The objective was to evaluate the toxicity and feasibility of intraperitoneal (IP) infusion of tumor-specific cytotoxic T-lymphocytes (CTL) as therapy for recurrent ovarian cancer, and to determine if repetitive cycles of CTL generation and infusion measurably increases the host’s ovarian cancer immune response. In this study, seven subjects with recurrent ovarian cancer confined to the peritoneal cavity underwent up to 4 cycles, each cycle beginning with a leukapheresis for collection of precursor lymphocytes, which were stimulated in vitro with MUC1, a tumor-specific antigen found commonly in ovarian cancer cells. The resulting new CTL for each cycle were re-introduced into the host via IP infusion. Immunological parameters (killer cells, cytokine production, memory T-lymphocytes and natural killer (NK) cells) were studied. Toxicity, CA-125, and survival data were also evaluated. The tumor marker CA-125 was non statistically significantly reduced after the first month of immunotherapy. However, after that, it rose. Killer cells, cytokine production and memory T-lymphocytes increased after the first cycle of stimulation, but plateaued or reduced thereafter. The percent of NK cells inversely correlated with other immune parameters. Median survival was 11.5 months. One subject is free of disease since December, 2000. Multiple cycles, beyond one cycle, of T-cell stimulation followed by adoptive T cell infusion, may not enhance the in vivo immune response.

Adoptive T cell immunotherapy using autologous lymphocytes is a viable treatment for patients with cancer and requires participation of Ag-specific CD4 and CD8 T cells. Here, we assessed the immunotherapeutic effects of autologous MUC1 peptide-stimulated CD4 + effector cells following adoptive transfer in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 + / Th1 effector cell generation, we show that three monthly treatment cycles of peripheral blood T cell restimulation and intraperitoneal re-infusion selectively modulated endogenous T cell-mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient survived long-term for nearly 16 months with recurrent disease and two patients expired within 3-5 months following final infusion. Although PBL from all patients showed elevated MUC1 cytolytic activity following therapy, such responses did not correlate with therapeutic efficacy. Long-term survivors showed elevated levels of systemic memory (CD45RO) and naïve (CD45RA) CD3/CD4/CD25 + T cells when compared to that of pre-treatment levels and similarly-treated short-term survivors. Such cells co-expressed different levels of Foxp3 and CTLA-4 that resulted in progressively lower systemic Foxp3/CTLA-4 memory T cell ratios that further correlated with disease-free survival. Lastly, these patients showed elevated levels of MUC1-specific T cells expressing the CCR5 and CCR1 chemokine receptors and the chemokine CCL4 associated with Th1 cell differentiation/memory. We suggest that effective Disclosures: This manuscript has not been published elsewhere and has not been submitted simultaneously for publication elsewhere.None of the authors have any potential financial conflict of interest related to this manuscript Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. immunotherapy with autologous MUC1-stimulated CD4 + effector cells induce differential levels of systemic "Ag-experienced" and "Ag-inexperienced" CD4/CD25 + TReg cell subpopulations that influence long-term tumor immunity in ovarian cancer patients. NIH Public Access

MUC1 is a glycoprotein found at the secretory poles of normal cells but is hypoglycosylated on the entire surface of cell membranes of adenocarcinomas. In order to determine the influence on the immune response of peptide context for epitope presentation, peripheral blood mononuclear cells (PBMC) from patients with adenocarcinomas, were stimulated with MUC1 peptides derived from the 20 amino acids (aa) long sequence that is characteristic of the MUC1 Variable Number of Tandem Repeats (VNTR). In the seven peptides tested, the T-cell tumor-specific epitope (cTSE) was surrounded by variable numbers of aa and repeated up to 5 times in the same peptide. The results of this study indicate that cultures stimulated with peptide 610 (GSTAPPAHGVTS APDTRPAP) showed the highest specific killing of the MUC1expressing breast cancer MCF-7 cells. Peptide 610 is also superior to the other peptides in inducing better production of the type 1 cytokines, tissue necrosis factor • and interferon Á. In conclusion, context of the epitope and not sequence alone determines immunogenicity.

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Adoptive T cell therapy for cancer patients optimally requires participation of CD4 T cells. In this phase I/II study, we assessed the therapeutic effects of adoptively transferred IL-10 and IFN-γ-producing CD4 effector cells in patients with recurrent ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 effector cell generation, we show that 3 monthly treatment cycles of autologous T cell restimulation and local intraperitoneal re-infusion modulated T cell mediated immune responses that were associated with enhanced patient survival. One patient remains disease free, another patient experienced prolonged survival for nearly 16 months with recurrent disease and two patients expired within 3–5 months following final infusion. Prolonged survivors showed elevated levels of systemic CD3+CD4+CD25+ and CD3+CD4+CD25− T cells when compared to that of pre-treatment levels and similarly treated short-term survivors. Such cell populations among these patients contained variable levels of “Inducible” Tr1 (CD4+CD25−FoxP3−IL-10+) and “Natural” (CD4+CD25+CD45RO+FoxP3+) TReg cell numbers and ratios that were associated with prolonged and/or disease-free survival. Moreover, peptide-restimulated T cells from these patients showed an elevation in both IFN-γ production, memory cell phenotype and select TNF family ligands associated with enhanced T cell survival and apoptosis-inducing activities. This suggests that intraperitoneally-administered Th1-like cells, producing elevated levels of IL-10, may require and/or induce differential levels of distinct systemic TReg subpopulations that influence, in part, long-term tumor immunity and enhanced memory/effector CD4-mediated therapeutic potentials. Furthermore, treatment efficacy and enhanced memory cell phenotype did not appear to be dependent on TReg cell numbers but upon ratios of “Inducible” and “Natural” TReg subpopulations.

Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)‐stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF‐7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony‐stimulating factor, gamma‐interferon and interleukin‐10) was 45 × 10−8 m (1 μg/ml); except for tumour necrosis factor (TNF)‐alpha which was 22 × 10−8 m (0.5 μg/ml). The unwashed MC were superior to washing them with Ficoll–Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 × 10−8 m (1 μg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.

Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

The influence of the number of apheresis-stimulation-infusion(s) cycles, and the time in culture before the infusion (one vs. two weeks), on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTL) was investigated in a phase I/II clinical adoptive immunotherapy trial. Two previously treated metastatic breast cancer patients with no evidence of disease, in complete remission (CR), were enrolled. Each apheretic peripheral blood mononuclear cell (PBMC) sample was stimulated twice with MUC-1 before infusion back into the patients. Killer T-cells responses against MUC-1-expressing MCF-7 (CTL), nonspecific natural killer (NK) and lymphokine-activated killer (LAK) target cell lines, as well as, cytokine production were measured before each infusion. Patients received 2 infusions per month for 4 months. There were no tumor recurrences or toxicity. CTL, NK and LAK cells, type 1 cytokine, gamma-interferon (G-INF), and CD4(+) and CD8(+) memory T-lymphocytes were initially generated, produced or induced, respectively, and then declined. The CTL, NK and LAK cells were only induced at the first infusion of the first month. Thus, maintaining PBMC in culture longer than the first infusion was of no benefit with regards to retaining functional killer T-cells. In conclusion, this study implies that one treatment is optimal.