Antibody Internalization Studied Using a Novel IgG Binding Toxin Fusion

J Immunol Methods. 2007 Apr 10;321(1-2):41-59. doi: 10.1016/j.jim.2007.01.008. Epub 2007 Feb 6.

Abstract

Targeted therapy encompasses a wide variety of different strategies, which can be divided into direct or indirect approaches. Direct approaches target tumor-associated antigens by monoclonal antibodies (mAbs) binding to the relevant antigens or by small-molecule drugs that interfere with these proteins. Indirect approaches rely on tumor-associated antigens expressed on the cell surface with antibody-drug conjugates or antibody-based fusion proteins containing different kinds of effector molecules. To deliver a lethal cargo into tumor cells, the targeting antibodies should efficiently internalize into the cells. Similarly, to qualify as targets for such drugs newly-discovered cell-surface molecules should facilitate the internalization of antibodies that bind to them. Internalization can be studied be several biochemical and microscopy approaches. An undisputed proof of internalization can be provided by the ability of an antibody to specifically deliver a drug into the target cells and kill it. We present a novel IgG binding toxin fusion, ZZ-PE38, in which the Fc-binding ZZ domain, derived from Streptococcal protein A, is linked to a truncated Pseudomonas exotoxin A, the preparation of complexes between ZZ-PE38 and IgGs that bind tumor cells and the specific cytotoxicity of such immunocomplexes is reported. Our results suggest that ZZ-PE38 could prove to be an invaluable tool for the evaluation of the suitability potential of antibodies and their cognate cell-surface antigens to be targeted by immunotherapeutics based on armed antibodies that require internalization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases / genetics
  • ADP Ribose Transferases / metabolism*
  • ADP Ribose Transferases / pharmacology
  • Antibody Affinity
  • Antigens, Neoplasm / immunology
  • Antigens, Neoplasm / metabolism*
  • Antineoplastic Agents / immunology
  • Antineoplastic Agents / metabolism*
  • Antineoplastic Agents / pharmacology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology
  • Bacterial Proteins / metabolism*
  • Bacterial Toxins / genetics
  • Bacterial Toxins / metabolism*
  • Bacterial Toxins / pharmacology
  • Cell Line, Tumor
  • Cell Membrane / metabolism
  • Cell Survival / drug effects
  • Cloning, Molecular
  • Cytoplasm / metabolism
  • Dose-Response Relationship, Drug
  • Endocytosis*
  • Enzyme-Linked Immunosorbent Assay
  • Exotoxins / genetics
  • Exotoxins / metabolism*
  • Exotoxins / pharmacology
  • Humans
  • Immunoglobulin G / metabolism
  • Immunotoxins / genetics
  • Immunotoxins / immunology
  • Immunotoxins / metabolism*
  • Immunotoxins / pharmacology
  • Inhibitory Concentration 50
  • Microscopy, Confocal
  • Mucin-1
  • Mucins / immunology
  • Mucins / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Staphylococcal Protein A / genetics
  • Staphylococcal Protein A / immunology
  • Staphylococcal Protein A / metabolism*
  • Transfection
  • Virulence Factors / genetics
  • Virulence Factors / metabolism*
  • Virulence Factors / pharmacology

Substances

  • Antigens, Neoplasm
  • Antineoplastic Agents
  • Bacterial Proteins
  • Bacterial Toxins
  • Exotoxins
  • Immunoglobulin G
  • Immunotoxins
  • MUC1 protein, human
  • Mucin-1
  • Mucins
  • Recombinant Fusion Proteins
  • Staphylococcal Protein A
  • Virulence Factors
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa