Directing ricin-based immunotoxins with targeting affibodies and KDEL signal peptide to cancer cells effectively induces apoptosis and tumor suppression

<jats:title>Abstract</jats:title><jats:p>The plant toxin ricin, especially its cytotoxic A chain (RTA), can be genetically engineered with targeting ligands to develop specific anti-cancer recombinant immunotoxins (RITs). Here, we used affibody molecules targeting two cancer biomarkers, the receptors HER2 and EGFR, along with the KDEL signal peptide to construct two cancer-specific ricin-based RITs, HER2Afb-RTA-KDEL and EGFRAfb-RTA-KDEL. The affibodies successfully provided target-specificity and subsequent receptor-mediated endocytosis and the KDEL signal peptide routed the RITs through the retrograde transport pathway, effectively delivering RTA to the cytosol as well as avoiding the alternate recycling pathway that typical cancer cells frequently have. The in vivo efficacy of RITs was enhanced by introducing the albumin binding domain (AlBD) to construct AlBD/HER2Afb/RTA-KDEL. Systemic administration of AlBD-containing RITs to tumor-bearing mice significantly suppressed tumor growth without any noticeable side-effects. Collectively, combining target-selective affibody molecules, a cytotoxic RTA, and an intracellularly designating peptide, we successfully developed cancer-specific and efficacious ricin-based RITs. This approach can be applied to develop novel protein-based ???magic bullets??? to effectively suppress tumors that are resistant to conventional anti-cancer drugs.</jats:p> <jats:p><jats:bold>Graphical Abstract</jats:bold></jats:p&gt

Lactate oxidase/catalase-displaying nanoparticles efficiently consume lactate in the tumor microenvironment to effectively suppress tumor growth

<jats:title>Abstract</jats:title><jats:p>The aggressive proliferation of tumor cells often requires increased glucose uptake and excessive anaerobic glycolysis, leading to the massive production and secretion of lactate to form a unique tumor microenvironment (TME). Therefore, regulating appropriate lactate levels in the TME would be a promising approach to control tumor cell proliferation and immune suppression. To effectively consume lactate in the TME, lactate oxidase (LOX) and catalase (CAT) were displayed onto <jats:italic>Aquifex aeolicus</jats:italic> lumazine synthase protein nanoparticles (AaLS) to form either AaLS/LOX or AaLS/LOX/CAT. These complexes successfully consumed lactate produced by CT26 murine colon carcinoma cells under both normoxic and hypoxic conditions. Specifically, AaLS/LOX generated a large amount of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> with complete lactate consumption to induce drastic necrotic cell death regardless of culture condition. However, AaLS/LOX/CAT generated residual H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>, leading to necrotic cell death only under hypoxic condition similar to the TME. While the local administration of AaLS/LOX to the tumor site resulted in mice death, that of AaLS/LOX/CAT significantly suppressed tumor growth without any severe side effects. AaLS/LOX/CAT effectively consumed lactate to produce adequate amounts of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> which sufficiently suppress tumor growth and adequately modulate the TME, transforming environments that are favorable to tumor suppressive neutrophils but adverse to tumor-supportive tumor-associated macrophages. Collectively, these findings showed that the modular functionalization of protein nanoparticles with multiple metabolic enzymes may offer the opportunity to develop new enzyme complex-based therapeutic tools that can modulate the TME by controlling cancer metabolism.</jats:p> <jats:p><jats:bold>Graphical Abstract</jats:bold></jats:p&gt

Myricetin improves endurance capacity and mitochondrial density by activating SIRT1 and PGC-1α

1132Nsciescopu

Dual-liganded protein nanoparticles displaying TRAILs and EGFR binding affibodies enhance therapeutic efficacy against EGFR overexpressing triple negative breast cancers

Triple-negative breast cancer (TNBC) is a highly aggressive and difficult-to-treat cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). Due to the absence of these receptors, standard hormonal therapy and targeted therapy with HER2 are not effective for TNBC. Therefore, there is an urgent need for the development of TNBC-specific therapies. TNBCs often highly express the epidermal growth factor receptor (EGFR), which makes it an attractive target for TNBC treatment. Herein, we constructed a protein nanoparticle that polyvalently displays the cancer-specific apoptotic protein TNF-related apoptosis-inducing ligands (TRAILs) and EGFR-binding affibodies on a lumazine synthase protein nanoparticle (AaLS/TRAIL/EGFRAfb) and evaluated its therapeutic efficacy against EGFR-overexpressing TNBCs. AaLS/TRAIL/EGFRAfb tightly binds to the surface of TNBC cells, allowing for frequent and consistent interactions between TRAIL molecules on the protein cage and death receptors on TNBC cells, ultimately resulting in effective apoptotic cell death and tumor growth suppression. AaLS/TRAIL/EGFRAfb is a promising candidate for further development as a targeted therapeutic agent for TNBC treatment. This research highlights the potential of protein nanoparticle technology to develop new, effective, and targeted cancer therapies

Developing Protein-based injectable hydrogels using PCNA and SpyCatcher for controlled cargo load and release

Three-dimensionally crosslinked hydrogels have been widely developed for various biomedical applications such as scaffolds, drug delivery, and biomaterial implants. However, many hydrogels use chemical crosslinkers that limit their applications due to their potential toxicity and non-biodegradabiliy. To overcome these limitations, protein-based hydrogels have emerged as promising alternatives. Herein, we designed two different non-fibrous recombinant protein building blocks; a core trimeric proliferating cell nuclear antigen (PCNA) and a bridging SpyCatcher protein (SC) dimer. We genetically introduced SpyTag peptide (ST) to the (PCNA) to form ST-PCNA to serve as the core building blocks, and fused two SCs to form SC-SC as the connecting building blocks. Simple mixing of these two building block proteins (ST-PCNA and SC-SC) spontaneously led to the PCNA-based 3D networks, facilitating rapid gelation and resulting in stable protein-based hydrogel formations both in vitro and in vivo. The newly formed protein-based hydrogels possessed a 3D porous network structure that allows for control of cargo loading and release, making them well-suited for use as sustained release systems. To take advantage of the attractive properties of protein-based hydrogels, we evaluated injectability of hydrogels and the controlled loading and sustained release of various cargos, including fluorescent dyes, therapeutic drugs, and proteins, both in vitro and in vivo. These non-fibrous protein building block-based hydrogels may offer new opportunities to develop biocompatible and versatile platforms for sustained cargo delivery, scaffold formation, and implantable biomaterials

Engineering protein cage nanoparticles as tunable multifunctional nanoplatforms

Protein cage nanoparticles are biomolecule-based supramolecular biopolymers and attractive candidates for nano-scale cargo delivery vehicles. While the interior surfaces of the protein cages have been used for encapsulation, attachment and synthesis of organic and inorganic materials, their exterior surfaces have been used for multivalent presentations of molecules, including affinity tags, antibodies, fluorophores, carbohydrates, nucleic acids, and peptides, for molecular targeting and hierarchical structure formation. Using these natural properties, we utilized protein cage nanoparticles as virus-mimic antigen-delivery nanoplatforms and evaluated their efficacy in inducing DC-mediated antigen-specific immune responses and subsequent melanoma tumor rejection in vivo. We also implemented them as intravascular magnetic resonance T contrast conjugates via site-selective attachment of Gd(III)-chelating agents and used them for in vivo MR imaging visualizing a mouse???s intravascular system, including the carotid, mammary arteries, and the superficial vessels of the head at an isotropic resolution of 250 ??m. Targeted drug and/or probe delivery using nanoparticles can achieve the localized and enhanced treatment of diseases, minimizing side-effects, and target-specific diagnosis of symptoms even at early stage. A wide range of target-specific ligands, including antibodies, peptides, and affibody molecules, have been used. Affibody molecules, however, are genetically engineered antibody mimics that exhibit high specificity and affinity toward their targets and prove the potential for diagnostic applications in biotechnology and therapeutic developments in biomedicine. By applying a recently developed bacterial superglue, SpyTag/SpyCatcher (ST/SC) protein ligation system to affibody molecules and protein cage nanoparticles, multiple displays of two or more targeting ligands on a polyvalent single nanoparticle were achieved

Selective and Effective Cancer Treatments using Target???Switchable Intracellular Bacterial Toxin Delivery Systems

Targeted cancer therapies have been extensively tested with the purpose to selectively suppress tumor growth and to avoid harming healthy tissue. However, failure to escape endosomes upon receptor???mediated endocytosis is a major obstacle limiting the efficacy of targeted cancer therapeutics. Here, novel target???switchable intracellular toxin delivery systems (TiTDS) are presented which use the catalytic and translocation domain of diphtheria toxin (dtA???T) as an intracellular toxin delivery platform and affibody molecules targeting human epidermal growth factor receptor 2 or epidermal growth factor receptor (HER2Afb or EGFRAfb) as target???specific ligands. The intracellular toxin delivery platform and the affibody molecules are genetically fused with SpyCatcher (SC) protein and SpyTag (ST) peptide, respectively, to generate dtA???T???SC and ST???HER2Afb or ST???EGFRAfb modules. These modules can be individually purified and post???translationally ligated to produce dtA???T/HER2Afb or dtA???T/EGFRAf. dtA???T/HER2Afb and dtA???T/EGFRAfb can selectively bind to their corresponding target cancer cells, efficiently enter the cells through receptor???mediated endocytosis, successfully escape endosomes, and release toxins into the cytosol. They exhibit high target???specific cytotoxicity in vitro and can significantly reduce tumor masses in vivo. TiTDS is a promising targeted cancer therapy platform because of its high target specificity, effective intracellular delivery of active toxins with improved therapeutic efficacy, and target switchability

Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue

The selective detection of specific cells of interest and their effective visualization is important but challenging, and fluorescent cell imaging with target-specific probes is commonly used to visualize cell morphology and components and to track cellular processes. Multiple displays of two or more targeting ligands on a polyvalent single template would make it possible to construct versatile multiplex fluorescent cell imaging probes that can visualize two or more target cells individually without the need for a set of individual probes. To achieve this goal, we used encapsulin, a new class of protein cage nanoparticles, as a template and implanted dual targeting capability by presenting two different affibody molecules on a single encapsulin protein cage nanoparticle post-translationally. Encapsulin was self-assembled from 60 identical subunits to form a hollow and symmetric spherical structure with a uniform size. We genetically inserted SpyTag peptides onto the encapsulin surface and prepared various SpyCatcher-fused proteins, such as fluorescent proteins and targeting affibody molecules. We successfully displayed fluorescent proteins and affibody molecules together on a single encapsulin in a mix-and-match manner post-translationally using bacterial superglue, the SpyTag/SpyCatcher ligation system, and demonstrated that these dual functional encapsulins can be used as target-specific fluorescent cell imaging probes. Dual targeting protein cage nanoparticles were further constructed by ligating two different affibody molecules onto the encapsulin surface with fluorescent dyes, and they effectively recognized and bound to two individual targeting cells independently, which could be visualized by selective colors on demand