Expression, refolding, and purification of recombinant human granzyme B

Characterization of a recombinant granzyme B derivative as a “restriction” protease

Cleavage of fusion proteins on the affinity resins using the TEV protease variant

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.

Cyanogen bromide cleavage of proteins in salt and buffer solutions

Recently we saw a link between activation of Interferon Regulatory Factor (IRF)-3 and expression of IFN-γ, as well as Granzyme B (GrB) from T cells during viral infections in C57Bl/6 (B6) and IRF3KO mice. To investigate this further, we stimulated splenic mononuclear cells (SMC) from B6 and IRF3KO mice in vitro with anti-CD3±anti-CD28± poly I:C, a TLR3/IRF3 agonist. We show during responses with anti-CD3/anti-CD28 that IRF3 is activated within 24 h in B6 SMC and expression of IFN-γ, IL-17 and GrB is impaired in IRF3KO SMC. Adding poly I:C with anti-CD3/anti-CD28 accelerated IRF3 activation and significantly increased IFN-γ, but not IL-17 or GrB, from IRF3KO SMC. Interestingly, Foxp3 expression and percentage of Foxp3+ T cells were significantly lower in unstimulated IRF3KO SMC compared with B6 SMC. To determine the contribution of IRF3 activation in T cells vs APCs, B6 and IRF3KO bone marrow dendritic cells (DCs; 3×104) were mixed in various combinations with WT or IRF3KO purified T cells (3×105), as well as anti-CD3±anti-CD28± poly I:C. After 48 h, IFN-γ, IL-17, and GrB expression was greatest in cultures where both B6 DCs and B6 T cells were mixed. IFN-γ, IL-17 and GrB decreased when either IRF3KO DCs or IRF3KO T cells were part of the mixture. Least production of IFN-γ, IL-17, and GrB occurred when DCs and T cells were from IRF3KO mice. These results suggest that IRF3 in both APCs and T cells is required to achieve optimum production of IFN-γ, IL-17, and GrB during T cell responses. Moreover, the data unexpectedly show that IRF3 is activated during T cell immune responses without a TLR3 agonist. The factor(s) responsible for IRF3 activation during APC/T cell responses is unclear but appears to be a soluble factor whose expression is stimulated through CD28.

The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.

VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature. The serine protease granzyme B (GrB) is capable of inducing intense cellular apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. Here we report on the development and characterization of GrB-Fc-VEGF, which fuses the two domains via an IgG heavy chain Fc-fragment. We hypothesized that this modification, resulting in a relatively high molecular weight (133 kDa vs 80 kDa) would result in improved in vivo circulation and efficacy. VEGF inter-chain dimerization domains were modified such that dimerization occurred only through the Fc domain. GrB-Fc-VEGF121was expressed in HEK-293E cells under serum-free conditions and purified from the media by immobilized metal affinity chromatography (25 mg/L). Western blotting confirmed incorporation of both VEGF121 and GrB moieties into the construct. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of human GrB. In vitro studies of GrB-Fc-VEGF121 suggests improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Thus, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. In vitro cytotoxicity against an expanded panel of cells, internalization, and in vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2396.

Echinococcus granulosus (sensu lato), a cestode that is endemic in Egypt, causes cystic echinococcosis (CE), a significant but neglected zoonotic disease that is prevalent throughout the world. Infected hydatid cysts are classified as fertile or non-fertile based on the presence of protoscoleces; nevertheless, the mechanism of non-fertile CE cysts remains unknown. The study aimed to assess whether granzyme B (GrB) expression and CD4 + /CD8 + could be related to the induction of non-fertile CE cysts. A total of fifty-eight individuals diagnosed with visceral hydatid cysts were selected, and they were further divided according to cyst fertility into fertile and non-fertile. Immunohistochemistry for CD4, CD8, and GrB was done. According to the results, hydatid cysts are common in adults and have no gender preference. The same clinical and laboratory data were shared by patients with fertile and non-fertile cysts (p = 0.186). GrB expression was not impacted by the fibrous deposition inside the hydatid cyst wall (p = 0.85); however, GrB was significantly correlated with the inflammatory density (p = 0.005). GrB expression was also found to be significantly higher in non-fertile cysts (p = 0.04). GrB expression is positively correlated with CD4 and CD8 expression. In conclusion, the expression of GrB in hydatid cysts may exacerbate the inflammatory response and impede cyst fertility while not affecting the fibrous deposition in the cyst wall.

Perforin and granzyme B (GB) are the main constituents of cytotoxic T-lymphocyte granules, and they have important roles in preventing the initiation and progression of cancer. The aim of this study was to compare the expression of CD8(+) /perforin(+) double-staining and GB(+) cells, by immunohistochemistry, in primary oral cavity squamous cell carcinoma (OCSCC), lip squamous cell carcinoma (LSCC), non-dysplastic leukoplakia (LK), dysplastic LK, actinic cheilitis (AC), oral lichen planus (LP) and normal oral mucosa. Our results showed a higher expression of CD8(+) /perforin(+) and GB(+) cells in LSCC when compared with the samples of OCSCC, non-dysplastic and dysplastic LK, AC, oral LP and normal oral mucosa. In addition, increased CD8(+) /perforin(+) and GB(+) cell numbers were observed in all pre-malignant lesions (non-dysplastic LK, dysplastic LK, AC) when compared with the control. Perforin and GB proteins may contribute to antitumoural immunity, leading to the direct killing of tumour cells; however, it seems to occur more effectively in LSCC than OCSCC.

The recently emerged Hendra virus is an enveloped, single-stranded, negative-sense RNA virus which is classified as a BSL-4 pathogen. Both the viral attachment (G) and fusion (F) proteins are required for fusion of the viral membrane with the host cell membrane, an obligate early step in infection. Hendra F must undergo proteolytic processing to be fusogenically active, and this unique cleavage event involves trafficking of F to the plasma membrane and subsequent endocytosis and cleavage by the endosomal/lysosomal protease cathepsin L. While cleavage of other paramyxovirus fusion proteins occurs following specific sequences, cathepsin L lacks a specific substrate recognition sequence. As Hendra F cleavage by cathepsin L occurs in acidic endosomal compartments, protonation of residues near the cleavage site could modulate F cleavage. The ectodomain of F contains only two histidine residues, H102 and H372. Mutation of H102, directly upstream of the cleavage site, to an alanine did not significantly alter cleavage or function. Modeling Hendra F on to the prefusion structure of HPIV5 F indicates that H372 is in close proximity to H102, thus local conformational changes driven by histidine protonation could alter the cleavage site. We will therefore examine processing and function of the Hendra F mutants H372A and H102A/H372A to define the possible role of histidine protonation in Hendra F cleavage. This work is supported by grants from the National Institutes of Health and the March of Dimes.

The cytokine TWEAK, a TNF superfamily member, and its cognate receptor, Fn14, have emerged as potentially valuable targets for cancer therapy since their expression has been frequently identified as independent negative prognostic indicators in a variety of tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This human protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14 targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 containing either WTCYS or EACYS mutants of GrB, and a human scFv targeting Fn14 were expressed in HEK293E and suspension CHO cells, harvested under serum-free conditions and purified to homogeneity. Both constructs display high-level protein production (>50 mg/L), enhanced serum stability and impressive cytotoxic activity (IC50 < 20 nM) against Fn14 positive cells. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic GrB substrate, was comparable to that of human GrB for the WTCYS while the EACYS variant showed a 50% reduction in specific activity. On the other hand, in vitro stability studies showed that EACYS was more stable after 48h incubation in human serum, retaining over 60% of its initial specific activity. Against a panel of 20 human cell lines expressing Fn14, both constructs showed high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) and were two to over a hundred times more potent than free GrB. Mechanistic studies demonstrated that GrB-Fc-IT4 constructs activated caspase cascades and cytochrome C related pro-apoptotic mechanisms in keeping with the known intracellular functions of GrB in target cells. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 fusion proteins exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½ α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2 β = 35 hours). Toxicity studies in mice demonstrated that the MTD is above 100 mg/kg total dose. Murine orthotropic tumor model efficacy and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Yunli Zhao, Kathryn E. Ruisaard, Jeffrey A. Winkles, Michael G. Rosenblum. Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 575.

Anti-VEGF therapy is a key therapeutic in several cancer treatment regimens. However, none of the current therapeutics are directly cytotoxic against tumor cells. VEGF121 is the smallest isoform in the VEGF-A family of cytokines that binds to VEGF receptors R-1 and R-2. Both receptors are over-expressed on several tumor cells and the endothelium of tumor vasculature but not normal vasculature. We hypothesized that delivery of a cytotoxic payload through VEGFR-2 into tumor cells and tumor neovasculature would significantly enhance cancer therapy. Granzyme B is a key effector in immune-mediated cell killing through both caspase-dependent and -independent mechanisms. We previously reported the development of a fusion protein composed of granzyme B (GrB) and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein incorporating a human IgG heavy chain Fc fragment for dimerization to increase the molecular weight and improve in vivo circulation and targeting potential. We expressed and purified GrB-Fc-VEGF121 from HEK-293E cells under serum-free conditions with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. We characterized this construct against both human and mouse VEGFR+ cell lines as this ligand cross-reacts to both species. GrB-Fc-VEGF121 showed in vitro cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2, while receptor-negative cells demonstrated IC50 levels in the high micromolar range. GrB-Fc-VEGF121 internalized into VEGFR-2+ tumor and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR2+ cells at the respective IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability studies indicated a gradual protein loss of GrB-Fc-VEGF121 with an overall stability of about 50% over 96 hours. No toxicity was observed in mice treated with a total dose of 475 mg/kg (IP, QOD x 5), indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model (20 mg/kg GrB-Fc-VEGF121 x 5) resulted in significant growth inhibition of established tumors compared to vehicle controls. The number of CD31+ blood vessels and Ki-67+ proliferating tumor cells decreased significantly in treated tumors compared to controls. Pharmacokinetic studies are underway and will be reported. Thus, GrB-Fc-VEGF121 significantly reduces tumor growth in vivo with no observed toxicity against normal tissues. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5974.

VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature, as well as several tumor cells. While anti-VEGF therapy is a key therapeutic in several cancer treatment regimens, none of the current therapeutics (bevacizumab, VEGF-trap, tyrosine kinase inhibitors) are directly cytotoxic against tumor cells. The serine protease Granzyme B (GrB) is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We previously reported the development and characterization of a fusion protein composed of GrB and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein that incorporates a human IgG heavy chain Fc fragment for dimerization which increases the molecular weight and improves in vivo circulation and targeting potential. GrB-Fc-VEGF121 was expressed in HEK-293E cells under serum-free conditions and purified from the conditioned medium with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. In vitro studies of GrB-Fc-VEGF121 showed cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2. Receptor-negative cells demonstrated IC50 levels in the high micromolar range. Immunofluorescence and confocal microscopy demonstrated that GrB-Fc-VEGF121 readily internalized into VEGFR-2+ tumor cells and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR-2+ endothelial and tumor cells at the IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall stability of about 50% over 96 hours. No toxicity was observed in BALB/c mice treated with a total 475 mg/kg given IP every other day over 5 doses, indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. We observed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells in treated tumors compared to controls. Thus, GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. This tumor- and vascular-targeting agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6315.

Cancer treatment has improved in the past decades due to advances in targeted therapies and surgical techniques. Immunotherapy, for example, has greatly improved outcomes for people with several types of cancer that typically have a poor prognosis. However, the response rate depends on the type of cancer being treated, with an average response rate between 20 to 50%. Thus, novel approaches to overcome such therapeutic challenges are warranted. Emerging therapies include fusion proteins and immunoconjugates that deliver a cytotoxic payload to the tumor cell with more specificity and potentially less avenues for resistance than exerted by the antibody or toxin alone. Carcinoembryonic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in a high percentage of solid tumors including colon and rectum, lung, prostate, ovary, thyroid, or liver. Thus, oncologic therapies against CEA may promote anti-tumor activity while sparing off-target toxicity to unaffected normal tissue. Our newly developed construct, designated GrB-Fc-huMFE, is a completely human fusion protein that contains the single-chain variable fragment huMFE antibody recognizing a juxta-membrane epitope of CEA remaining after proteolytic cleavage of CEA from the cell surface. The construct rapidly internalized into target cells resulting in granzyme B (GrB)-mediated mitochondrial depolarization and irreversible activation of both apoptotic and necrotic cascade mechanisms of cell death. In vitro cytotoxicity of GrB-Fc-huMFE against an initial subset of CEA+ tumor cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar IC50 cytotoxicity against CEA-negative cell lines. Significantly, GrB-Fc-huMFE cytotoxicity in vitro was unaffected by exogenously-added CEA extracellular domain at doses up to 1000 μg/ml, suggesting that the construct is not competitively absorbed by soluble CEA proteolytically released by tumors and present in the circulation of patients. An in vivo efficacy study against established CEA+ (A-549) tumors in nude mice resulted in significant growth inhibition compared to vehicle controls (1200 mm3 vs <40 mm3). Complete tumor regression was observed in 7/10 tumors in mice treated (IP) with doses of 100 mg/kg of GrB-Fc-huMFE in a Q3Dx5 dose administration schedule. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Pharmacokinetic, stability and in vitro tests against a broader panel of tumor cell lines are ongoing and will be reported. Altogether, GrB-Fc-huMFE appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical and eventual clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Walter N. Hittelman, Michael G. Rosenblum. Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6317.

The molecular mechanism involved in hepatocellular injury in viral hepatitis remains to be clarified. We investigated the in situ expression of effector molecules of cytotoxic T lymphocytes such as Fas-ligand (Fas-L), perforin and Granzyme B (Gr-B) immunohistochemically in liver tissues from 20 patients with chronic hepatitis B (CHB) and C (CHC). The degree of cell infiltration was analysed semi-quantitatively and compared with the histological activity index (HAI). Fas-L was expressed in both CD4 and CD8 T-cells in the portal tract as well as in the parenchyma. Immunostaining of serial sections demonstrated that mononuclear cells at interface hepatitis and focal necrosis were mainly Fas-L positive CD8 T-cells. On the other hand, the expression of perforin or Gr-B was limited to a few mononuclear cells in the portal tract and parenchyma. Semi-quantitative analysis showed a positive correlation between HAI and the grade of infiltration of CD8 T-cells or Fas-L-positive cells, while the correlation was not apparent between HAI and the number of Gr-B positive cells. The expression of these molecules was not different between types of viruses. These results suggest that Fas-L-positive CD8 T-cells play a major role in the pathogenesis of liver cell injury in chronic hepatitis.