Antigen-binding fragments of antibodies specific to the tumor-associated ganglioside GD2 are well poised to play a substantial role in modern GD2-targeted cancer therapies, however, rapid elimination ...from the body and reduced affinity compared to full-length antibodies limit their therapeutic potential. In this study, scFv fragments of GD2-specific antibodies 14.18 were produced in a mammalian expression system that specifically bind to ganglioside GD2, followed by site-directed pegylation to generate mono-, di-, and tetra-scFv fragments. Fractionated pegylated dimers and tetramers of scFv fragments showed significant increase of the binding to GD2 which was not accompanied by cross-reactivity with other gangliosides. Pegylated multimeric di-scFvs and tetra-scFvs exhibited cytotoxic effects in GD2-positive tumor cells, while their circulation time in blood significantly increased compared with monomeric antibody fragments. We also demonstrated a more efficient tumor uptake of the multimers in a syngeneic GD2-positive mouse cancer model. The findings of this study provide the rationale for improving therapeutic characteristics of GD2-specific antibody fragments by multimerization and propose a strategy to generate such molecules. On the basis of multimeric antibody fragments, bispecific antibodies and conjugates with cytotoxic drugs or radioactive isotopes may be developed that will possess improved pharmacokinetic and pharmacodynamic properties.
Rapid uptake of nanoparticles by mononuclear phagocyte system (MPS) significantly hampers their therapeutic efficacy. Temporal MPS blockade is one of the few ways to overcome this barrier - the ...approach rediscovered many times under different names but never extensively used in clinic. Using meta-analysis of the published data we prove the efficacy of this technique for enhancing particle circulation in blood and their delivery to tumours, describe a century of its evolution and potential combined mechanism behind it. Finally, we discuss future directions of the research focusing on the features essential for successful clinical translation of the method.
Neuroblastoma is a pediatric solid cancer of heterogeneous clinical behavior. The unique features of this type of cancer frequently hamper the process of determining clinical presentation and ...predicting therapy effectiveness. The tumor can spontaneously regress without treatment or actively develop and give rise to metastases despite aggressive multimodal therapy. In recent years, immunotherapy has become one of the most promising approaches to the treatment of neuroblastoma. Still, only one drug for targeted immunotherapy of neuroblastoma, chimeric monoclonal GD2-specific antibodies, is used in the clinic today, and its application has significant limitations. In this regard, the development of effective and safe GD2-targeted immunotherapies and analysis of other potential molecular targets for the treatment of neuroblastoma represents an important and topical task. The review summarizes biological characteristics of the origin and development of neuroblastoma and outlines molecular markers of neuroblastoma and modern immunotherapy approaches directed towards these markers.
Targeted anticancer therapeutics offer the advantage of reducing cytotoxic side effects to normal cells by directing the cytotoxic payload selectively to cancer cells. Designed ankyrin repeat ...proteins (DARPins) are promising non‑immunoglobulin‑based scaffold proteins for payload delivery to cancer‑associated molecular targets. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40‑60% of prostate cancers (PCs) and is associated with metastasis, increased risk of PC recurrence and resistance to treatment. Here, we investigated the use of DARPin Ec1 for targeted delivery of
exotoxin A variant (LoPE) with low immunogenicity and low non‑specific toxicity to EpCAM‑expressing prostate cancer cells. Ec1‑LoPE fusion protein was radiolabeled with tricarbonyl technetium‑99m and its binding specificity, binding kinetics, cellular processing, internalization and cytotoxicity were evaluated in PC‑3 and DU145 cell lines. Ec1‑LoPE showed EpCAM‑specific binding to EpCAM‑expressing prostate cancer cells. Rapid internalization mediated potent cytotoxic effect with picomolar IC
values in both studied cell lines. Taken together, these data support further evaluation of Ec1‑LoPE in a therapeutic setting in a prostate cancer model
.
Biocomputing based on particle disassembly Nikitin, Maxim P; Shipunova, Victoria O; Deyev, Sergey M ...
Nature nanotechnology,
09/2014, Letnik:
9, Številka:
9
Journal Article
Recenzirano
Nanoparticles with biocomputing capabilities could potentially be used to create sophisticated robotic devices with a variety of biomedical applications, including intelligent sensors and theranostic ...agents. DNA/RNA-based computing techniques have already been developed that can offer a complete set of Boolean logic functions and have been used, for example, to analyse cells and deliver molecular payloads. However, the computing potential of particle-based systems remains relatively unexplored. Here, we show that almost any type of nanoparticle or microparticle can be transformed into autonomous biocomputing structures that are capable of implementing a functionally complete set of Boolean logic gates (YES, NOT, AND and OR) and binding to a target as result of a computation. The logic-gating functionality is incorporated into self-assembled particle/biomolecule interfaces (demonstrated here with proteins) and the logic gating is achieved through input-induced disassembly of the structures. To illustrate the capabilities of the approach, we show that the structures can be used for logic-gated cell targeting and advanced immunoassays.
When combined with immunotherapy, image-guided targeted delivery of chemotherapeutic agents is a promising direction for combination cancer theranostics, but this approach has so far produced only ...limited success due to a lack of molecular targets on the cell surface and low therapeutic index of conventional chemotherapy drugs. Here, we demonstrate a synergistic strategy of combination immuno/chemotherapy in conditions of dual regioselective targeting, implying vectoring of two distinct binding sites of a single oncomarker (here, HER2) with theranostic compounds having a different mechanism of action. We use: (i) PLGA nanoformulation, loaded with an imaging diagnostic fluorescent dye (Nile Red) and a chemotherapeutic drug (doxorubicin), and functionalized with affibody ZHER2:342 (8 kDa); (ii) bifunctional genetically engineered DARP-LoPE (42 kDa) immunotoxin comprising of a low-immunogenic modification of therapeutic Pseudomonas exotoxin A (LoPE) and a scaffold targeting protein, DARPin9.29 (14 kDa). According to the proposed strategy, the first chemotherapeutic nanoagent is targeted by the affibody to subdomain III and IV of HER2 with 60-fold specificity compared with nontargeted particles, while the second immunotoxin is effectively targeted by DARPin molecule to subdomain I of HER2. We demonstrate that this dual targeting strategy can enhance anticancer therapy of HER2-positive cells with a very strong synergy, which made possible 1000-fold decrease of effective drug concentration in vitro and a significant enhancement of HER2 cancer therapy compared to monotherapy in vivo. Moreover, this therapeutic combination prevented the appearance of secondary tumor nodes. Thus, the suggested synergistic strategy utilizing dual targeting of the same oncomarker could give rise to efficient methods for aggressive tumors treatment.
Smart nanomaterials, contrast nanoparticles and drug nanocarriers of advanced targeting architecture were designed for various biomedical applications. Most of such agents demonstrate poor ...pharmacokinetics in vivo due to rapid elimination from the bloodstream by cells of the mononuclear phagocyte system (MPS). One of the promising methods to prolong blood circulation of the nanoparticles without their modification is MPS blockade. The method temporarily decreases macrophage endocytosis in response to uptake of a low-toxic non-functional material. The effect of different factors on the efficiency of macrophage blockade in vivo induced by solid nanomaterials has been studied here. Those include: blocker nanoparticle size, ζ-potential, surface coating, dose, mice strain, presence of tumor or inflammation. We found that the blocker particle coating type had the strongest effect on MPS blockade efficiency, which allowed to prolong functional particle blood circulation half-life 18 times. The mechanisms capable of regulation of the MPS blockade have been demonstrated, which can promote application of this phenomenon in medicine for improving delivery of diagnostic and therapeutic nanomaterials.
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Tumour microenvironment hinders nanoparticle transport deep into the tissue precluding thorough treatment of solid tumours and metastatic nodes. We introduce an anticancer drug delivery concept ...termed FlaRE (Flash Release in Endothelium), which represents alternative to the existing approaches based on enhanced permeability and retention effect. This approach relies on enhanced drug-loaded nanocarrier accumulation in vessels of the target tumour or metastasised organ, followed by a rapid release of encapsulated drug within tens of minutes. It leads to a gradient-driven permeation of the drug to the target tissue. This pharmaceutical delivery approach is predicted by theoretical modelling and validated experimentally using rationally designed MIL-101(Fe) metal-organic frameworks. Doxorubicin-loaded MIL-101 nanoparticles get swiftly trapped in the vasculature of the metastasised lungs, disassemble in the blood vessels within 15 minutes and release drug, which rapidly impregnates the organ. A significant improvement of the therapeutic outcome is demonstrated in animal models of early and late-stage B16-F1 melanoma metastases with 11-fold and 4.3-fold decrease of pulmonary melanoma nodes, respectively.
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The fluorescent protein KillerRed, a new type of biological photosensitizer, is considered as a promising substitute for current synthetic photosensitizes used in photodynamic therapy ...(PDT). However, broad application of this photosensitiser in treating deep-seated lesions is challenging due to the limited tissue penetration of the excitation light with the wavelength falling in the visible spectral range. To overcome this challenge, we employ upconversion nanoparticles (UCNPs) that are able to convert deep-penetrating near infrared (NIR) light to green light to excite KillerRed locally, followed by the generation of reactive oxygen species (ROS) to kill tumour cells under centimetre-thick tissue. The photosensitizing bio-nanohybrids, KillerRed-UCNPs, are fabricated through covalent conjugation of KillerRed and UCNPs. The resulting KillerRed-UCNPs exhibit excellent colloidal stability in biological buffers and low cytotoxicity in the dark. Cross-comparison between the conventional KillerRed and UCNP-mediated KillerRed PDT demonstrated superiority of KillerRed-UCNPs photosensitizing by NIR irradiation, manifested by the fact that ∼70% PDT efficacy was achieved at 1-cm tissue depth, whereas that of the conventional KillerRed dropped to ∼7%.
KillerRed is a protein photosensitizer that holds promise as an alternative for the existing hydrophobic photosensitizers that are widely used in clinical photodynamic therapy (PDT). However, applications of KillerRed to deep-seated tumours are limited by the insufficient penetration depth of the excitation light in highly scattering and absorbing biological tissues. Herein, we reported the deployment of upconversion nanoparticles (UCNPs) to enhance the treatment depth of KillerRed by converting the deep-penetrating near-infrared (NIR) light to upconversion photoluminescence and activating the PDT effect of KillerRed under deep tissues. This work demonstrated clear potential of UCNPs as the NIR-to-visible light converter to overcome the light penetration limit that has plagued PDT application for many years.
Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked ...success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.
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