Background and aims Present antitumoral therapies against solid neoplasms are often not sufficient to control the course of the disease. The expression of tumor associated antigen (TAA) led to the development of monoclonal antibodies (mAbs) based therapies wich use naked or armed antibodies for control of cancer progression. Armed antibodies therapies could use toxin conjugated or radiolabeled reagents. One putative target for conjugated mAb may be the Prostate Stem Cell Antigen (PSCA), a member of the “GPI-anchored protein”. PSCA is expressed at low level in normal prostate tissue and overexpressed in prostate, pancreatic and bladder cancer. We studied an anti PSCA mAbs and its variable portion scFv which could be used “naked” or “armed” with radionuclide/cytotoxic molecule for passive immunotherapy; the conjugation of these mAbs with fluorophore/radioisotope could also allow their use for “in vivo” imaging. Material and methods Anti-PSCA mAb clone1 was obtained by hybridoma technology and characterized by flow cytometry, Elisa and Western blot. Using degenerate primers we amplified the variable heavy (VH) and light chain (VL) sequence of mAb clone1 . VH and VL were cloned in the pHEN2 plasmid. The single chain variable fragment (scFv) was produced in E. coli HB2151 bacterial strain and purified on NiNTA column. scFv clone1 was tesetd by flow citometry and ELISA. An antibody internalization assay was performed with Trypan Blue which is able to quench about 50-60% surface-bound fluorescence. A recombinant immunotoxin was obtained by genetically joining scFv clone1 and the catalytic domain of Pseudomonas Endotoxin A (PE40). Results Anti-PSCA mAb clone1 is able to recognize the native form of PSCA by flow cytometry and shows on SW780 (PSCA+) a MFI (Mean Fluorescence Intensity) of 554 similar to the positive control (a validated anti-PSCA antibody MFI=587). The specificity is confirmed on PC3 cells (PSCA-) MFI=224, similar to the negative control (MFI=189). If SW780 cells are fixed with paraformaldehyde 2% before the cytometric assay, the MFI is 412, 7.5 time higer than negative control (MFI=64); the ability of the mAbs to recognize fixed cells is important for the possible use of mAb clone1 in diagnosis by immunohistochemistry (IHC). Moreover mAb clone 1 is able to recognize hPSCA present in prostate and pancreatic tumor tissue lysates by Western blot, whereas no signal is detected in normal tissues used as negative control. When the scFv derived from clone1 is assayed on recombinant PSCA by ELISA, the scFv/background ratio is 4.25. The cytometric analysis performed on SW780 cells shows the ability of scFv to stain cells if it is pre-incubated with anti-myc antibody; this pre-incubation of the scFv with anti myc- Ab increases the affinity/avidity because the scFv becomes as divalent. The antibody internalization assay shows that after incubation of cells at 37°C the PSCA-Ab complex is internalized and this property can be exploited to deliver intracellularly acting cytotoxic agents. For this reason we are cloning a recombinant scFv-PE40 which will be assessed on SW780 and PSCA negative cells to analyze the cytotoxic activity and specificity. Conclusions We have developed anti-PSCA mAb which could be used for imaging or passive immunotherapy; moreover we have obtained a scFv fragment. Preliminary results show that this scFv can recognize PSCA Ag expressed on tumor cells. We are planing to improve the affinity and to analyze the efficacy of a conjugate scFv-PE40.

Characterization of a new anti PSCA monoclonal antibody and scFv fragment for diagnosis and immunotherapy of prostate, pancreatic and bladder carcinoma.

CREMONESE, Giorgia;FRACASSO, Giulio;CINGARLINI, Sara;ANSELMI, Cristina;GIGLIO, BARBARA;CETTO, Gianluigi;COLOMBATTI, Marco
2008

Abstract

Background and aims Present antitumoral therapies against solid neoplasms are often not sufficient to control the course of the disease. The expression of tumor associated antigen (TAA) led to the development of monoclonal antibodies (mAbs) based therapies wich use naked or armed antibodies for control of cancer progression. Armed antibodies therapies could use toxin conjugated or radiolabeled reagents. One putative target for conjugated mAb may be the Prostate Stem Cell Antigen (PSCA), a member of the “GPI-anchored protein”. PSCA is expressed at low level in normal prostate tissue and overexpressed in prostate, pancreatic and bladder cancer. We studied an anti PSCA mAbs and its variable portion scFv which could be used “naked” or “armed” with radionuclide/cytotoxic molecule for passive immunotherapy; the conjugation of these mAbs with fluorophore/radioisotope could also allow their use for “in vivo” imaging. Material and methods Anti-PSCA mAb clone1 was obtained by hybridoma technology and characterized by flow cytometry, Elisa and Western blot. Using degenerate primers we amplified the variable heavy (VH) and light chain (VL) sequence of mAb clone1 . VH and VL were cloned in the pHEN2 plasmid. The single chain variable fragment (scFv) was produced in E. coli HB2151 bacterial strain and purified on NiNTA column. scFv clone1 was tesetd by flow citometry and ELISA. An antibody internalization assay was performed with Trypan Blue which is able to quench about 50-60% surface-bound fluorescence. A recombinant immunotoxin was obtained by genetically joining scFv clone1 and the catalytic domain of Pseudomonas Endotoxin A (PE40). Results Anti-PSCA mAb clone1 is able to recognize the native form of PSCA by flow cytometry and shows on SW780 (PSCA+) a MFI (Mean Fluorescence Intensity) of 554 similar to the positive control (a validated anti-PSCA antibody MFI=587). The specificity is confirmed on PC3 cells (PSCA-) MFI=224, similar to the negative control (MFI=189). If SW780 cells are fixed with paraformaldehyde 2% before the cytometric assay, the MFI is 412, 7.5 time higer than negative control (MFI=64); the ability of the mAbs to recognize fixed cells is important for the possible use of mAb clone1 in diagnosis by immunohistochemistry (IHC). Moreover mAb clone 1 is able to recognize hPSCA present in prostate and pancreatic tumor tissue lysates by Western blot, whereas no signal is detected in normal tissues used as negative control. When the scFv derived from clone1 is assayed on recombinant PSCA by ELISA, the scFv/background ratio is 4.25. The cytometric analysis performed on SW780 cells shows the ability of scFv to stain cells if it is pre-incubated with anti-myc antibody; this pre-incubation of the scFv with anti myc- Ab increases the affinity/avidity because the scFv becomes as divalent. The antibody internalization assay shows that after incubation of cells at 37°C the PSCA-Ab complex is internalized and this property can be exploited to deliver intracellularly acting cytotoxic agents. For this reason we are cloning a recombinant scFv-PE40 which will be assessed on SW780 and PSCA negative cells to analyze the cytotoxic activity and specificity. Conclusions We have developed anti-PSCA mAb which could be used for imaging or passive immunotherapy; moreover we have obtained a scFv fragment. Preliminary results show that this scFv can recognize PSCA Ag expressed on tumor cells. We are planing to improve the affinity and to analyze the efficacy of a conjugate scFv-PE40.
PSCA; antibody; scFv
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/340885
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