Imelda S. Quinlin

CYTOTOXIC T-LYMPHOCYTE IMMUNOTHERAPY FOR OVARIAN CANCER: A PILOT STUDY

The objective was to evaluate the toxicity and feasibility of intraperitoneal 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, 7 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 mucin 1, a tumor-specific antigen found commonly in ovarian cancer cells. The resulting new CTL for each cycle were reintroduced into the host by intraperitoneal infusion. Immunologic parameters (killer cells, cytokine production, memory T lymphocytes, and natural killer cells) were studied. Toxicity, CA-125, and survival data were also evaluated. The tumor marker CA-125 was nonstatistically 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 natural killer 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 1 cycle, of T-cell stimulation followed by adoptive T-cell infusion, may not enhance the in vivo immune response.

Tumor Burden Influences Cytotoxic T Cell Development in Metastatic Breast Cancer Patients—A Phase I/II Study

The influence of tumor burden on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTL) was investigated in a phase I/II clinical adoptive immunotherapy trial. Four previously treated metastatic breast cancer patients, two with macroscopic disease and two 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. CTL responses against MCF-7 cell line and cytokine production were measured before infusion. Patients received two monthly CTL infusions and were monitored for toxicity, tumor response as well as tumor marker levels. The CTL generated from patients with high tumor burdens had less cytokine production and lower cytotoxicity of MCF-7 than the CTL of patients in CR. The differences between the two groups were observed after the two MUC1 in vitro stimulations of the cells obtained in first apheresis. This difference increased after the two MUC1 stimulations of the cells obtained in the second apheresis. The cytotoxicity function was sustained from the first infusion to the second apheresis only for the patients in CR. This suggests that tumor burden had an inverse effect on the function of the generated CTL.

Generation of MUC1‐stimulated Mononuclear Cells using Optimizied Conditions

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.

MHC-Unrestricted Lysis of MUC1-Expressing Cells by Human Peripheral Blood Mononuclear 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.

Retention of immunogenicity produced by mucin1 peptides with glycosylation site substitutions

Mucin1 (MUC1) with altered glycosylation behaves as an antigen unique to adenocarcinomas (ADCs). As a step toward DNA vaccines, the goal of this work was to determine whether MUC1 peptides substituted with an asparagine at O-linked glycosylation sites, might expose MUC1 peptide backbone to serve as immunogens to generate cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells of patients with ADCs. Substitution of some or all tyrosine and serine residues by asparagine in MUC1 did not inhibit the generation of mucin-specific CTLs. This suggests that MUC1 tandem repeat altered sequences to prevent O-linked glycosylation may be useful as DNA vaccines with tumor specificity.

Dendritic cells enhance the activity of human MUC1-stimulated mononuclear cells against breast cancer

Dendritic cells (DCs) are among the most potent antigen-presenting cells (APCs), stimulating peripheral blood mononuclear cells (PBMCs) to generate antigen-specific cytotoxic T lymphocytes (CTLs). The objectives of this study were to determine if interleukin (IL)-4 is beneficial or detrimental for the generation of human DCs in vitro and to understand whether DCs generated in vitro in the presence or absence of IL-4 stimulate the killing of adenocarcinoma cells by CTLs in vivo. Mucin 1 (MUC1), a glycoprotein found on the surface of adenocarcinoma cells was used to load DCs. MUC1-loaded DCs generated in the absence of IL-4 were superior to their counterparts produced with IL-4 in stimulating PBMCs to kill human breast cancer MCF-7 cells in vitro. A corollary in vivo protection experiment was performed by injecting immunodeficient NOD-SCID mice with MCF-7 cells s.c. and MUC1-loaded CTLs, PBMCs, or DCs generated in the absence of IL-4, i.p. Mice that received CTLs and MUC1-loaded DCs on days 0, 2, 4, 9, 14 and 19 were completely protected against the development of MCF-7-derived tumors, while other schedules conferred lower protection. Therefore, tumor antigen-loaded DCs enhance the efficacy of adoptive CTL transfer, and should thus be considered for combinatorial immunotherapeutic regimens.

Number of treatment cycles influences development of cytotoxic T cells in metastatic breast cancer patients - a phase I/II study.

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.

TGFα‑PE38 enhances cytotoxic T‑lymphocyte killing of breast cancer cells

The aim of the present study was to determine whether the combination of two modalities of immunotherapy, targeting two different tumor antigens, may be feasible and non-toxic, yet enhance the killing of a human breast cancer cell line. The first modality was tumor growth factor α-Pseudomonas exotoxin 38 (TGFα-PE38), which specifically targets and kills tumor cells that express the epidermal growth factor receptor. The second modality was mucin-1 (MUC1)-specific cytotoxic T lymphocytes (CTLs), generated by MUC1 stimulation of peripheral blood mononuclear cells, to target the human breast cancer cell line, MCF7. TGFα-PE38 exhibited specific lysis of the MCF7 cells in a concentration- and time-dependent manner. TGFα-PE38 did not kill the normal hematopoietic stem cells or CTLs. Furthermore, TGFα-PE38 was not inhibitory for the growth or differentiation of the normal human hematopoietic stem cells into erythroid and myeloid colonies. In addition, TGFα-PE38 did not inhibit the killing function of CTLs, either when preincubated or co-incubated with CTLs. Finally, therapeutic enhancement was observed, in that TGFα-PE38 and CTLs were additive in the specific lysis of the MCF7 cells. These two modalities of immunotherapy may be beneficial for humans with breast cancer with or without other therapies, including autologous hematopoietic stem cell transplantation, specifically for purging cancer cells from hematopoietic stem cells prior to transplantation.