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

Abstract

Many 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.

Impact of Tumor HER2/ERBB2 Expression Level on HER2-Targeted Liposomal Doxorubicin-Mediated Drug Delivery: Multiple Low-Affinity Interactions Lead to a Threshold Effect

“…Initial binding enhances the diffusive rate of encounter with additional HER2 by factor a. Each individual receptorscFv interaction is characterized by dissociation constant K D_LR (28). Because of the large size of liposomes (100 nm in diameter), total binding is limited by physical space on the cell surface.…”

“…Second, targeting of HER2 results in preferential accumulation of doxorubicin within HER2-positive tumor cells and reduced nonspecific uptake by macrophages and other phagocytic cells (26). Previous work has also shown that HER2-tPLD has little or no uptake into human cardiomyocytes, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of PLD (22) We and others have shown that targeting HER2-overexpressing cells with HER2-tPLD results in superior activity relative to PLD in preclinical models expressing high levels of HER2 (22,28,29). Correspondingly, the absence of a targeting effect has been shown in HER2-negative models where activity was similar to PLD (29).…”

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…Selfefficient internalization of antibodies against Her2/neu has been described both for trastuzumab (Herceptin) [48][49][50] and F5. 51 Other receptors that are known to induce internalization are the EGF 52 and transferrin receptors. 53 Direct selection of internalizing antibodies can be made from phage display libraries, as not all antibodies against a selected target will promote internalization.…”

Nuclisome—targeting the tumor cell nucleus

“…For targeting against EGFR we have employed the natural ligand EGF as targeting agent, whereas for targeting against HER2 a single-chains fragment (F5) [31] have been used.…”