Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures

Abstract

Several coupling methods for binding antibodies (Ab) to liposomes have previously been developed. We were interested in examining if some of these methods would be suitable for attaching Ab to long-circulating formulations of liposomes (SL), sterically stabilized with poly(ethylene glycol) (PEG). We studied three 'classical' coupling methods in which Ab was attached at the bilayer surface of SL, and two new coupling methods in which Ab was attached at the PEG terminus. Parameters examined including binding efficiency, antibody surface density, the ability of the immunoliposomes to remote-load the anticancer drug doxorubicin, and the specific binding of the resulting immunoliposomes to target cells. The non-covalent biotin-avidin coupling method resulted in low Ab densities at the cell surface, as did a coupling in method in which maleimide-derivatized Ab was attached to the liposome surface through a thiolated phospholipid incorporated into the liposomes. The low levels of Ab achieved in these method was likely due to interference by PEG with the access of the Ab to the liposome surface. However, when a maleimide-derivatized Ab was coupled to thiolated PEG, moving the coupling reaction away from the liposome surface, very high coupling efficiencies were achieved, and these immunoliposomes achieved good specific binding to their target cells. Oxidizing the Fc region of the Ab and coupling it to the PEG terminus through a hydrazone bond was a less efficient coupling method, but had the advantage of retaining Ab orientation. Efficient remote-loading of doxorubicin was found for immunoliposomes in which Ab was attached at the PEG terminus.

“SMART” Drug Delivery Systems: Double-Targeted pH-Responsive Pharmaceutical Nanocarriers

“…Monoclonal antibody (mAb) 2G4 was produced and purified in our laboratory. Also, pNP-PEG 3400 -PE was synthesized and purified according to an established method in our laboratory (27) Developing the method of coupling oxidized antibody to the PEG terminus through a hydrazone bond as suggested by Hansen et al (28), we devised our own scheme of conjugate reaction for synthesis of pH-cleavable PEG-PE. The reaction was performed in two steps: first, the activation of mPEG 2000 -CHO with PDPH, and second, the conjugation of 1,2-dipalmitoylsn-glycero-3-phosphothioethanol (sodium salt) (PE-SH) to activated mPEG-CHO.…”

Development of a Platform of Antibody-Presenting Liposomes

“…The affinity coupling of IgGs to ZZ domains presents several major advantages; not only it is highly efficient and complete, but it involves un-modified IgGs, avoids possible alteration by chemical modification, and maintains an optimal orientation of IgGs towards their target site [20,21]. In comparison, the covalent coupling of IgGs to lipid hydrazides via carbohydrate residues located in Fc domains constitutes another approach allowing the control of the IgG orientation, yet with low yield [15]. Another advantage of the non-covalent binding of Anx5ZZ to IgGs is that the reaction is complete in near stoichiometric conditions and thus does not require purification procedures, in contrast to most methods of protein derivatization which require an excess of crosslinkers in order to achieve a satisfactory yield, and consequently a purification step for eliminating un-reacted molecules.…”

“…The Anx5ZZ fusion protein constitutes therefore a simple, fast and versatile adaptor system for linking IgGs to liposomes, in a plug-and-use fashion. In comparison, several studies have reported variable coupling yields (from 100 to 10%) for the formation of immunoliposomes by covalent conjugation [15,[30][31][32], together with the need of excess lipid reactive groups to reach reasonable coupling rates.…”

“…This strategy of covalent coupling presents however several drawbacks, principally the lack of control of the site of protein modification which involves commonly their most accessible and/or most reactive protein amino groups, so that their molecular recognition properties may be altered due to chemical modification and/or non-optimal orientation [14]. The only method which ensures a favorable IgG orientation and preserves their binding domains involves IgG anchoring via carbohydrate residues located in their Fc domain to lipid molecules exposing hydrazide groups; this strategy presents however a low efficiency [15]. Furthermore, chemical modification of proteins requires often conditions which may be detrimental for protein integrity, such as low or high pH or the use of oxidants.…”

Immunoliposomes: A Multipurpose Strategy in Breast Cancer Targeted Therapy

“…The linking of antibodies to liposomes has been intensely studied from different perspectives (see review (Hansen, Kao et al, 1995)). Most of these techniques have been used in different cellular and animal models.…”