Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis

AbstractMany targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles

An analytical procedure for determining the structures of molecules of the type (PNX2)4

Abstract not availabl

Interaction of clathrin with liposomes: pH-dependent fusion of phospholipid membranes induced by clathrin

AbstractClathrin, dissociated from coated vesicles of bovine brain and purified by gel chromatography, was found to interact with the lipid bilayer as shown by the spontaneous release of encapsulated fluorescent dye in liposome. Clathrin-induced dye release was enhanced at acidic pH in phosphatidylserine-containing vesicles. A strong correlation between dye release and fusion of liposomes was observed. In general, when there was a fast release of encapsulated dye induced by clathrin, a pH-dependent, clathrin-induced fusion was observed. Clathrin did not induce either dye release or fusion of egg phosphatidylcholine liposomes. The self-association of clathrin at low pH diminished the fusogenic activity. Fusion induced by clathrin at low pH c̀ould be stopped at pH above 5.0 and resumed by lowering the pH below 5.0. This suggests that the interaction of clathrin with phospholipid membranes can be regulated by pH

Polyamines as Modulators of Membrane Fusion: Aggregation and Fusion of Liposomes

We have studied the effect of the polyamines (spermine, spermidine, and putrescine) on the aggregation and fusion of large (approximately 100 nm in diameter) unilamellar liposomes in the presence of 100 mM NaCl, pH 7.4. Liposome fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents, and the release of contents was followed by carboxyfluorescein fluorescence. Spermine and spermidine at physiological concentrations aggregated liposomes composed of pure phosphatidylserine (PS) or phosphatidate (PA) and mixtures of PA with phosphatidylcholine (PC) but did not induce any fusion. However, liposomes composed of mixtures of acidic phospholipids, cholesterol, and a high mole fraction of phosphatidylethanolamine could be induced to fuse by spermine and spermidine in the absence of divalent cations. Putrescine alone in the physiological concentration range was ineffective for both aggregation and fusion of these liposomes. Liposomes made of pure PC did not aggregate in the presence of polyamines. Addition of aggregating concentrations of spermine caused a drastic increase in the rate of Ca2+-induced fusion of PA liposomes and a large decrease in the threshold Ca2+ concentration required for fusion. This effect was less pronounced in the case of PS or PA/PC vesicles. Preincubation of PA vesicles with spermine before the addition of Ca2+ resulted in a 30-fold increase in the initial rate of fusion. We propose that polyamines may be involved in the regulation of membrane fusion phenomena accompanying cell growth, cell division, exocytosis, and fertilization. © 1983, American Chemical Society. All rights reserved