No abstract

A great challenge in nanodiagnostics is the identification of new strategies aimed to optimize the detection of primary breast cancer and metastases by the employment of target-specific nanodevices. At present, controversial proof has been provided on the actual importance of surface functionalization of nanoparticles to improve their in vivo localization at the tumor. In the present paper, we have designed and developed a set of multifunctional nanoprobes, modified with three different variants of a model antibody, that is, the humanized monocolonal antibody trastuzumab (TZ), able to selectively target the HER2 receptor in breast cancer cells. Assuming that nanoparticle accumulation in target cells is strictly related to their physicochemical properties, we performed a comparative study of internalization, trafficking, and metabolism in MCF7 cells of multifunctional nanoparticles (MNP) functionalized with TZ or with alternative lower molecular weight variants of the monoclonal antibody, such as the half-chain (HC) and scFv fragments (scFv). Hence, to estimate to what extent the structure of the surface bioligand affects the targeting efficiency of the nanoconjugate, three cognate nanoconstructs were designed, in which only the antibody form was differentiated while the nanoparticle core was maintained unvaried, consisting of an iron oxide spherical nanocrystal coated with an amphiphilic polymer shell. In vitro, in vivo, and ex vivo analyses of the targeting efficiency and of the intracellular fate of MNP-TZ, MNP-HC, and MNP-scFv suggested that the highly stable MNP-HC is the best candidate for application in breast cancer detection. Our results provided evidence that, in this case, active targeting plays an important role in determining the biological activity of the nanoconstruct.

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.

No abstract

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 30% of human breast cancers. One of the mechanisms by which tumor cell proliferation can be inhibited consists in hampering HER2 dimerization by targeting its extracellular domain with specific antibodies. In recent clinical practice, a valuable alternative to entire IgGs resides in the use of smaller molecules, such as single-chain variable fragments (scFv), developed for selective molecular targeting. In this paper, we report on the production and purification of a soluble anti-HER2 scFv antibody secreted by Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at the 3'-end was inserted into the expression vector pPICZα and transformed in P. pastoris. The highest expression level was obtained in presence of 0.5% methanol and 0.8% glycerol in the culture medium after 48 h of induction. The use of P. pastoris proved very valuable as an expression system, allowing the isolation of 10 mg/L of highly purified antibody, remarkably higher than previously reported data. The functionality of purified anti-HER2 scFv was assessed by cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer cells, showing good affinity and high selectivity for the target membrane receptor. These findings confirm that P. pastoris is a suitable host for high level expression of antibody fragments and highlight the potential role of scFv800E6 in diagnostic and therapeutic application.