Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model

Abstract

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.

Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

“…Therefore, the enzymatic activity of each construct was determined by a colorimetric assay (13), and the relative activity to Gb-2.44 was calculated. All IC 50 s were corrected by this factor ( Table 1).…”

“…The concept of recombinant immunotoxins has been extensively investigated in the field of targeted tumor therapy with various bacterial and human effector domains (30)(31)(32), including Gb (11,13,27,33). After binding to a disease-specific cell surface antigen, these immunotoxins are internalized, e.g., by receptor-mediated endocytosis, released from endosomal compartments into the cytoplasm, and efficiently kill the malignant cell by their catalytic activity.…”

“…Activity was restored by the enzymatic removal of this peptide using 0.02 U of recombinant enterokinase (Novagen; Merck) per g of protein (12). The restored enzymatic activity was confirmed using a colorimetric activity assay (13). Parasite growth was specifically inhibited by activated Gb, with a half-maximal inhibitory concentration (IC 50 ) of 1,590 nM (95% confidence interval [95% CI], 1,197 to 2,112 nM, calculated using the Hill equation in GraphPad Prism version 5).…”

Human microtubule‐associated protein tau mediates targeted killing of CD30+ lymphoma cells in vitro and inhibits tumour growth in vivo

“…In addition to human granzyme B, human RNases, such as angiogenin, have been used to replace nonhuman toxins (Hetzel et al, 2008;Stahnke et al, 2008;Mathew & Verma, 2009;Schiffer et al, 2013). The specific cytotoxicity of human angiogenin towards CD30-overexpressing HL-derived cell lines has been demonstrated by fusion with the CD30 ligand (Huhn et al, 2001).…”

“…In 2008, an entirely human granzyme B-based CFP directed against CD64 was found to be toxic for an acute myeloid leukaemia (AML)-related cell line and primary AML cells with IC 50 values ranging between 1Á7 and 17 nmol/l (Stahnke et al, 2008). Recently, our group also reported on an improved version of granzyme B, which shows resistance against the endogenous inhibitor serpin B9 and thus overcomes one potential escape strategy of CD30 + lymphoma cells (Schiffer et al, 2013). In addition to granzyme B, the human RNase angiogenin was shown to be specifically cytotoxic towards CD30-overexpressing HL-derived cell lines, when fused to the CD30 ligand (Huhn et al, 2001).…”