Nuclear Medicine and Biology
Volume 36, Issue 4 , Pages 395-402, May 2009

A simplified suite of methods to evaluate chelator conjugation of antibodies: effects on hydrodynamic radius and biodistribution

  • Fares Al-Ejeh

      Affiliations

    • Experimental Therapeutics Laboratory, Hanson Institute, Adelaide SA 5000, Australia
    • ,
  • Jocelyn M. Darby

      Affiliations

    • Experimental Therapeutics Laboratory, Hanson Institute, Adelaide SA 5000, Australia
    • ,
  • Benjamin Thierry

      Affiliations

    • Ian Wark Research Institute, University of South Australia, Adelaide SA 5000, Australia
    • ,
  • Michael P. Brown

      Affiliations

    • Experimental Therapeutics Laboratory, Hanson Institute, Adelaide SA 5000, Australia
    • Department of Medical Oncology, Royal Adelaide Hospital Cancer Centre, and School of Medicine, University of Adelaide, Adelaide SA 5000, Australia
    • Corresponding Author InformationCorresponding author. Department of Medical Oncology, Royal Adelaide Hospital, MDP 11, Level 4, East Wing, North Terrace, Adelaide SA 5000, Australia. Tel.: +61 8 8222 4398; fax: +61 8 8222 4358.

Received 13 November 2008; received in revised form 18 December 2008; accepted 13 January 2009. published online 31 March 2009.

Abstract 

Introduction

Antibodies covalently conjugated with chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) are required for radioimmunoscintigraphy and radioimmunotherapy, which are of growing importance in cancer medicine.

Method

Here, we report a suite of simple methods that provide a preclinical assessment package for evaluating the effects of DOTA conjugation on the in vitro and in vivo performance of monoclonal antibodies. We exemplify the use of these methods by investigating the effects of DOTA conjugation on the biochemical properties of the DAB4 clone of the La/SSB-specific murine monoclonal autoantibody, APOMAB®, which is a novel malignant cell death ligand.

Results

We have developed a 96-well microtiter-plate assay to measure directly the concentration of DOTA and other chelators in antibody-chelator conjugate solutions. Coupled with a commercial assay for measuring protein concentration, the dual microtiter-plate method can rapidly determine chelator/antibody ratios in the same plate. The biochemical properties of DAB4 immunoconjugates were altered as the DOTA/Ab ratio increased so that: (i) mass/charge ratio decreased; (ii) hydrodynamic radius increased; (iii) antibody immunoactivity decreased; (iv) rate of chelation of metal ions and specific radioactivity both increased and in vivo, (v) tumor uptake decreased as nonspecific uptake by liver and spleen increased.

Conclusion

This simplified suite of methods readily identifies biochemical characteristics of the DOTA-immunoconjugates such as hydrodynamic diameter and decreased mass/charge ratio associated with compromised immunotargeting efficiency and, thus, may prove useful for optimizing conjugation procedures in order to maximize immunoconjugate-mediated radioimmunoscintigraphy and radioimmunotherapy.

Keywords: Radioimmunoconjugates, Radioimmunoscintigraphy, Radioimmunotherapy, DOTA/antibody ratio, Hydrodynamic radius

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PII: S0969-8051(09)00008-0

doi:10.1016/j.nucmedbio.2009.01.001

Nuclear Medicine and Biology
Volume 36, Issue 4 , Pages 395-402, May 2009

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