MUC1 immunotherapy against a metastatic mammary adenocarcinoma model: Importance of IFN-gamma

Abstract Immunotherapy using mucin 1 (MUC1) linked to oxidised mannan (MFP) was investigated in an aggressive MUC1+ metastatic tumour, DA3-MUC1 because, unlike many MUC1+ tumour models, DA3-MUC1 is not spontaneously rejected in mice making it an alternative model for immunotherapy studies. Further, DA3-MUC1 cells are resistant to lysis by anti-MUC1 cytotoxic T cells (CTLs). The inability of DA3-MUC1 tumours to be rejected in naïve mice as well as vaccination to MUC1 was attributed to a deficiency of expression of MHC class I molecules on the tumour cell surface. In vitro and in vivo analysis of subcutaneous tumours and lung metastases demonstrated that DA3-MUC1 tumour cells have a low expression (&lt; 6%) of MHC class I which can be upregulated (&gt; 90%) following culturing with IFN-γ. Results from flow cytometry analysis and immunoperoxidase staining indicated that the in vitro up-regulation of MHC class I could be maintained for up to seven days in vivo, without affecting the expression levels of MUC1 antigen. Interestingly, MUC1-specific CTL that lyse DA3-MUC1 targets in vitro were induced in MFP immunised mice but failed to protect mice from a DA3-MUC1 tumour challenge. These results highlight the importance of MHC class I molecules in the induction of anti-tumour immunity and the MFP immune response.</jats:p

Selective inhibition of T suppressor-cell function by a monosaccharide

Interactions between regulatory T lymphocytes and other cells are assumed to occur at the level of the cell surface. T cells which suppress the generation of specifically effector cells have been described as having antigenic, idiotypic, allotypic and I-region specificity1−4. Other T suppressor cells generated by in vitro cultivation with or without mitogenic stimulation5,6 have suppressive activity for T and B cells but no specificity can be assigned to them. These T suppressor cells (Ts) inhibit various lymphoid functions—this either reflects their polyclonal origin or indicates that the structures recognized by the Ts receptors must be common for many cell types. Carbohydrates on cell membrane-inserted glycoproteins or glycolipids might function as specific ligands for recognition by cellular receptors or soluble factors. Almost all cell-surface proteins of mammalian cells are glycosylated. There is evidence for lectin-like carbohydrate binding proteins not only in plants7 but also in toxins8, viruses9, prokaryotic cells10 and even mammalian cells, including T cells11. A functional role for these lectin-like proteins has been described for slime moulds and suggested for the selective association of embryonic cells12,13. We report here that addition of a monosaccharide can counteract the effect of T suppressor cells during the generation of alloreactive cytotoxic T cells (CTLs) in vitro

Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21

The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH). Six breast cancer patients immunized with this conjugate plus the immunological adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the β turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells. © 1999 Cancer Research Campaig

RECOMBINANT SOLUBLE HUMAN FC-GAMMA-RII - PRODUCTION, CHARACTERIZATION, AND INHIBITION OF THE ARTHUS REACTION

A recombinant soluble form of human Fc gamma RII (rsFc gamma RII) was genetically engineered by the insertion of a termination codon 5' of sequences encoding the transmembrane domain of a human Fc gamma RII cDNA. Chinese hamster ovary cells were transfected with the modified cDNA and the secreted rsFc gamma RII purified from the tissue culture supernatant (to > 95%, assessed by SDS-PAGE) using heat aggregated human immunoglobulin G (IgG) immunoaffinity chromatography. The IgG-purified rsFc gamma RII was relatively homogeneous (approximately 31,000 M(r)) whereas the total unpurified rsFc gamma RII secreted into the tissue culture supernatant was heterogeneous relating to N-linked glycosylation differences. Functional in vitro activity of the rsFc gamma RII was demonstrated by: (a) ability to bind via the Fc portion of human IgG and mouse IgG (IgG2a > IgG1 > > IgG2b); (b) complete inhibition of binding of erythrocytes sensitized with rabbit IgG to membrane-bound Fc gamma RII on K562 cells; and (c) inhibition of the anti-Leu4-induced T cell proliferation assay. Blood clearance and biodistribution studies show the rsFc gamma RII was excreted predominantly through the kidney in a biphasic manner, with an alpha-phase (t1/2 approximately 25 min) and a beta-phase (t1/2 approximately 4.6 h); the kidneys were the only organs noted with tissue-specific accumulation. In vivo, the administration of rsFc gamma RII significantly inhibited the immune complex-mediated inflammatory response induced by the reversed passive Arthus reaction model in rats. There was a specific and dose-dependent relationship between the amount of rsFc gamma RII administered, and the reduction in the size and severity of the macroscopic inflammatory lesion. Histological analysis of the skin showed a diffuse neutrophil infiltrate in both control and rsFc gamma RII-treated rats, however the perivascular infiltrate and the red cell extravasation was less intense in the rsFc gamma RII-treated group. It is likely that complement activation leads to neutrophil chemotaxis, but neutrophil activation via Fc gamma RII, which results in inflammatory mediator release, is inhibited. The data indicate that rsFc gamma RII is a potential therapeutic agent for the treatment of antibody or immune complex-mediated tissue damage

Demonstration of 2 Ovarian Tumor Associated Antigens in Primary and Metastatic Breast, Gastric, and Colonic Tumors

The presence of two ovarian tumor-associated antigens (TAAs) defined by monoclonal antibodies BC3 and CC4 in primary and metastatic breast, gastric, and colonic malignant tumors was determined by immunohistochemistry. The BC3 TAA was present in all gastric, colonic, and breast tumors, typically with a large proportion of tumor cells expressing antigen. The CC4 TAA was present in most of these tumors, generally with a lower proportion of tumor cells expressing antigen compared with that of BC3. Both of these TAAs were found in some epithelial cells in normal breast, stomach, and colon, however, the location of the two TAAs in normal tissue was different. With the use of both antibodies, an increase in the number of tumor cell-positive lymph nodes was found in patients with breast, gastric, colonic, and ovarian tumors, including detection of micrometastases in nodes from patients considered node negative on purely morphologic grounds. Immunohistochemical detection of micrometastases derived from adenocarcinomas appears to be superior to purely morphologic detection

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for Golgi localization

C1 - Journal Articles RefereedThe Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase

Up to 15-year clinical follow-up of a pilot Phase III immunotherapy study in stage II breast cancer patients using oxidized mannan-MUC1

Background: Targeting antigens to dendritic cell receptors has recently become a popular approach to inducing effective immune responses against cancer antigens. Almost 20 years ago, however, we demonstrated that targeting the mannose receptor on macrophages and dendritic cells leads to strong cellular immune responses. We conducted numerous human clinical trials demonstrating the effectiveness of oxidized mannan–MUC1 (M-FP) in MUC1+ adenocarcinoma patients. In one trial, the 5–8-year follow-up of breast cancer patients vaccinated with M-FP was published previously; we now report here the 12–15-year follow-up. Details regarding the preparation of the vaccine, inclusion and exclusion criteria, immunotherapy and follow-up schedule, were published previously. Results: The follow-up at 12–15 years showed that the recurrence rate in patients receiving placebo was 60% (nine of 15). In those receiving immunotherapy (M-FP), the rate was 12.5% (two of 16). The time of recurrence in the placebo group ranged from 7 to 180 months (mean: 65.8 months) and in the two patients of the vaccine group, the recurrence appeared at 95 and 141 months (mean: 118 months) after surgery. These findings are statistically significant (p = 0.02 for survival and p = 0.009 for percentage of patients cancer-free). All patients injected with M-FP showed no evidence of toxic effects or signs of autoimmunity during the 12–15-year follow-up. Discussion: The preliminary evidence indicates that M-FP is beneficial in the overall survival of early-stage breast cancer patients. This long-term clinical follow-up of patients strongly supports the necessity for a large Phase III study of direct M-FP injection in early-stage breast cancer patients, to evaluate immunotherapy as an adjuvant treatment for breast cancer. </jats:p