 912 | www.immunotherapy-journal.com r 2010 Lippincott Williams & Wilkins
J Immunother Volume 33, Number 8, October 2010 Abstracts and evaluated for the production of cytokines. The PA2024 culture supernatants at each treatment week displayed a substantial increase in both APC activation-associated cytokines (IL-1-alpha, IL-10, IL-12p70, IFN-gamma and TNF-alpha) and T cell activation-associated cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-gamma and TNF-alpha), which increased at the second and third infusions relative to the first. In contrast, the GM-CSF culture supernatants had comparatively diminished levels of both sets of cytokines at all treatments. In additional experiments, both CD4+ and CD8+ T cells generated by culture with either PA2024 or GM- CSF were assessed by flow cytometry for the expression of CD134 (OX40), CD137 (4-1BB), CD278 (ICOS) and CD279 (PD-1). Cells from the culture with PA2024, but not with GM-CSF, displayed a generalized pattern of enhanced expression of the T cell activation markers at the second infusion compared to the first and third. These data indicate that T cell activation and enhanced cytokine expression are a consequence of priming after the first infusion. These effects are not driven by GM-CSF, as T cell activation and enhanced cytokine production were only observed in PA2024 cultures. patients: 187 (41.3%) were HLA-A2+ and 266 (58.7%) were HLA-A2-. A similar median OS was observed for HLA-A2+ and HLA-A2- patients treated with ipilimumab across studies (Table 1). 1 The adverse events associated with ipilimumab treatment are primarily immune-related, which reflect its immune-based mechanism of action. 1 Ipilimumab-associated irAEs can be severe and life-threatening, but most are reversible using product-specific treatment guidelines. 1 Safety analyses on data from all treated patients in the phase II trials and in patients treated with ipilimumab alone in the phase III trial showed that irAEs occur at similar frequencies in HLA-A2+ and HLA-A2 patients (Table 1). These retrospective analyses show that HLA-A2 and HLA-A2+ patients with advanced melanoma have a similar survival benefit with a similar toxicity profile, from ipilimumab therapy. The data suggest that HLA-A2 subtype does not impact survival outcomes with ipilimumab in advanced melanoma patients. Reference: 1. Hodi FS, et al. N Engl J Med. 2010. [Epub ahead of print]. Impact of HLA-A2 Status on Ipilimumab Efficacy and Safety: Pooled Data from Four Phase II Trials in Advanced Melanoma Jedd D. Wolchok*, Jeffrey S. Weberw, Omid Hamidz, Celeste Lebbe´ y, Michele MaioJ, Dirk Schadendorf z, Veerle de Pril#, Ramy Ibrahim**, Axel Hoos**, Steven J. O’Dayz. *Memorial Sloan-Kettering Cancer Center, New York, NY; w H. Lee Moffitt Cancer Center, Tampa, FL; zThe Angeles Clinic and Research Institute, Santa Monica, CA; yInstitut Gustave Roussy, Villejuif, France; JUniversity Hospital of Siena, Siena, Italy; zUniversity Hospital Essen, Essen, Germany; #Bristol-Myers Squibb, Brainel’Alleud, Belgium; **Bristol-Myers Squibb, Wallingford, CT. Ipilimumab, which blocks cytotoxic T-lymphocyte antigen-4 to potentiate an antitumor T cell response, has demonstrated an improvement in overall survival (OS) in a phase III, randomized controlled trial in previously treated patients with advanced melanoma. 1 In this trial, ipilimumab at 3 mg/kg, with or without an HLA-A2-restricted gp100 peptide vaccine, was compared to gp100 alone. The mechanism of action of this gp100 vaccine required that all patients be HLA-A2+. However, the mechanism of action of ipilimumab suggests that HLA status has no impact on its clinical activity. We analyzed data from phase II trials to determine if HLA-A2 status impacts ipilimumab efficacy and safety. Data were pooled from 4 completed phase II trials (CA184-004, - 007, -008, and -022) for previously treated patients with advanced melanoma who received ipilimumab at 0.3, 3 or 10 mg/kg, and were analyzed comparing HLA-A2+ and HLA-A2 patients. In phase II studies, HLA-A2 typing was performed for biomarker analyses but was not used to restrict eligibility. Using a PCR-based assay, on-study HLA-A2 typing was performed on 453 previously treated NY-ESO-1-Specific CD8 T Cell Response in NY-ESO-1 Seropositive Metastatic Melanoma Patients Treated with Ipilimumab Correlates with Clinical Benefit Jianda Yuan*, Sacha Gnjaticw, Matthew Adamow*, Brian Ginsberg*, Teresa S. Rasalan*, Erika Ritterw, Humilidad F. Gallardo*, Yinyan Xu*, Evelina Pogorilerz, Katherine S. Panageasz, Arvin Yang*z, Stephanie Terzulli*z, Gerd Ritterw, Lloyd J. Oldw, James P. Allison*, Jedd D. Wolchok*z. *Ludwig Center for Cancer Immunotherapy; w Ludwig Institute of Cancer Research, NY Branch; zMedicine, MSKCC, New York, NY. Background: Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), has been shown to elicit durable immunologic and clinical responses in patients with metastatic melanoma. Our lab has demonstrated that ipilimumab enhances B and T cell immunity to NY-ESO-1, a prototypical cancer-testes antigen expressed in melanoma. Methods: In order to better characterize the association between immune response and clinical outcome, sera from 100 advanced melanoma patients treated with ipilimumab at Memorial Sloan- Kettering Cancer Center were analyzed for NY-ESO-1 seropositivity. In addition, pre- and/or post-therapy peripheral blood mononuclear cells from all NY-ESO-1 seropositive patients without the purification of CD4+ and CD8+ T cells were assayed for NY-ESO-1-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining following a 10-day in vitro stimulation with NY-ESO-1 overlapping peptides. Results: 20 out of the 100 patients were found to be seropositive at any time-point (6 seroconverted), with a trend toward these patients experiencing more frequent clinical benefit (11/20; 55%) than seronegative patients (25/80; 31%), P = 0.067. Within the seropositive subgroup with the availability of suitable specimens, TABLE 1. Phase III Pooled Phase II Data Survival HLA-A2 + (N = 137) HLA-A2 + (N = 187) HLA-A2 (N = 266) Median OS, 10.1 (8.0-13.8) 9.3 (7.4-11.5) 11.4 (9.3-15.1) months (95% CI) irAEs, no. (%) HLA-A2 + (N = 131) HLA-A2 + (N = 218)* HLA-A2 (N = 311)* Skin 57 (43.5) 97 (44.5) 153 (49.2) Gastrointestinal 38 (29.0) 65 (29.8) 119 (38.3) Hepatic 5 (3.8) 9 (4.1) 23 (7.4) Other 6 (4.6) 5 (2.3) 18 (5.8) CI indicates confidence interval; HLA, human leukocyte antigen. *Includes previously treated and untreated patients who received ipilimumab. r 2010 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 913
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ABSTRACTS VACCINE COMBINATIONS Abst
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