S. Shahinian et Jr. Silvius, A NOVEL STRATEGY AFFORDS HIGH-YIELD COUPLING OF ANTIBODY FAB' FRAGMENTS TO LIPOSOMES, Biochimica et biophysica acta. Biomembranes, 1239(2), 1995, pp. 157-167
A new assay for the production of reactive sulfhydryl-bearing antibody
Fab' fragments has been utilized to develop conditions affording high
efficiencies of coupling of mouse and rabbit IgG-derived Fab' fragmen
ts to lipid vesicles containing maleimidyl-functionalized phospholipid
s. Cysteine and mercaptoethylamine, but not dithiothreitol, reduce ant
ibody F(ab')(2) to Fab' fragments in very good yields under conditions
where overreduction to heavy and light chains is minimized. Surprisin
gly, however, a large fraction of the Fab' fragments generated under t
hese conditions can lack maleimide-reactive sulfhydryl groups, as demo
nstrated using a maleimidyl-poly(ethylene glycol) conjugate to shift s
electively the electrophoretic mobility of the reactive sulfhydryl-bea
ring Fab' fragments, After modification of F(ab'), reduction condition
s specifically to maximize the yield of the latter fraction, it is pos
sible to achieve high and very reproducible coupling of functional Fab
' fragments to liposomes (equivalent to coupling of ca, 70% of total i
nput protein and almost 100% of the reactive sulfhydryl-bearing Fab' f
raction), A novel phospholipid-poly(ethylene glycol)-maleimide 'anchor
' allows particularly efficient coupling of Fab' fragments to liposome
s, even using relatively low liposome concentrations and molar percent
ages of the liposome-incorporated 'anchor' species. These results demo
nstrate that with appropriate optimization of the conditions for Fab'
production and liposome coupling, Fab' fragments can be coupled to lip
osomes with efficiencies comparable to or exceeding those reported for
coupling of intact antibodies. These results should facilitate the wi
der use of Fab' fragments asa potentially advantageous alternative to
intact antibodies for liposomal targeting in various applications.