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Granzyme B-mimicking nanozyme-containing nanovesicles for tumour-targeting delivery

ABSTRACT: Cytotoxic T lymphocytes play a crucial role in anti-tumour immunity, with granzyme B (GrB) being a decisive factor in this process. Developing a GrB-based delivery system to mimic T cell-based immunotherapy holds promise but remains challenging. Here, we present an artificial metalloenzyme (nanozyme) with GrB-like protease activity capable of inducing caspase-dependent cell apoptosis. The nanozyme is based on the site-specific binding of Pd(II) ions to recombinant human heavy chain apo-ferritin nanocages to generate a binuclear catalytic centre consisting of two Pd atoms with bridging cysteine ligands, monodentate methionine and histidine residues, and two water molecules. We show that encapsulation of the Pd-ferritin complex within membrane-fused lipid nanoparticles comprising surface-displayed single-chain antibodies affords GrB-mimicking nanozyme-based nanovesicles capable of receptor-mediated delivery of the nanozyme into the cytoplasm of tumour cells and induction of caspase-dependent apoptosis. This study provides valuable insights into the construction of nano-delivery systems with artificial GrB activity and presents a promising therapeutic option for solid tumours.

ORGANISM(S): Mus musculus

PROVIDER: GSE312940 | GEO | 2025/12/17

REPOSITORIES: GEO

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Project description:Background: Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer, including triple negative breast cancer, and represents an attractive therapeutic target. Methods: We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single chain antibody tethered through the IgG heavy chain (Fc) domain. The protein was expressed by HEK-293E cells. We assessed the construct’s GrB enzymatic activity, anti-EMP2 binding affinity, cytotoxicity against a panel of breast cancer cells, pharmacokinetics, toxicity profile, and in vivo efficacy. Results: GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+ breast cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with IC50 values below 100 nM for most positive lines. Ex vivo stability at an elevated temperature (37°C) indicated a half-life exceeding 96h while in vivo pharmacokinetics indicated a bi-exponential plasma clearance, with a moderate initial clearance (t1/2α = 18.4 h) and a much slower terminal clearance rate (t1/2β = 73.1 h). No significant differences between the vehicle control and the GrB-Fc-KS49 was detectable in a Chem16 toxicity panel. In vivo efficacy against a TNBC syngeneic mouse model EMT6 mouse model resulted in a reduction in cell proliferation compared to controls (p=0.02). Treatment using a second syngeneic model (4T1/FLuc) confirmed these results and resulted in a dramatic reduction in cell proliferation (p = 0.001) and increase in cell death by TUNEL (p=0.02) compared to controls. In addition to a significant impact on cell proliferation (p=0.005), GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor infiltrating CD45+ cells (p=0.002), and redistribution of tumor-associated macrophages (TAMs) which was confirmed by transcriptomic analysis of tumors post treatment. Conclusions: GrB-Fc-KS49 showed high specificity to EMP2 and cytotoxicity towards EMP2 positive cells. In vivo, GrB-Fc-KS49 reduced tumor burden by direct targeting as well as increasing recruitment of immune cells into the tumor microenvironment, suggesting that GrB-Fc-KS49 is a promising therapeutic for further development against breast cancer.

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Granzyme B-mimicking nanozyme-containing nanovesicles for tumour-targeting delivery

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