Anti-CD20 single chain variable antibody fragment–apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas

Abstract

A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

Use of Single-Chain Antibody Derivatives for Targeted Drug Delivery

“…Fluorescence-activated cell sorting analysis revealed that the fusion peptide bound to CD20-positive cell lines through its anti-CD20 scFv section. The fusion peptide was unable to bind the CD20-negative cell line (Jurkat) substantially (71). These results all …”

“…The major drawback correlated with rHDL-mediated drug delivery is the lack of specificity for the cells to which the drug is going to be delivered. Crosby and colleagues used an engineered protein scaffold to overcome the lack of specificity associated with rHDL-mediated drug delivery ( Figure 3E) (71). They designed a fusion peptide composed of an anti-CD20 scFv and human apo A-I and evaluated its capacity for curcumin delivery to either CD20-positive or CD20-negative cells.…”

Update on Nanotechnology-based Drug Delivery Systems in Cancer Treatment

“…NDs are distinguishable from conventional liposomes or vesicles in that they have scaffold proteins constituting an intrinsic structural element of ND; diameters ranging from 8-20 nm versus 60-250 nm for liposomes; and most importantly, unlike liposomes, ND are fully soluble in aqueous media. Building on this technology, Ryan et al (9) created a chimera based on an apolipoprotein-antibody fusion to direct ND particles to a specific cell surface antigen. Certain B-cell lymphomas including Mantle cell lymphoma (MCL), express CD20 as a cell surface protein (10).…”

“…Later, Crosby et al (9) demonstrated that the α-CD20 scFv-apoA-I-ND bound to CD20-positive non-Hodgkin's lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20.…”

“…Also, as stated earlier, based on HDL structure, Crosby et al (9) have built a new technology demonstrating a variety of applications including drug delivery to cancer cells. The nanodisk (ND) technology (27), is a formulation of HDL, and it contains scaffold protein apoA1, as well as a CD20-specific single chain variable fragment antibody which binds and specifically delivers hydrophobic anti-cancer drugs to CD20 expressing cells.…”