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Preparation and characterization of the antibody recognizing AMACR inside its catalytic center

Boris V Popov1, Gleb I Sutula1, Nikolay S Petrov1

  • 1Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia.

International Journal of Oncology
|January 19, 2018
PubMed

Insights

Researchers developed new mouse monoclonal antibodies targeting Alpha-methylacyl-CoA racemase (AMACR), an enzyme overexpressed in cancers. These antibodies bind to the AMACR catalytic center and can enter live cells, showing potential for anticancer therapy development.

Area of Science:

  • Biochemistry and Molecular Biology
  • Immunology
  • Cancer Research

Background:

  • Alpha-methylacyl-CoA racemase (AMACR) is crucial for fatty acid β-oxidation.
  • AMACR overexpression is linked to various carcinomas, making it a potential cancer target.
  • Inhibiting AMACR activity can suppress cancer cell growth.

Purpose of the Study:

  • To generate and characterize novel mouse monoclonal antibodies against human AMACR.
  • To identify the epitopes recognized by these antibodies.
  • To investigate the cellular localization and potential therapeutic efficacy of AMACR antibodies.

Main Methods:

  • Preparation and characterization of 20 mouse monoclonal antibodies against human AMACR.
  • Phage display biopanning to select mimotopes recognized by antibodies.
  • Computational analysis using Pepitope and AMACR crystal structure to determine epitope location.
  • Intracellular delivery of antibodies into HeLa cells using cationic lipid-based PULSin reagent.
  • Confocal microscopy for co-localization studies with peroxisomes.
  • Cell growth inhibition assays on HeLa and T98G cells.

Main Results:

  • Ten phage mimotopes were selected for each antibody type.
  • Epitopes recognized by AMACR antibodies are conformation-dependent sequences within the catalytic center.
  • Delivered antibodies co-localized with peroxisomes in live HeLa cells.
  • The in-house 6H9 antibody showed limited co-localization and did not inhibit cell growth.
  • Commercial 63340 antibody showed higher co-localization.

Conclusions:

  • Novel AMACR-specific antibodies were successfully generated and delivered into live cells.
  • Antibody epitopes are located within the AMACR catalytic center, suggesting potential for catalytic inhibition.
  • AMACR antibodies warrant further investigation as a potential therapeutic strategy for anticancer treatment.

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