, Volume 68, Issue 4, pp 979-990,
Open Access This content is freely available online to anyone, anywhere at any time.
Date: 16 Feb 2011

Targeting granzyme B to tumor cells using a yoked human chorionic gonadotropin

Abstract

Purpose

Luteinizing hormone receptor (LHR) is found in abundance on human ovarian, breast, endometrial and prostate carcinomas but at only low levels on non-gonadal tissues. To selectively kill LHR-expressing tumors, granzyme B (GrB) was linked to a protein in which both chains of human chorionic gonadotropin were yoked together (YCG).

Methods

GrB-YCG was expressed and secreted from insect Sf9 cells. Its GrB enzymatic activity and binding affinity for hLHR were then characterized. The differential cytotoxicity of GrB-YCG versus GrB alone was tested in a panel of LHR-expressing tumor cells by SRB assay, and the mechanisms involved in the cell death were investigated by confocal fluorescence microscopy, flow cytometry, and western blot analysis.

Results

GrB-YCG was successfully expressed and secreted from Sf9 insect cells and purified from cell culture supernatants. The serine protease activity of GrB-YCG was equivalent to that of human recombinant GrB. An in vitro hormone binding assay revealed that the GrB-YCG molecule also retained the ability to bind to the LHR receptor with an affinity similar to that of native hCG. Upon cell binding, GrB-YCG was rapidly internalized into LHR-expressing human ovarian cancer cells and produced selective and potent tumor cell killing by inducing apoptosis through activation of caspase-3.

Conclusions

These results validate LHR as a therapeutic target and indicate that delivery of the human pro-apoptotic enzyme GrB to tumor cells by yoked hCG has substantial selectivity and therapeutic potential for human tumors that express high levels of LHR such as ovarian carcinomas.

I. Kanatani and X. Lin contributed equally to this manuscript.