D. Kirpotin et al., STERICALLY STABILIZED ANTI-HER2 IMMUNOLIPOSOMES - DESIGN AND TARGETING TO HUMAN BREAST-CANCER CELLS IN-VITRO, Biochemistry, 36(1), 1997, pp. 66-75
Liposomes (70-100 nm) Of 1-paalmitoyl-2-oleoylphosphatidylcholine, cho
lesterol, and poly-(ethylene glycol) (PEG)-modified phosphatidylethano
lamine (PEG-DSPE) were conjugated to Fab' fragments of a humanized rec
ombinant MAb against the extracellular domain of HER2/neu to create st
erically stabilized Immunoliposomes (anti-HER2 SL) as a drug carrier t
argeting HER2-overexpressing cancers, Conjugation employed maleimide-t
erminated membrane-anchored spacers of two kinds: a short spacer, prov
iding attachment of Fab' close to the liposome bilayer, or a long spac
er, with Fab' attachment al the distal terminus of dhe PEG chain. Conf
ocal microscopy and spectrofluorometry of HER2-overexpressing breast c
ancer cells incubated with fluorescently labeled anti-HER2 SL prepared
with either spacer showed binding of liposomes is (8000-23 000 vesicl
es/cell) followed by endocytosis irate constant k(e) = 0.012-0.033 min
(-1)) via the coated-pit pathway, evidenced by intracellular acidifica
tion and colocalization with transferrin. Uptake of anti-HER2 immunoli
posomes by breast cancer cells with low HER2 expression, or after prei
ncubation of cells with free anti-HER2 Fab', was less than 0.2% and 4.
3%, respectively, of the uptake by HER2-overexpressing cells. Increasi
ng PEG-DSPE content (up to 5.7 mel %) in anti-HER2-SL prepared with th
e short spacer decreased liposome-cell binding affinity 60-100-fold, w
hile k(e) decreased only 2-fold; however, when Fab' fragments were con
jugated via a PEG spacer, both binding affinity and k(e) were unaffect
ed by PEG-DSPE content. Cell binding and internalization of anti-HER:!
immunoliposomes increased at higher surface density of conjugated Fab
' fragments, reaching plateaus at similar to 40 Fab'/liposome for bind
ing and similar to 10-15 Fab'/liposome for internalization. Uptake of
anti-HER:! immunoliposomes correlated with the cell surface density of
HER2 and significantly (p < 0.005) correlated with tile antiprolifera
tive effect of the targeting antibody but not with the total level of
cellular HER2 expression. The results obtained were used to optimize i
ll vivo preclinical studies anti-HER2 SL loaded with antineoplastic dr
ugs.