Improving the Therapeutic Potential of Human Granzyme B for Targeted Cancer Therapy

Abstract

Conventional cancer treatments lack specificity and often cause severe side effects. Targeted therapeutic approaches are therefore preferred, including the use of immunotoxins (ITs) that comprise cell-binding and cell death-inducing components to allow the direct and specific delivery of pro-apoptotic agents into malignant cells. The first generation of ITs consisted of toxins derived from bacteria or plants, making them immunogenic in humans. The recent development of human cytolytic fusion proteins (hCFP) consisting of human effector enzymes offers the prospect of highly-effective targeted therapies with minimal side effects. One of the most promising candidates is granzyme B (GrB) and this enzyme has already demonstrated its potential for targeted cancer therapy. However, the clinical application of GrB may be limited because it is inactivated by the overexpression in tumors of its specific inhibitor serpin B9 (PI-9). It is also highly charged, which means it can bind non-specifically to the surface of non-target cells. Furthermore, human enzymes generally lack an endogenous translocation domain, thus the endosomal release of GrB following receptor-mediated endocytosis can be inefficient. In this review we provide a detailed overview of these challenges and introduce promising solutions to increase the cytotoxic potency of GrB for clinical applications.

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

“…The fusion protein was effectively cleaved at the furin cleavage site within the endosome, translocated to cytosol to provoke cell death, and reduced tumor size in nude mice [17]. An optimal linker can provide many advantages for improved bioactivity and specificity of the fusion protein, potentially facilitating its activity by avoiding steric hindrance or conformational changes [13]. The furinsensitive polyarginine linker we used to connect p28 to GrB, enabled in vivo activation of the fusion protein in tumor cells, which may lead to reduced off-target binding and systemic toxicity of the drug protein [30].…”

“…The effector component of a CFP may include an RNase, kinase, protease or another chemotherapeutic agent. Granzyme B (GrB), a 32-KD human serine protease involved in the granule-mediated apoptosis of virus-infected or transformed tumor cells by effector cells of the innate and adaptive immune system, has several inherent advantages as an immunotoxin, including high cytotoxic efficacy, a broad portfolio of apoptosis inducing mechanisms and low immunogenicity [13]. Yet, natural inhibition by serpin B9, a high isoelectric point resulting in a positive surface charge contributing to non-specific binding to normal cells, and relatively slow release after uptake by the receptor-mediated endocytosis have limited the clinical application of GrB [14].…”

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

“…We recently summarized examples of tumor cells, which have been found to express PI-9 to evade granule-mediated killing and might, therefore, be difficult to treat with granzyme B-based immunotherapeutics [73]. Indeed, several studies have confirmed the direct correlation between PI-9 expression and the loss of granzyme B pro-apoptotic activity and cytotoxic lymphocyte activity in vitro and in vivo.…”

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

“…It is one of the most important effector molecules for the immune surveillance of virus-infected and transformed tumor cells [99] and is exceptional among the apoptosis-inducing enzymes used in immunotherapeutic approaches because it can induce apoptosis at multiple levels using different pathways [96,97,99]. Granzyme B is therefore more likely to overcome anti-apoptosis mechanisms that evolve in tumor cells and this makes it a potent and reliable inducer of apoptosis in hCFPs [100]. The potential of granzyme B in cancer therapy has been demonstrated by coupling it to antibodies and ligands that target CD64, gp240, ErbB2/Her2 (human epidermal growth factor receptor 2), hLHR (human luteinizing hormone receptor), LeY (Lewis Y antigen), EGFR (epidermal growth factor receptor), and CD30 [100][101][102].…”

“…Granzyme B is therefore more likely to overcome anti-apoptosis mechanisms that evolve in tumor cells and this makes it a potent and reliable inducer of apoptosis in hCFPs [100]. The potential of granzyme B in cancer therapy has been demonstrated by coupling it to antibodies and ligands that target CD64, gp240, ErbB2/Her2 (human epidermal growth factor receptor 2), hLHR (human luteinizing hormone receptor), LeY (Lewis Y antigen), EGFR (epidermal growth factor receptor), and CD30 [100][101][102]. Granzyme B-based hCFPs have been proven successful in ex vivo experiments [93] and in a xenograft mouse model [102].…”