Selection of aptamers with high affinity and high specificity against C595, an anti-MUC1 IgG3 monoclonal antibody, for antibody targeting.

Targeting of antibodies has found a number of applications in assays, anti-idiotypic therapies and vaccine design with a number of anti-idiotypic Abs generated and used in clinical applications, and some currently in clinical trials. Meanwhile, aptamers are a novel and particularly interesting targeting modality, with a unique ability to bind to a variety of targets. Aptamers offer unique benefits compared to other targeting agents, due to their high affinity and selectivity, relatively small size and in vitro synthesis, making them attractive alternatives to Abs and peptides. Aptamers have already been selected against a number of Abs for various applications. We now present a novel methodology for the selection of aptamers against Abs, which minimises the number of steps used and results in molecules that bind to the target Ab with high affinity and specificity. We have used the well-characterised anti-MUC1 monoclonal Ab C595 as an exemplar for raising aptamers against Abs. The methodology is based on the adsorption of the Ab to the surface of a PCR tube and the performance of SELEX selections in the PCR tube, based on elution steps resulting from the denaturation of the Ab on the first PCR amplification cycle. After 10 rounds of selection and amplification, selected aptamers have been characterised using a number of techniques, including fluorescence quenching, ELISA and competition ELISA procedures and a FRET type assay. Aptamers were found to bind their target Ab with a higher affinity than its natural antigenic peptide, as observed in fluorescent quenching and FRET experiments. Furthermore, they were able to displace the antigens from the antibody binding pocket in competition assays. This methodology offers the possibility of rapidly selecting aptamers for antibody targeting that could be used as diagnostic, imaging or therapeutic agents, or as recognition units in immunoassays, and can be potentially useful in raising aptamers against other protein targets.

Tissue and serum MUC1 mucin detection in breast cancer patients.

Tumor MUC1 expression as well as levels of MUC1, MUC1 circulating immune complexes (MUC1-CIC) and free antibodies against MUC1 (IgG and IgM-MUC1) were evaluated in 70 breast cancer patients with different stages of disease. Controls included: 135 serum samples from healthy women, normal mammary tissue samples (n = 7) and benign breast disease specimens (n = 6). In all assays, pre- and post-vaccination serum samples from breast cancer patients belonging to a vaccination protocol developed at the Memorial Sloan Kettering Cancer Center (New York, USA) were included as controls. Serum MUC1 was measured through Cancer Associated Serum Antigen test and CA15-3 test. Employing ELISA, MUC1-CIC-IgG/M were measured with either C595 or SM3 monoclonal antibodies (MAb) as catchers and also free antibodies against MUC1 (IgG and IgM) using 100mer peptide as catcher. Employing multivariate statistical analysis, results were correlated with age, tumor type, stage of disease and grade of differentiation. By quantitative immunohistochemistry using three anti-MUC1 core protein MAbs (C595, HMFG2 and SM3), tumor MUC1 was detected in 60/70 (86%) breast cancer specimens which reacted with at least one of these MAbs. High MUCI serum levels were detected in 14/67 (21%); IgG and IgM anti-MUC1 antibodies were found elevated in 32 and 14%, respectively, while IgG-MUC1-CIC-measured with C595 in 42% and IgM-MUC1-CIC in 54%; finally, SM3 was positive in 43 and 18%, respectively. Results of these studies demonstrate that in a group of breast cancer patients, MUC1 was detected both in tissue specimens as well as free in serum samples; furthermore, MUC1 can also circulate complexed with IgG and IgM antibodies; thus an accurate measurement should include free and complexed forms. On the other hand, immunohistochemical studies on breast cancer tissues may contribute to reveal different MUC1 glycoforms.