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.
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.
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.
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.