Monoclonal Antibodies Recognising Sialyl‐Tn: Production and Application to Immunochemistry

Abstract

In order to develop reagents that can detect the exposed sialyl-Tn antigen (NeuAcα2,6GaINAcα 1-O-Ser/Thr) on tumour-associated mucins, we have prepared monoclonal antibodies (mabs 3C2 and 301, both IgM) against ovine submaxillary mucin (OSM; >98% of glycans as sialyl-Tn). These mabs showed strong reactivity with OSM and bovine submaxillary mucin (BSM; 50% of glycans as sialyl-Tn) but did not react with desialylated OSM or BSM. Sialic acid at I mg/ml did not significantly inhibit mab binding to OSM, suggesting that the linkage to GalNAc may be important for mab binding. 3C2 and 3D I also showed similar reactivity to sialyl-Tn reactive mab Bn.3, and detected Bn.3 capturedOSM in a sandwich ELISA. In Western blotting of mucus from a patient with a mucinous ovarian tumour, the mabs reacted with high molecular weight (>200 kDa) species. In immunohistochemistry, these mabs showed strong reactivity with most cancers of the colon, lung, and stomach, and also some tumours of the ovary and breast. There was only limited reactivity in normal tissue from these sites. The antibodies should be useful reagents for the detection of the sialyl-Tn antigen in human cancers.

Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2–6 sialic acids

“…3 . BSM was used as target antigen since it predominantly contains STn moieties as well as to decrease the possibility of obtaining mAbs only with reactivity against the immunogen OSM 39 . The reactivity response curve was drawn by plotting absorbance against serial dilutions of L2A5 mAb.…”

Sialyl-Tn Polysaccharide A1 as an Entirely Carbohydrate Immunogen: Synthesis and Immunological Evaluation

“…Previous clinical studies of THERATOPE have indicated that the utilization of natural STn-expressing mucins for serological assays could lead to more clinically relevant data compared to that of synthetic STn glycoprotein conjugates. − It is well-known that the linkers used in synthetic conjugates will exhibit a certain level of influence on antibody–antigen recognition events. Both ovine submaxillary mucin (OSM) and bovine submaxillary mucin (BSM) predominantly contain STn moieties , and have become the preferred choices for serological assays. For the specificity of the antibody induced by STn-PS A1 ( 16 ) to be determined, sera from Jax C57BL/6 mice were collected and tested on BSM as shown in Figure .…”

“…Furthermore, the choice of adjuvant can affect the outcome of antibody production. Previous studies have confirmed that monophosphoryl lipid A (MPL), which is the major component of SAS, preserved most of the immunostimulatory activity of lipid A with a significant decrease in toxicity. , MPL is an agonist for TLR-4, which can increase the cellular immune response and is recommended in many types of mice immunizations. − TiterMax Gold (TMG), known as a “depot” adjuvant, is less toxic compared to SAS; however, several studies have reported that use of TMG can lead to inferior antibody production compare to MPL-containing vaccines. , This is most likely a direct result of TMGs ability to protect the antigen from both dilution and rapid degradation and elimination by the host rather than target a specific receptor. Although covalently incorporating specific receptor-based adjuvants directly on vaccine constructs has been done before, the field has yet to completely adopt this strategy due to the lack of a clear understanding.…”

“…63−66 It is well-known that the linkers used in synthetic conjugates will exhibit a certain level of influence on antibody−antigen recognition events. Both ovine submaxillary mucin (OSM) and bovine submaxillary mucin (BSM) predominantly contain STn moieties 18,67 and have become the preferred choices for serological assays. For the specificity of the antibody induced by STn-PS A1 ( 16) to be determined, sera from Jax C57BL/6 mice were collected and tested on BSM as shown in Figure 2.…”

“…71−73 TiterMax Gold (TMG), known as a "depot" adjuvant, is less toxic compared to SAS; however, several studies have reported that use of TMG can lead to inferior antibody production compare to MPL-containing vaccines. 67,68 This is most likely a direct result of TMGs ability to protect the antigen from both dilution and rapid degradation and elimination by the host rather than target a specific receptor. Although covalently incorporating specific receptorbased adjuvants directly on vaccine constructs has been done before, 74 the field has yet to completely adopt this strategy due to the lack of a clear understanding.…”