Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer

J Cancer Res Clin Oncol. 2017 Oct;143(10):2025-2038. doi: 10.1007/s00432-017-2472-9. Epub 2017 Jun 30.

Abstract

Purpose: Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA') was generated.

Methods: We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa.

Results: Alexa Fluor® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells.

Conclusions: Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.

Keywords: Immunotoxin (IT); Prostate cancer; Prostate stem cell antigen (PSCA); Pseudomonas exotoxin A (ETA′); SNAP-tag; Single-chain fragment variable (scFv).

MeSH terms

  • ADP Ribose Transferases / administration & dosage*
  • ADP Ribose Transferases / immunology
  • Antigens, Neoplasm / immunology*
  • Bacterial Toxins / administration & dosage*
  • Bacterial Toxins / immunology
  • Exotoxins / administration & dosage*
  • Exotoxins / immunology
  • Flow Cytometry
  • GPI-Linked Proteins / immunology
  • HEK293 Cells
  • Humans
  • Immunologic Tests
  • Immunotherapy / methods
  • Immunotoxins / administration & dosage*
  • Immunotoxins / immunology
  • Immunotoxins / pharmacokinetics
  • Male
  • Molecular Targeted Therapy
  • Neoplasm Proteins / immunology*
  • Prostatic Neoplasms / diagnosis*
  • Prostatic Neoplasms / immunology
  • Prostatic Neoplasms / therapy*
  • Pseudomonas aeruginosa Exotoxin A
  • Recombinant Fusion Proteins / administration & dosage*
  • Recombinant Fusion Proteins / immunology
  • Single-Chain Antibodies / administration & dosage
  • Single-Chain Antibodies / immunology
  • Virulence Factors / administration & dosage*
  • Virulence Factors / immunology

Substances

  • Antigens, Neoplasm
  • Bacterial Toxins
  • Exotoxins
  • GPI-Linked Proteins
  • Immunotoxins
  • Neoplasm Proteins
  • PSCA protein, human
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • Virulence Factors
  • ADP Ribose Transferases