Human monoclonal antibody developed against a metastasizing paraganglioma: preparation and preclinical studies.

A tumor cell line, named HS, was established from a bone metastasis of a patient with metastasizing paraganglioma. In vitro immunization of normal human peripheral blood mononuclear cells by coculturing with viable HS cells, followed by fusion with mouse myeloma cells, yielded a stable human/murine heterohybridoma producing the highly specific monoclonal anti-body KM-155. This MAB KM-155 is a member of the IgG3 subclass and shows no alpha GAL glycosylation that is specific for mouse but not for human antibodies. In pilot preclinical studies it could be demonstrated that MAB KM-155 is highly efficient in targeting a KM-155 antigen-expressing human tumor developing in nu/nu mice after xenografting. Moreover, the growth of KM-155 antigen-expressing human tumors in nu/nu mice was largely inhibited when the concentration of circulating MAB KM-155 was maintained at a high enough level by serial injections.

Optimizing production of human monoclonal IgG antibodies by in vitro-primed human PBMC: influence of CD56+ NK cell depletion.

Freshly isolated human peripheral blood mononuclear cells (PBMC) were immunomagnetically depleted of CD56+ cells. When these CD56- PBMC populations were cultured in the presence of autologous donor serum, polyclonal activation with IL-2 and pokeweed mitogen (PWM) generally resulted in exclusive production of IgG antibodies. Fusion with SP2/O-Ag14 mouse myeloma cells was highly efficient and yielded a great number of IgG-producing heterohybridomas. These conditions were used for in vitro immunization with viable human HT29 tumor cells. After fusion, an increase in hybridoma clones producing IgG monoclonal antibodies (MAb) with HT29 specificity showing a higher portion of MAb binding to the surface of viable HT29 cells was recorded. This immunizing efficiency was not observed with HT29 membrane protein fractions or HT29 proteins integrated into ISCOM particles. Investigations with human anti-alpha Gal antibodies showed that the IgG antibodies produced by the human/mouse heterohybridomas did not contain the mouse-specific Gal alpha 1-3Gal epitope.

Lack of expression of a 31/33kD surface protein on human colon carcinoma cells is a marker for metastasizing potential.

Two highly metastatic human tumor cell lines, SLU-M1 SLU-M2, were established by in vivo selection in Balb/c-nu/nu mice of SLU-1 xenotransplants derived from an adenocarcinoma of the sigmoid colon. Metastatic spread was screened by transplantation of tissues from various organs of s.c.-tumor-bearing nu/nu mice. A monoclonal antibody, mab ME6H2, prepared against a membrane fraction of HT29 cells, also derived from an adenocarcinoma of the colon, showed high 125I-mab ME6H2 binding only to HT29 and SLU-1 cells, whereas hardly any binding was recorded for SLU-M1 and SLU-M2 cells. All cells of the HT29 and SLU-1 populations exhibited a positive immunofluoresence (IF) but only 1-5% of the SLU-M2 and 10-15% of the SLU-M1 subpopulation. A number of other tumor cell lines did not express the ME6H2 target antigen except for line MCF7, derived from an adenocarcinoma of the breast, which showed an IF positive reaction of 100% of the cells but only 25% of mab binding compared to HT29 and SLU-1 cells. The data indicate that expression of the ME6H2 target antigen is adenocarcinoma-specific and lack of expression is a marker for the metastatic potential of these cells. Mab ME6H2 was rapidly internalized upon binding to viable HT29 cells, resulting in an enhancement of cell growth in vitro and tumor growth in vivo. The mab ME6H2-defined target antigen was isolated from cell lysates by antibody affinity chromatography and was identified as a double band in SDS-PAGE with 31kD and 33kD molecular mass usually present in equal amounts.