Genetically engineered CD80–pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide–MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols

Plants with genetically encoded autoluminescence

Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Comparison of Colorimetric and Fluorometric Chemosensors for Protein Concentration Determination and Approaches for Estimation of Their Limits of Detection

Here, we present a direct comparison of different dyes and assays for the determination of protein concentrations. We compared the classical Bradford assay with two modern assays based on the fluorogenic dyes QuDye and ProteOrange and showed that the Bradford reagent achieved excellent results in the determination of protein concentrations as compared with more modern rivals. We also showed that standard approaches for determining the limit of detection (LoD) and limit of quantification (LoQ) may not work correctly with the tested dyes. We proposed a new approach that extends the standard algorithm for LoD and LoQ determination. This approach works well with both classical colorimetric and fluorogenic dyes, as well as with nontrivial fluorescent probes

Self-assembling complexes of quantum dots and scFv antibodies for cancer cell targeting and imaging.

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex

Visualization of epithelial cell adhesion molecule-expressing renal cell carcinoma xenografts using designed ankyrin repeat protein Ec1 labelled with Tc-99m and I-125

The upregulation of epithelial cell adhesion molecule (EpCAM) expression, found in a substantial fraction of renal cell carcinomas (RCCs), renders it a potential molecular target for the treatment of disseminated RCC. However, the heterogeneous expression of EpCAM necessitates first identifying the patients with sufficiently high expression of EpCAM in tumors. Using the specific radionuclide-based visualization of EpCAM might enable such identification. The designed ankyrin repeat protein, Ec1, is a small (molecular weight, 18 kDa) targeting protein with a subnanomolar affinity to EpCAM. Using a modified Ec1, a tracer was developed for the radionuclide-based visualization of EpCAM in vivo, i.e., an EpCAM-visualizing designed ankyrin repeat protein (EVD). EVD was labelled with either technetium-99m using technetium tricarbonyl or with iodine-125 (as a surrogate for iodine-123) by coupling it to para-[I-125]iodobenzoyl ([I-125]PIB) groups. Both the I-125-labelled EVD (I-125-EVD) and Tc-99m-labelled EVD (Tc-99m-EVD) bound specifically to EpCAM-expressing SK-RC-52 renal carcinoma cells. The binding affinity (K-D value) of Tc-99m-EVD to SK-RC-52 cells was 400 +/- 28 pM. The tracers' uptake in SK-RC-52 xenografts at 3 h after injection was 5.2 +/- 1.4%ID/g for I-125-EVD and 6.0 +/- 1.4%ID/g for Tc-99m-EVD (no significant difference). These uptake values in SK-RC-52 xenografts were significantly higher (P<0.001) than those in Ramos lymphoma xenografts (used as EpCAM-negative control). The tumor-to-blood uptake ratio was significantly higher for Tc-99m-EVD (25 +/- 6) compared with that of I-125-EVD (14 +/- 3). However, I-125-EVD was associated with higher tumor-to-liver, tumor-to-salivary gland, tumor-to-spleen and tumor-to-intestinal wall ratios. This makes it the preferable tracer for visualizing EpCAM expression levels in the frequently occurring abdominal metastases of RCC

Photoluminescent hybrid inorganic-protein nanostructures for imaging and sensing in vivo and in vitro

The last decade has seen an exciting confluence of biophotonics with nanotechnology, with nanoparticle-based investigations in life sciences ranging from fundamental biological research to clinical therapeutics. This chapter is focused on photoluminescent nanoparticle-based "bioprobes," designed and built to visualize and probe specific biological processes. Unlike organic dyes and fluorescent proteins, photoluminescent nanoparticles are highly photostable and, depending on the nanomaterial design, exhibit narrow and/or tuneable emission spectra, thereby greatly extending the capabilities of the existing fluorophores. Inorganic materials show photoluminescence independent of environmental conditions, including pH and temperature, while their chemically active surface area provides docking platforms for anchoring targeting and/or cargo biomolecules. Moreover, some types of nanoparticles exhibit unique properties, including enhanced detection/imaging contrast due to long photoluminescence lifetime, and/or an "anti-Stokes" emission wavelength shift. In this review, selected key types of such nanoparticle-based bioprobes are discussed: quantum dots, fluorescent nanodiamonds, nanorubies and upconversion nanoparticles. The chapter aims to demonstrate the power of this bottom-up bio-nanophotonics approach for biological sensing and imaging. The design of photoluminescent nanoparticles, surface activation and bioconjugation are discussed, in addition to the deployment and application of selected bioconjugate structures for specific internalization in cells and living biological tissue.40 page(s

Epithelial cell adhesion molecule-targeting designed ankyrin repeat protein-toxin fusion Ec1-LoPE exhibits potent cytotoxic action in prostate cancer cells

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 Pseudomonas 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 IC50 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 in vivo