Oriented EpCAM presentation improves detection of anti EpCAM antibodies

Proc Amer Assoc Cancer Res, Volume 45, 2004 4588 The Epithelial Cell Adhesion Molecule (EpCAM) is over-expressed on most cancers of epithelial origin and therefore represents a valid target molecule for immunotherapy. IGN101, igeneon’s lead project in phase III studies, targets EpCAM positive cells by active immunization with the mouse mAb17-1A adsorbed to aluminumhydroxide. MAb17-1A is considered to be a mimotopic antibody, because it is specific for EpCAM and at the same time induces anti-idiotypic antibodies that are also specific for EpCAM, be it with low affinity. Using conventional ELISA and BIACore techniques also mAb17-1A is described as a low affinity anti EpCAM antibody. In this report we describe a new set up for the detection of EpCAM reactivity with both ELISA and BIACORE. The methods are using orientated presentation of recombinant EpCAM (rEpCAM), through its carboxyl terminal deca-histidine tag as immobilization site. Surface plasmon resonance data (BIACore) indicated that this orientated presentation of rEpCAM greatly improves mAb17-1A binding, probably by “unshielding” of the binding epitope, and the affinity of mAb17-1A should now be considered to be in the range of 2*10-8 Kd. The BIACORE set-up was transferred to an ELISA plate system and further optimized using a human anti-human EpCAM mAb, spiked into human serum. First results of the rEpCAM ELISA with serum from cancer patients show that also natural occurring anti-EpCAM antibodies are dependent on the correct orientation of the target antigen. We are currently comparing the results of the cancer patients to a control group of normal healthy human donors and in addition we will correlate the anti-EpCAM immune response induced by immunization with IGN101 to the results obtained after sequential affinity purification using a mAb17-1A coated column in the first step and rEpCAM coated CH-Sepharose 4B in the second step. Efforts to specify the epitopes of EpCAM, using overlapping linear peptides, which are recognized by the IGN101-induced immune response, are also ongoing.