Monoclonal antibody 16D10 to the C-terminal domain of the feto-acinar pancreatic protein binds to membrane of human pancreatic tumoral SOJ-6 cells and inhibits the growth of tumor xenografts

Abstract

Feto-acinar pancreatic protein (FAPP) characterized by mAbJ28 reactivity is a specific component associated with ontogenesis and behaves as an oncodevelopment-associated antigen. We attempted to determine whether pancreatic tumoral SOJ-6 cells are expressed at their surface FAPP antigens and to examine if specific antibodies directed against these FAPP epitopes could decrease the growth of pancreatic tumors in a mice model. For this purpose, we used specific antibodies against either the whole FAPP, the O-glycosylated C-terminal domain, or the N-terminal domain of the protein. Our results indicate that SOJ-6 cells expressed at their surface a 32-kDa peptide corresponding to the C-terminal domain of the FAPP. Furthermore, we show, by using endoproteinase Lys-C or geldanamycin, a drug able to impair the FAPP secretion, that this 32-kDa peptide expressed on the SOJ-6 cell surface comes from the degradation of the FAPP. Finally, an in vivo prospective study using a preventative tumor model in nude mice indicates that targeting this peptide by the use of mAb16D10 inhibits the growth of SOJ-6 xenografts. The specificity of mAb16D10 for pancreatic tumors and the possibility to obtain recombinant structures of mucin-like peptides recognized by mAb16D10 and mAbJ28 are promising tools in immunologic approaches to cure pancreatic cancers.

Figure 1

Immunodetection of proteins present in extracellular medium and cell lysate of SOJ-6 cells. Subconfluent SOJ-6 cells were cultured in Opti-MEM medium for 24 hours. Then the cell culture medium was withdrawn and the cells were harvested, lysed, and centrifuged to eliminate insoluble materials. Proteins present in the soluble fraction were separated on SDSPAGE and electrotransferred onto nitrocellulose membranes. The membranes were finally probed with the polyclonal antibody to BSDL, pAbL64, in cell culture medium (lane 1) and in the soluble fraction of cell lysate (lane 2). Arrows indicate the apparent molecular mass of detected proteins or peptides.

Figure 2

SOJ-6 surface reactivity with antibodies to FAPP. Panel A: Subconfluent SOJ-6 cells were fixed with paraformaldehyde (3%) then probed with different antibodies pAbL64 (lane 2), mAbJ28 (lane 3) mAb16D10 (lane 4), and pAbAntipeptide (lane 5) before immunofluorescence microscopy analyses. Lane 1 shows the nonspecific binding of secondary FITC-labeled antibody. Panel B: Cell surface antigen expression was analyzed by flow cytometry using pAbL64 (lane 2), mAbJ28 (lane 3), mAb16D10 (lane 4), and pAbAntipeptide (lane 5) and compared to controls (lane 1), which shows the nonspecific binding of secondary FITC-labeled antibodies.

Figure 3

Immunodetection of biotinylated cell surface antigens in SOJ-6 cells. SOJ-6 cells were grown until confluence, and membrane proteins and peptides were biotinylated and lysed. Then biotinylated membrane proteins were purified on streptavidin-agarose affinity column. The material eluted with 0.1 M acetic acid was separated on SDS-PAGE and electrotransferred onto nitrocellulose membranes, which were finally probed with antibodies: antibiotin (lane 1; 0.5 µg of protein), pAbL64 (lane 2; 5 µg of protein), mAbJ28 (lane 3; 5 µg of protein), mAb16D10 (lane 4; 5 µg of protein), pAbAntipeptide (lane 5; 5 µg of protein), and antibodies to Glut-1 (lane 5; 5 µg of protein). Arrow indicates the apparent molecular mass of detected peptides.

Figure 4

Immunodetection of biotinylated cell surface antigens purified on agarose-immobilized antibodies to FAPP. Membrane peptides of SOJ-6 cells were first biotinylated and purified on streptavidin-agarose affinity column (Figure 3). (A) The material eluted with acetic acid was purified on agaroseimmobilized pAbL64 affinity column and the material eluted was separated on SDS-PAGE and electrotransferred onto nitrocellulose membranes. Then membranes were probed with mAbJ28. (B) Conversely, the material eluted with acetic acid was purified on agarose-immobilized mAbJ28 affinity column and the material eluted was separated on SDS-PAGE and electrotransferred onto nitrocellulose membranes. Then membranes were probed with pAbL64. The detection of immunoprecipitated biotinylated peptides was performed using Lumi-Light^plus Western Blotting kit. Arrow indicates the apparent molecular mass of detected peptide.

Figure 5

Effect of geldanamycin on the rate of FAPP secretion by SOJ-6 cells. Subconfluent SOJ-6 cells were incubated in fresh RPMI in the absence (empty column) or presence (dashed column) of 3 µM geldanamycin and further incubated for 6 hours. At the end of incubation, the culture medium was removed. The secretion of FAPP was analyzed by SDS-PAGE and Western blot analysis using pAbL64 (A), and the quantification of this Western blot analysis is performed by the NIH Program (B). The FAPP activity was also determined (C) using 4-nitrophenyl hexanoate as substrate. This experiment was done in triplicate and results are presented as mean ± SD.

Figure 6

Endoproteinase Lys-C treatment of BSDL, secreted FAPP, and recombinant C-terminal peptide of FAPP. (A) FAPP obtained from the serum-free culture medium of SOJ-6 cells was treated (+) or mock-treated (-) with endoproteinase Lys-C (2% by weight), overnight at 37C, then analyzed on SDS-PAGE and Western blot analysis using pAbL64. Lane 1 represents untreated FAPP present in the SOJ-6 cell culture medium. Lane 2 represents mock-treated FAPP of the SOJ-6 cell culture medium. Lane 3 represents FAPP of the SOJ-6 cell culture medium treated with endoproteinase Lys-C. Lanes 4 and 5 represent FAPP of the SOJ-6 cell culture medium treated with endoproteinase Lys-C and immunodetection using mAbJ28 and mAb16D10, respectively. (B) Pure human pancreatic BSDL was treated and analyzed under identical conditions. Lane 1 represents native BSDL, lane 2 represents mock-treated BSDL, and lane 3 represents BSDL treated with endoproteinase Lys-C. Lane 4 represents BSDL treated with endoproteinase Lys-C and immunodetection using mAbJ28. (C) The recombinant C-terminal peptide of FAPP obtained from CHO-C2-F3 cells transfected with the pSecCterFAPP was treated (+) or mocked-treated (-) with endoproteinase Lys-C, overnight at 37°C, then analyzed on SDS-PAGE and Western blot analysis using pAbL64. Lane 1 represents recombinant C-terminal peptide, lane 2 represents mocktreated recombinant C-terminal peptide, and lane 3 represents recombinant C-terminal peptide treated with endoproteinase Lys-C. Lanes 4 and 5 represent recombinant C-terminal peptide treated with endoproteinase Lys-C and immunodetection using mAbJ28 and mAb16D10, respectively. Arrows indicate the apparent molecular mass of detected peptides.

Figure 7

Antitumor effect of mAb16D10 on SOJ-6 xenograft growth rates in a preventative model. SOJ-6 cells were injected subcutaneously into the right flank of 6-week-old nude mice (n = 5) at day 0. Mice were injected intraperitoneally with 0.5 mg of mAb16D10 (assay group, triangle) or with PBS vehicle only (control group, diamond) starting 1 day prior to tumor cell inoculation. Data are expressed as mean tumor volume ± SEM.