Novel Single-Chain Fv′ Formats for the Generation of Immunoliposomes by Site-Directed Coupling

Abstract

Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active targeting of entrapped compounds to diseased areas. Single-chain Fv fragments (scFv) represent the smallest part of an antibody containing the entire antigen-binding site. They can be coupled in a defined and site-directed manner through genetically engineered cysteine residues, for example, those added at the C-terminus. Here, we have performed a comparative analysis of various scFv' variants with cysteine residues present at the end of a C-terminal extension of varying length and composition (HC variants) or introduced in the linker sequence connecting the variable heavy and light chain domain (LC variants). Using a scFv fragment directed against fibroblast activation protein (FAP) as a model antibody, we could show that all variants can be employed for the generation of active immunoliposomes, although the presence of three additional cysteine residues in one scFv' molecule resulted in decreased binding of immunoliposomes compared to that of immunoliposomes generated with scFv' molecules containing only one additional cysteine residue. In order to further improve the scFv' format by reducing the number of additional amino acid residues, we also generated molecules with the hexahistidyl-tag incorporated into the linker sequence together with a cysteine residue either at position 1 or 3 of the linker sequence (LCH variants). These newly designed scFv' molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules to a minimum.

Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody

“…In addition to these parameters, cell compartments such as supernatant, pf, and cytoplasmic fractions (cfs) were analyzed to detect protein content. In previous studies, Messerschmidt et al 34 and Baum et al 35 used pABC4 vector in combination with TG1 bacteria to produce scFv by growing for 3 hours post induction at room temperature (23°C).…”

“…34,37,41,42 Briefly, all lipids were mixed in a round bottom flask and dissolved in chloroform:methanol (9:1 v/v). A thin and uniform lipid film was obtained by evaporating the solvents under pressure for 30 minutes at 42°C.…”

“…The mixture was then extruded 17-20 times under pressure through a series of polycarbonate filters ranging from 200 to 50 nm to obtain a uniform suspension of particles. Size and polydispersity index (PDI) 31,34,41,43 were determined by dynamic light scattering 43 on a Zetasizer ® Nano ZS (Malvern Instruments, Worcestershire, UK). Liposomes were subjected to Rouser phosphate assay 44 where phospholipid was determined and a total lipid amount was calculated after cholesterol correction.…”

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…Site-specific coupling can be achieved in scFv through the introduction of cysteine residues in specific sites within the scFv construct. The location and the number of cysteine residues have been shown to impact the coupling efficiency to liposomes and the binding avidity of the resultant SIL [72].…”

“…The stability of scFv can also be measured by stressed incubation, where the scFv is incubated in conditions similar to its intended application (e.g., in serum at 37°C) followed by evaluation of the remaining activity or the amount of protein aggregation after the incubation [104]. In addition, dynamic light scattering has been used to assess the thermal stability of scFv [72]. In this method, the incubation temperature is gradually increased until the melting point of the scFv is reached and the intensity of the light scattering dramatically increases.…”