Cu
rrent limitations to applications of monoclonal antibody (mAb) ta
rgeted isotope gene
rato
rs in
radioimmunothe
rapyinclude the low mAb labeling yields and the nonspecific
radiation of no
rmal tissues by nonta
rgeted
radioimmunoconjugates (RIC). Radiotoxicity occu
rs in no
rmal o
rgans that metabolize
radiolabeled p
roteins andpeptides, p
rima
rily live
r and kidneys, o
r in
radiosensitive o
rgans with p
rolonged exposu
re to the isotope f
rom theblood, such as the bone ma
rrow. Actinium-225 nanogene
rato
rs also have the p
roblem of
released
![](/images/gifcha<font color=)
rs/alpha.gif" BORDER=0>-emittingdaughte
rs. We developed two new bifunctional chelating agents (BCA) in o
rde
r to add
ress these issues. Thiol-maleimide conjugation chemist
ry was employed to inc
rease the efficiency of the mAb
radiolabelings by up to8-fold. In addition, one bifunctional chelating agent inco
rpo
rated a cleavable linke
r to alte
r the catabolism of the
![](/images/gifcha<font color=)
rs/alpha.gif" BORDER=0>-pa
rticle-emitting mAb conjugate. This linke
r was designed to be sensitive to cathepsins to allow
release andclea
rance of the chelated
radiometal afte
r inte
rnalization of the
radioimmunoconjugate into the cell. We compa
redthe p
rope
rties of the cleavable conjugate (mAb-DOTA-G3FC) to noncleavable const
ructs (mAb-DOTA-NCS and mAb-DOTA-SH). The cleavable RIC was able to
release 80% of its
radioactive payload when incubatedwith pu
rified cathepsin B. The catabolism of the const
ructs mAb-DOTA-G3FC and mAb-DOTA-NCS wasinvestigated in vit
ro and in vivo. RIC integ
rity was
retained at 85% ove
r a pe
riod of 136 h in mouse se
rum invivo
. Both conjugates we
re deg
raded ove
r time inside HL-60 cells afte
r inte
rnalization and in mouse live
r invivo. While we found that the
rates of deg
radation of the two RICs in those conditions we
re simila
r, the amountsof the
radiolabeled p
roduct
residues we
re diffe
rent. The cleavable mAb-DOTA-G3FC conjugate yielded a la
rge
rp
ropo
rtion of f
ragments below 6kDa in size in mouse live
r in vivo afte
r 12 h than the DOTA-NCS conjugate.Biodist
ribution studies in mice showed that the mAb-DOTA-G3FC const
ruct yielded a highe
r live
r dose andp
rolonged live
r retention of
radioactivity compa
red to the mAb-DOTA-NCS conjugate. The accumulation inthe live
r seemed to be in pa
rt caused by the maleimide functionalization of the antibody, since the noncleavablemAb-DOTA-SH maleimide-functionalized cont
rol conjugate displayed the same biodist
ribution patte
rn. These
results p
rovide an insight into the catabolism of RICs, by demonst
rating that the
release of the
radioisotope f
roma RIC is not a sufficient condition to allow the
radioactive moiety to clea
r f
rom the body. The exc
retion mechanismsof
radiolabeled f
ragments seem to constitute a majo
r limiting step in the chain of events leading to thei
r clea
rance.