Targeted delivery of anti-CD19 liposomal doxorubicin in B-cell lymphoma: A comparison of whole monoclonal antibody, Fab′ fragments and single chain Fv
Introduction
Conventional anticancer drugs have many adverse effects resulting from non-selective toxicity and distribution of the drug to normal cells. Liposomes were one of the first nanoparticulate drug delivery systems to show increased delivery of small molecule anticancer drugs to solid tumors [1], [2]. Drug-loaded liposomes with diameters in the range of 100 nm can accumulate in solid tumors via the enhanced permeability and retention (EPR) effect [3], [4], which occurs when nanoparticles extravasate from the circulation into tumors through gaps in the tumor vasculature endothelium [5]. Once there, the liposomes release their contents at a rate that is determined by the physical properties of the liposome and the drug [6]. The ability of liposomes to localize in tumors via the EPR effect depends in part on their having long circulation half-lives (T1/2 on the order or 24 h or longer), which can be achieved by grafting polyethylene glycol (PEG) to the surface of the liposomes (i.e., Stealth® liposomes, SL). A Stealth® (PEGylated) liposomal formulation of the anticancer drug doxorubicin (DXR), Doxil/Caelyx®, which is approved in the treatment of Kaposi's cancer and ovarian cancer, has been in clinical use for over a decade [7], [8].
Stealth® liposomal drugs can be specifically targeted to cancer cells by the surface conjugation of one or more of a variety of ligands against tumor antigens that are uniquely expressed or over-expressed on the tumor cells. Ligands used for targeting include whole monoclonal antibodies (mAb) or their fragments, e.g., Fab′ or single chain Fv (scFv) [9], [10]. Although no immunoliposomes are yet in clinical use, extensive pre-clinical research activity is taking place in this area.
Previously published studies from this laboratory have shown that treatment of murine models of human B lymphoma with Stealth® immunoliposomal doxorubicin (SIL–DXR), targeted via the FMC63 anti-CD19 mAb or its Fab′ fragment, resulted in increased survival times relative to free DXR or untargeted Stealth® liposomal DXR (SL–DXR) [11]. The CD19 antigen is an attractive target for delivery of liposomal anticancer drugs since it is an internalizing antigen exclusive to B cells that is expressed in most types of B-lymphoid malignancies [12]. Internalization of the antibody–CD19 complex and its recycling back to the cell surface from the endosomal compartment has been shown to be important for delivery of liposomal DXR [13], [14].
At the pre-clinical development stage of antibody-targeted liposomes it is important to have a clear understanding of the advantages and disadvantages of the use of whole mAb vs. Fab′ or scFv fragments as targeting ligands. Factors to be considered include the ease of production, yield, ease of purification, stability, affinity and avidity, immunogenicity and pharmacokinetics/biodistribution (PK/BD) [15]. Improved circulation half-lives and therapeutic effects have been observed when SIL conjugated with anti-CD19 Fab′ fragments were compared to SIL conjugated with the parental whole mAb, since Fab′-targeted liposomes avoid Fc-mediated clearance [11], [16], [17]. These experiments have now been extended to include a comparison with scFv fragments, since the smaller fragments may have advantages such as further reductions in immunogenicity and ease of production. The present study compared SIL targeted via the anti-CD19 HD37 mAb, its Fab′ fragment and an scFv construct, all of which bind to the same epitope on CD19. The current study examined the in vitro binding and cytotoxicity, the in vivo pharmacokinetics and biodistribution (PK/BD), and therapeutics of all three formulations of anti-CD19 SIL, compared to untargeted liposomes.
Section snippets
Materials
Hydrogenated soy phosphatidylcholine (HSPC) and methoxypoly(ethylene glycol) (MW 2000), covalently linked to distearoylphosphadidylethanoloamine (mPEG2000-DSPE), were generous gifts from ALZA Corporation, Inc. (Mountain View, CA). Cholesterol (Chol) and maleimide-derivatized PEG2000-DSPE (Mal-PEG2000-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Bio-Rad Protein Assay Reagent was purchased from Bio-Rad Laboratories (Mississauga, ON). 2-iminothiolane (Traut's Reagent), RPMI 1640
Results
In some initial experiments with the scFv fragment stored at − 20 °C, the scFv-targeted liposomes failed to bind to B cells and it was suspected that the fragment was not stable to frozen storage. This was confirmed by FACS (not shown). In an attempt to increase the storage stability, and in the interests of simplifying the preparations of the SIL, immediately after purification of the scFv, we coupled it to Mal-PEG2000-DSPE micelles before storage at − 20 °C. This resulted in retention of the
Discussion
For the clinical development of immunoliposomal anticancer drugs, it is important to compare different antibody constructs in order to determine the construct that has the most beneficial combination of properties leading to ease of production, while not compromising the therapeutic activity and toxicity profiles. To the best knowledge of the authors, this is the first study to compare the PK and therapeutic effects of SIL–DXR targeted by three different antibody constructs with identical
Conclusions
This study has shown that targeting SIL–DXR with HD37-CCH or HD37 Fab′ is at least as effective as SIL–DXR targeted with HD37 mAb in an animal model of B-cell malignancies, in spite of significant differences in pharmacokinetics. This suggests that the choice of the targeting construct can be based, in part, on other considerations such as ease of production and purification, suitable levels of stability and reduced immunogenicity. Single chain Fv are expected to be preferred over Fab′ and mAb
Acknowledgements
The authors thank Dr. Bernd Doerken of the Berlin Humboldt University for permission to use the HD37 mAb, obtained via Dr. Ellen Vitetta of the University of Texas Southwestern Medical Center and Dr. Georg Fey of the University of Erlangen-Nuremberg. The authors also thank Dr. Sergei Kiprianov of Affimed Therapeutics for providing the HD37 scFv constructs, and for helpful discussions. Dr. Georg Fey provided much support and helpful discussions for this project. The authors thank Elaine Moase
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