Expression of tissue-restricted self-antigens in the thymus, termed promiscuous gene expression, imposes T cell tolerance and protects from autoimmune diseases. This antigen pool also includes various types of tumor-associated antigens (TAA) previously thought to be secluded from the immune system. The scope of promiscuous gene expression has been defined by mRNA analysis at the global level of isolated medullary thymic epithelial cells (mTECs). Information at the protein level on the frequency of mTECs expressing a given antigen, on coexpression patterns, and post-translational modifications is largely missing. We report here promiscuous expression at the protein level of two TAA, MUC1 and CEA, in situ and in purified human mTECs. Both antigens are expressed in 1% to 3% of mTECs, either individually or coexpressed in the same cell. Using a panel of anti-MUC1 monoclonal antibodies recognizing different post-translational modifications, i.e., glycoforms of MUC1, we show that only fully glycosylated forms of MUC1 and the differentiation-dependent glycoforms were detected on mTECs, but not the cancerassociated glycoforms. Our findings imply that MUC1 and CEA are amenable to central tolerance induction, which might, however, be incomplete in case of tumor cell-restricted MUC1 glycoforms. Knowledge of these subtleties in promiscuous gene expression may, in the future, assist the selection of T cell tumor vaccines for clinical trials. [Cancer Res 2007;67(8):3919-26]

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SummaryPresent therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.Keywords: multiple myeloma, Mucin-1, glycoforms, immunotherapy, tumour-associated antigens. Flow cytometryBM samples were prepared for flow cytometry according to standard procedures. All incubations were at room temperature and samples were washed in between. Briefly: following lysis of erythrocytes, cells were incubated with a series of anti-MUC1 mAbs (van Leeuwen et al, 2006) or isotype control (BD Biosciences, San Diego, CA, USA). Subsequently, cells were incubated with a PE-conjugated rabbit anti-mouse IgG antibody (Dako, Glostrup, Denmark), after which unoccupied binding sites were blocked by incubation with normal mouse serum. Finally, cells were stained with CD38-fluorescein isothiocyanate (FITC), CD45-peridinin chlorophyll (PerCP), CD34-allophycocyanin (APC) or CD11c-APC, CD33-FITC (all: BD Biosciences), followed by fixation, and acquisition of 10 6 events for each sample using a FACSsort cytometer (BD Biosciences). Staining intensities for MUC1 were defined by the mean fluorescence intensity (MFI) ratios (MFI specific antibody/MFI isotype control). An MFI ratio that was greater than the average MFI ratio of control cells (CD34 + cells present in BM from MM patients) + 2 standard deviations (SD) was considered as positive for MUC1 expression. Results and discussionThe monoclonal PC causing MM can have an immature and mature phenotype based on their differential expression of CD45 (Schneider et al, 1997;Kumar et al, 2003). As demonstrated in Fig 1A, et al, 1996). BM from all MM patients had a MPC population, while 19 patients also had an IPC subset. The histograms in Fig 1C show that the different glycoforms of MUC1 were detected on IPC as well as MPC, with a similar expression profile. None of the groups 2 and 3 mAbs reacted with control cells or BM cells from normal donors, including non-malignant PC, although a subset of these cells were stained occasionally by antibodies from group 1 (data not shown). Figure 2A demonstrates that MPC from 59% and 36% of the MM patients bound antibodies from groups 2 and 3, respectively, while 73% were positive for group 1 antibodies. A similar trend was observed for IPC although less samples reacted with antibodies from groups 2 and 3 (Fig 2B). Staining of PC by group 2 and/or 3 antibodies was always accompanied by intense recognition by antibodies from grou...

The limited response rate of cancer patients treated with dendritic cell (DC)-based vaccines indicates that vast improvements remain necessary. In many murine tumour models it has been demonstrated that the use of innate triggers (e.g. TLR triggers) in the maturation of DC results in higher efficacy. However, as few of these innate triggers are generated clinical grade, there remains a great necessity to fill the gap between fundamental mouse studies and a clinical trial in humans. In the present study we used a TLR2/4-agonist (FMKp which is available clinical grade) in combination with IFN-y (FIcocktail) in the maturation of elutriated monocyte-derived DC and compared it with the most used DC in current clinical trials (TNF -a/PGE-2, i.e, TP-cocktail). In addition to the assessment of CD4+ T cell polarizing capacity, we compared the quantity and intrinsic quality of induced CD8+ T cells of 2 different DC maturation protocols with all cells from the same donor. Besides differences in the cytokine profile, which could be coupled to increased Thl and Th17 polarization, we demonstrate in this study that FMKp/IFN-y matured DC are twice as effective in inducing cytotoxic T cells against known tumor antigens. Both DCs induced phenotypically equivalent effector memory CD8+ T cells that did not show a significant difference in their intrinsic capacity to kill tumor cells. These findings point to the therapeutic applicability of FI-DC as superior inducers of functional antigen-specific T cells. Their increased chemokine secretion is suggestive of a mechanism by which these DC may compensate for the limited migration observed for all ex vivo cultured DC when applied in patients.Dendritic cells (DC) are very efficient initiators of potent immune responses in vivo. These cells are therefore attractive tools for cellular immunotherapy in the treatment of cancer and viral infections that evade the regular immune surveillance (1-3). As a consequence, a range of methods to generate large amounts of DC ex vivo has become available. They can be generated from a variety of sources, but DC differentiation from monocytes with a growth factor cocktail containing IL-4 and GM-CSF is the current gold standard (4-5).Translating these culture methods into good manufacturing practice guidelines has allowed the introduction of DC immunotherapy into the

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.

Antibodies are commonly used in cancer immunotherapy because of their high specificity for tumor-associated antigens. The binding of antibodies can have direct effects on tumor cells but also engages natural killer (NK) cells via their Fc receptor. Mucin 1 (MUC1) is a highly glycosylated protein expressed in normal epithelial cells, while the under-glycosylated MUC1 epitope (MUC1-Tn/STn) is only expressed on malignant cells, making it an interesting diagnostic and therapeutic target. Several anti-MUC1 antibodies have been tested for therapeutic applications in solid tumors thus far without clinical success. Herein, we describe the generation of fully humanized antibodies based on the murine 5E5 antibody, targeting the tumor-specific MUC1-Tn/STn epitope. We confirmed that these antibodies specifically recognize tumor-associated MUC1 epitopes and can activate human NK cells in vitro. Defucosylation of these newly developed anti-MUC1 antibodies further enhanced antigen-dependent cellular cytotoxicity (ADCC) mediated by NK cells. We show that endocytosis inhibitors augment the availability of MUC1-Tn/STn epitopes on tumor cells but do not further enhance ADCC in NK cells. Collectively, this study describes novel fully humanized anti-MUC1 antibodies that, especially after defucosylation, are promising therapeutic candidates for cellular immunotherapy.

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