Unconjugated, toxin-conjugated or radiolabeled antibodies (Ab) recognizing tumor-associated antigens have proven beneficial for solid and hematolymphoid neoplasms. A suitable antigen for targeted therapy could be Prostate Stem Cell Antigen (PSCA), a cell surface-antigen expressed at low levels in normal prostate and over expressed in prostate, pancreatic and bladder carcinoma. Anti PSCA mAb was obtained by hybridoma technology and a recombinant version in single chain format (scFv) was generated by cloning the variable heavy (VH) and light chain (VL) sequences in the expression vector pHEN-2. mAb and scFv ability to recognize native PSCA was assessed by flow cytometry on SW780 (PSCA+) cells. The mAb demonstrated a good specificity and affinity, whereas the scFv showed a lower staining compared to whole mAb. The affinity/avidity of scFv was partially recovered making it divalent by cross-linking myc Tag by pre-incubation with anti myc-Ab. mAb was also able to recognize the glycosylated PSCA on prostate and pancreatic neoplastic tissue lysates by Western Blot. The antibody internalization assay demonstrated that the Ab-PSCA complex was internalized. This property was exploited for intracellular delivering of cytotoxic agents. The sequence coding for the Pseudomonas aeruginosa exotoxin A mutant PE40 was cloned at the 3′-end of the scFv yielding an anti-PSCA recombinant immunotoxin. Analysis of cytotoxic activity and specificity of scFv-PE40 are on-going

Anti PSCA Monoclonal Antibody and scFv-Fragment for Immunotherapy of Prostate, Pancreatic and Bladder Carcinoma

CREMONESE, Giorgia;FRACASSO, Giulio;CINGARLINI, Sara;ANSELMI, Cristina;BOSCAINI, Anita;CETTO, Gianluigi;COLOMBATTI, Marco
2009

Abstract

Unconjugated, toxin-conjugated or radiolabeled antibodies (Ab) recognizing tumor-associated antigens have proven beneficial for solid and hematolymphoid neoplasms. A suitable antigen for targeted therapy could be Prostate Stem Cell Antigen (PSCA), a cell surface-antigen expressed at low levels in normal prostate and over expressed in prostate, pancreatic and bladder carcinoma. Anti PSCA mAb was obtained by hybridoma technology and a recombinant version in single chain format (scFv) was generated by cloning the variable heavy (VH) and light chain (VL) sequences in the expression vector pHEN-2. mAb and scFv ability to recognize native PSCA was assessed by flow cytometry on SW780 (PSCA+) cells. The mAb demonstrated a good specificity and affinity, whereas the scFv showed a lower staining compared to whole mAb. The affinity/avidity of scFv was partially recovered making it divalent by cross-linking myc Tag by pre-incubation with anti myc-Ab. mAb was also able to recognize the glycosylated PSCA on prostate and pancreatic neoplastic tissue lysates by Western Blot. The antibody internalization assay demonstrated that the Ab-PSCA complex was internalized. This property was exploited for intracellular delivering of cytotoxic agents. The sequence coding for the Pseudomonas aeruginosa exotoxin A mutant PE40 was cloned at the 3′-end of the scFv yielding an anti-PSCA recombinant immunotoxin. Analysis of cytotoxic activity and specificity of scFv-PE40 are on-going
PSCA; immunotherapy
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/340889
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