Human Granzyme B Based Targeted Cytolytic Fusion Proteins

Hlongwane, Precious; Mungra, Neelakshi; Madheswaran, Suresh; Akinrinmade, Olusiji A.; Chetty, Shivan; Barth, Stefan

doi:10.3390/biomedicines6020072

Cited by 27 publications

(27 citation statements)

References 61 publications

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“…41 Granzyme B-based immunotoxins have been generated with therapeutic effect in preclinical models in vivo. 42 In this line, the granulysin-based immunotoxin described here should be devoid of immunogenicity due to the cytotoxic payload, since granulysin is also a human protein. Toxicity associated with immunotoxins can be either nonspecific or targeted.…”

Section: Discussionmentioning

confidence: 97%

Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin

et al. 2019

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Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of intratumoral injection of recombinant granulysin using in vivo models of breast cancer and multiple myeloma. In the present work we have developed a granulysin gene fusion to the anticarcinoembryonic antigen (CEA/CEACAM5) single chain Fv antibody fragment MFE23. Both granulysin and the granulysin-based immunotoxin were expressed in Pichia pastoris. The immunotoxin specifically recognized CEA, purified or expressed on the cell surface. Moreover, the bioactivity of the immunotoxin against several CEA + cell lines was higher than that of granulysin alone. Granulysin and the immunotoxin were tested as a treatment in in vivo xenograft models in athymic mice. When injected intratumorally, both granulysin and the immunotoxin were able to inhibit tumor growth. Furthermore, systemic administration of the immunotoxin demonstrated a decrease in tumor growth in a CEA + tumor-bearing mouse model, whereas granulysin did not exhibit a therapeutic effect. This is the first granulysin-based immunotoxin and the present work constitutes the proof of concept of its therapeutic potential.

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Section: Discussionmentioning

confidence: 97%

Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin

et al. 2019

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“…2). 33 Compared with toxins from bacteria and plants, hCFPs have lower immunogenicity and cytotoxicity, with higher tumor selectivity and infiltration ability. At present, most of the human toxins used in research are granzyme B (GrB) and ribonucleases (RNases).…”

Section: Mechanism Of Cancer Therapy By Immunotoxinsmentioning

confidence: 99%

“…(6) the caspase/mitochondrial independent pathway was initiated; (7) the caspase-dependent pathway was initiated. 33 recombinant humanized anti-EpCAM antibody scFv and PE. 42 Once it binds to EpCAM on the surface of cancer cells, it is internalized to the cytoplasm to induce apoptosis.…”

Section: Immunotoxins In Clinical Trialsmentioning

confidence: 99%

“…Compared with heterologous toxins, hCFPs have lower immunogenicity and cytotoxicity, while having higher tumor selectivity and infiltration ability. At present, most of the human toxins used in research are GrB [33][34][35][36][37] and RNases. [38][39][40] Granzyme B GrB is a serine protease found in cytoplasmic granules of NK cells and cytotoxic T cells.…”

Section: Human Toxinsmentioning

confidence: 99%

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Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

Xin

Chen

Wang

et al. 2019

Pharmaceutical Fronts

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Immunotoxins are proteins that consist of a protein toxin conjugated to a specific targeting moiety. The targeting moiety is usually an antibody or ligand, such as monoclonal antibody, antibody fragment, a cytokine, or a growth factor. The toxins usually come from plant toxins, bacterial toxins, or human-origin cytotoxic elements. Nearly all toxins work by enzymatically inhibiting protein synthesis. After binding to antigens or receptors on target cell surfaces, immunotoxins are internalized and translocated to the cytosol where they can kill the cells. Immunotoxins have demonstrated high cytotoxicity to cancer cells and to date two immunotoxins have been approved by U.S. Food and Drug Administration on the markets for the treatment of hematological tumors: Lumoxiti and Ontak. Many other molecules are under development or clinical trials for different forms of cancer. Although immunotoxins exhibit great potency in xenograft model systems and early clinical trials, there are obstacles that limit successful treatments, including immunogenicity, nonspecific toxicity, and poor penetration. However, efforts are underway to address these problems. In this review, we summarize immunotoxins currently in clinical trials for either hematological tumors or solid tumors, outline the design of immunotoxins utilizing variety of components, and discuss the prominent examples of redesigned immunotoxins with reduced immunogenicity and nonspecific toxicity, as well as the strategies in manufacturing immunotoxins. With further improvements, it is anticipated that immunotoxins will play an increasing role in cancer therapy.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Momeni¹,

Reshadmanesh²,

Fakhravar³

et al. 2019

COR

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Keywords: Granzyme B p28 azurin breast cancer fusion protein targeting A B S T R A C T Targeting tumor cells via multiple pathways promises the emergence of a new era in cancer therapy.Consisting of a cell-binding ligand and a cytotoxic moiety, cytolytic fusion proteins can selectively bind and kill target cells with minimal adverse effects. We designed a novel immunoproapoptotic fusion protein, p28-fur-GrB, composed of the cancer-specific azurin-derived cell penetrating peptide, p28, and a mutant version of human serine protease granzyme B. The two moieties were genetically fused by a furin sensitive linker, allowing in vivo cleavage and activation of the immunotoxin after cell entry. Synthesized coding gene of the recombinant protein was cloned and expressed in HEK293T cells, and nickel chromatography was applied for protein purification. After in vitro furin cleavage and primary analyses of SDS-PAGE, Western blotting, GrB activity and ELISA binding assay, the fusion protein was tested for its cytotoxicity on various breast cancer cell lines. Suppression of cell proliferation and viability was evaluated using the WST-1 assay. Furthermore, DNA fragmentation was measured as an indication of apoptotic effects of the fusion protein on treated cells. Based on our results, p28-fur-GrB was efficiently cleaved by furin and showed high GrB activity and binding affinity after cleavage. Following 72h of incubation with IC50 values of the fusion protein, significant cytotoxic effects of 80.6%, 77.1%, 74% and 69.6% were recorded for BT-474, MCF7, SK-BR-3 and MDA-MB-231 tumor cells, respectively. Proliferative potential of MCF 10A normal cells was not affected by the treatment. Analysis of the rate of apoptosis in treated cells confirmed our cytotoxicity results. We concluded that p28-fur-GrB is a potent anti-tumor agent with high cytotoxicity against breast cancer cells.

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Human Granzyme B Based Targeted Cytolytic Fusion Proteins

Cited by 27 publications

References 61 publications

Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin

Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin

Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

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

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