Angiogenin Mutants as Novel Effector Molecules For the Generation of Fusion Proteins With Increased Cytotoxic Potential

Abstract

Human cytolytic fusion proteins (hCFPs) are therapeutically efficacious recombinant polypeptides comprising a target cell-specific binding component and a human effector domain that induces apoptosis. Compared with former generations of immunotoxins, which contain immunogenic cytotoxic domains derived from bacteria or plants, hCFPs contain solely human proteins that do not induce an immune response, thus avoiding the development of neutralizing antibodies. Here, we investigated the suitability of human angiogenin (Ang) mutants as effector domains. We engineered 3 different Ang variants that outperformed the wild-type enzyme by replacing amino acid residues with key roles in the protein's catalytic activity and its interaction with the ribonuclease inhibitor RNH1. The cytotoxic potential of these mutants was compared with wild-type Ang by fusing each to the CD64-specific single-chain variable fragment H22. All hCFPs were successfully expressed in HEK293T cells and purified from the cell culture supernatant by immobilized metal ion affinity chromatography. The Ang mutant-based hCFPs showed normal binding activity towards human interferon-γ-stimulated CD64 HL-60 cells and activated human macrophages isolated from peripheral blood mononuclear cells, but increased cytotoxicity based on reduced affinity towards RNH1 and higher ribonucleolytic activity.

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

“…Further analysis showed that this variant was 10-25 times more cytotoxic than the wild-type enzyme towards the human promyelocytic leukemia cell line HL-60 when fused to H22(scFv) [242]. EC 50 values in the picomolar range were observed against human pro-inflammatory macrophages, which represents a five-fold higher efficacy than ETA′-based immunotoxins [197,242]. This example clearly demonstrates the potential of modified human angiogenin variants that are resistant to the inhibitor RNH1.…”

“…The Ang G85R/G86R variant was shown to have a 10 6 -fold lower affinity for RNH1 than the wild-type enzyme [178]. Further analysis showed that this variant was 10-25 times more cytotoxic than the wild-type enzyme towards the human promyelocytic leukemia cell line HL-60 when fused to H22(scFv) [242]. EC 50 values in the picomolar range were observed against human pro-inflammatory macrophages, which represents a five-fold higher efficacy than ETA′-based immunotoxins [197,242].…”

“…Based on our previous studies, we expected a synergistic improvement by combining the lower affinity for RNH1 with the higher RNase activity. Instead, the resulting enzyme showed extraordinary RNase activity but lower cytotoxicity than the individual Ang G85R/G86R mutant because the affinity for RNH1 was restored to a near wild-type level [242]. The advantage of more efficient substrate cleavage was therefore offset by the greater susceptibility to inhibition.…”

“…Therefore, the removal of Q117 was believed to favor the accessibility and cleavage of tRNA, producing more tiRNA and increasing the cytotoxicity of the enzyme. The angiogenin Q117G variant was therefore fused to H22(scFv) doubling the cytotoxicity of the hCFP compared to the equivalent construct containing wild-type angiogenin [242].…”

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

“…One drawback limiting the therapeutic impact of immunotoxins containing bacterial or plant toxins is their immunogenicity, particularly when repeated administration is necessary. This has been addressed by the development of a new generation of immunotoxins containing human cytolytic enzymes such as the serine protease granzyme B, the ribonuclease angiogenin, or the microtubule-associated protein tau (MAPTau) [21, 27–31]. We have developed mutated versions of these effector proteins to increase their potency, e.g.…”