Human anti-PEM (polymorphic epithelial mucin) antibodies produced by specific B-lymphocytes isolated from tumor-draining lymph nodes

PEM is a high molecular weight transmembrane glycoprotein expressed at the apical surface of several siplex epithelia. The extracellular domain of the protein core of PEM is made up of contiguous repetitions of a 20 amino-adds sequence named tandem repeat (TR). Tumor cells of epithelial histotype express new PEM glycoforms with shorter O-linked sugar side chains resulting in the exposition of new antigenic determinants and in an increase of immunogenicity of this self-protein. Experimental and clinical evidence shows that PEM is able to evocate cellular and humoral immune response in patients with PEM-expressing tumors. We first describe a humoral immune response in ovarian cancer patients and established B-cell clones producing human anti-PEM antibodies. These recognize the TR aminoacid sequence APPAH, which is distinct from the one immunodominant in mice, APDTRPAP. Antibody producing B-cell clones were obtained by infection of tumor draining lymph node B-cells. However, EBV methodology presents some limits, such as low efficiency/frequency of immortalization, prevalence of the IgM isotype and shut-off of immunoglobulin production. B-cell expressing anti-TR antibodies were isolated to increase the specificity of the B-cell population and to detect me potential IgG immune response towards this antigen in cancer patients. B-cells expressing membrane bound immunoglobulins were selected form freshly dissected lymph nodes using as a 'catcher' the 60mer peptide corresponding to 3TR, i.e. the minimum number of TRs able to assume the protein conformation and containing every possible linear epitope. The rescue of labelled B-cells was performed using an immunomagnetic method based on biotin-conjugate 60mer peptide and streptavidin-coated magnetic microbeads (Miltenyi). Highly sensitive immunoassay was used to test culture supernatants from such selected EBV immortalized B-lymphocytes for specificity and isotype. The immunoglobulins were detected by their ability to bind the biotin-conjugate 60mer linked to streptavidin-coated plates. Our results show that the described procedure allows the isolation of PEM specific B-lymphocytes. The immunoglobulins produced by these B-cell clones are both of IgM and IgG isotype. These results indicate the presence of a memory humoral immunity against the TR of PEM and confirm the role of PEM in the anti-tumor response of cancer patients.