Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T-cell migration

MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1-specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription–polymerase chain reaction (RT–PCR) or Northern blot analysis in these cells. The profile of activated T-cell reactivity with different MUC1-specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2-based O-glycans, as opposed to the core 1 structures that dominate in the cancer-associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin-like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites

Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies