Targeted human cytolytic fusion proteins at the cutting edge: harnessing the apoptosis-inducing properties of human enzymes for the selective elimination of tumor cells

Abstract

Patient-specific targeted therapy represents the holy grail of anti-cancer therapeutics, allowing potent tumor depletion without detrimental off-target toxicities. Disease-specific monoclonal antibodies have been employed to bind to oncogenic cell-surface receptors, representing the earliest form of immunotherapy. Targeted drug delivery was first achieved by means of antibody-drug conjugates, which exploit the differential expression of tumor-associated antigens as a guiding mechanism for the specific delivery of chemically-conjugated chemotherapeutic agents to diseased target cells. Biotechnological advances have expanded the repertoire of immunology-based tumor-targeting strategies, also paving the way for the next intuitive step in targeted drug delivery: the construction of recombinant protein drugs consisting of an antibody-based targeting domain genetically fused with a cytotoxic peptide, known as an immunotoxin. However, the most potent protein toxins have typically been derived from bacterial or plant virulence factors and commonly feature both off-target toxicity and immunogenicity in human patients. Further refinement of immunotoxin technology thus led to the replacement of monoclonal antibodies with humanized antibody derivatives, including the substitution of non-human toxic peptides with human cytolytic proteins. Preclinically tested human cytolytic fusion proteins (hCFPs) have proven promising as non-immunogenic combinatory anti-cancer agents, however they still require further enhancement to achieve convincing candidacy as a single-mode therapeutic. To date, a portfolio of highly potent human toxins has been established; ranging from microtubule-associated protein tau (MAP tau), RNases, granzyme B (GrB) and death-associated protein kinase (DAPk). In this review, we discuss the most recent findings on the use of these apoptosis-inducing hCFPs for the treatment of various cancers.

Critical Issues in the Development of Immunotoxins for Anticancer Therapy

“…92 To prevent the hypersensitivity and intrinsic immunogenicity associated with nonhuman toxins, human endogenous cytotoxic proteins have been utilized as an alternative payload in fourth-generation ITs. 21,93 Such ITs have been generated based on GrB, 94,95 angiogenin, 96 pancreatic RNase A, 8 microtubule-associated protein tau, 97 and deathassociated protein kinase 98 and have shown in vitro and in vivo antitumor activities. Nonetheless, the cytotoxicity of human enzymatic toxins is compromised by endogenous inhibitors in the cytosol: GrB is suppressed by serine protease inhibitor B9 (serpin B9 or PI9), 22 and pancreatic RNase A by the ribonuclease inhibitor (RI) protein.…”

“…Nonetheless, the in vivo antitumor activity of the human enzyme variantebased ITs is much lower than that of PE38-and DT-based ITs. 21,61,93 Furthermore, human cytotoxic proteins lack a translocation domain for cytosolic access; therefore, their endosomal escape into the cytosol after endocytosis is inefficient, restricting the potency. These limiting factors may explain the absence of clinical trials of human cytotoxic-proteinebased ITs until now.…”

“…For example, granzymes, including GrB, rely on another protein (perforin) for cytosolic access. 93,95 Accordingly, the transfer of human cytotoxic proteins into the cytosol is the most difficult part in the design of ITs. For example, evaluation of RI-evasive human pancreatic RNase A as a toxin fused to several tumor-specific human IgGs has not exerted any antiproliferative or cytotoxic effects against several antigen-positive tumor cell lines even at high concentrations, mainly because of inefficient cytosolic translocation.…”

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

“…Therefore, further ADC refinement should ideally produce immunoconjugates, which are nonimmunogenic and non-toxic in their native administered state, with toxicity only unleashed when internalized into targeted tumor cells. Protein engineering was consequently allowing to replace small molecule toxins by cytotoxic proteins originally derived from plants and bacteria in so-called immunotoxins (ITs) and later by replacing these highly immunogenic protein toxins by human apoptosis inducing enzymes to generate targeted human cytolytic fusion proteins (hCFPs) for cancer therapy [32][33][34]. In spite of their initial preclinical promises, enzymes to be used for the generation of hCFPs might be blocked by the activity of their natural inhibitors upregulated in tumor cells to allow escape from immune responses [32,34,35].…”

P28-Fur-Grb, A Novel Fusion Protein with Enhanced Targeted Cytotoxicity Against Breast Cancer

“…A novel candidate is cytolytic fusion proteins (CFPs) comprised of a ligand targeting cancer cell biomarkers fused to apoptosis-inducing effector proteins [3]. Nearly all previously developed CFPs include antibody fragments as their cellbinding moiety; although, other tumor-specific proteins have also been discovered for this purpose [4,5].…”