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

CD64: An Attractive Immunotherapeutic Target for M1-type Macrophage Mediated Chronic Inflammatory Diseases

“…The third generation of anti-CD64 based fusion proteins are entirely made-up of components of human origin to prevent immunogenicity related clinical drawbacks [ 62 ]. One of such human cytolytic fusion proteins developed consist of the human granzyme B (Gb) effector molecule.…”

The Fc‐alpha receptor is a new target antigen for immunotherapy of myeloid leukemia

“…The cytotoxic efficacy of CD89(scFv)‐ETA' was promising, with EC 50 values ranging from 0.2 to 3 nM. Nevertheless, the control IT based on H22(scFv) achieved a significantly lower EC 50 value of 0.04 nM although this can be explained by the twofold to threefold higher density of CD64 on the surface of the same cells, allowing more ITs to be internalized. There was also only a distant correlation between the number of apoptotic cells and the cell viability, which probably reflects the number of early‐apoptotic cells and metabolically active cells.…”

“…The open reading frame for the anti‐human CD89(scFv) was capped with 5′ Sfi I and 3′ Not I restriction sites using adapted PCR primers. Afterwards, the product was ligated into the pMT vector, already containing the ETA' sequence using the Sfi I and Not I sites . The plasmid was amplified using the Escherichia coli ( E. coli ) strain DH5α (New England Biolabs) and successful cloning was confirmed by test digestion and sequencing.…”

Advances in epidermal growth factor receptor specific immunotherapy: lessons to be learned from armed antibodies

“…Photoimmunotheranostic treatment is a new treatment strategy combining the diagnostic and tumor shrinkage properties of antibody photoimmunoconjugates (APCs), which specifically accumulate into targeted tumors and induce their selective destruction upon irradiation with a specific light source (Figure 4) [9,[156][157][158][159][160]. This novel cancer treatment approach offers promising opportunities in improving cancer detection and monitoring post-treatment responses [161].…”