Human Cytolytic Fusion Proteins: Modified Versions of Human Granzyme B and Angiogenin Have the Potential to Replace Bacterial Toxins in Targeted Therapies against CD64+ Diseases

Abstract

Targeted therapies for the treatment of cancer, but also inflammation and autoimmune diseases will reduce major side effects accompanied with conventional treatment modalities. The immunotoxin concept uses bacterial or plant toxins, coupled to antibodies or natural ligands targeting cancer cells. Initially, immunotoxins suffered from drawbacks like nonspecific cytotoxicity. Even the third generation of immunotoxins comprised of truncated antibodies and modified effector molecules experienced clinical set-backs due to immune responses. Long-term treatment of cancer and non-life-threatening chronic inflammatory diseases requires their complete ‘humanization’. This lead to evaluating human cytolytic fusion proteins (hCFPs), based on human apoptosis-inducing proteins. Lacking an endogenous translocation domain dramatically reduces the cell-death inducing capacity of such proteins. Here, we report on optimizing hCFPs, based on the anti-CD64 single chain variable fragment H22(scFv), specifically eliminating CD64+ macrophages and malignant progenitor cells. We replaced the bacterial toxin in H22(scFv)-ETA' with the pro-apoptotic human granzyme B or angiogenin. Translocation was promoted by a sophisticated adapter containing a membrane transfer peptide (MTD) flanked by endosomal and cytosolic cleavable peptides, thus achieving in vitro cytotoxic activity comparable to bacterial immunotoxins. We demonstrate for the first time that optimized hCFPs, based on granzyme B or angiogenin, can compete with classical ETA-based immunotoxins.

Keywords

• immunotherapy
• immunotoxin
• humanization
• CD64
• granzyme B
• angiogenin
• endosomal release