Background. Ferroptosis is a type of non-apoptotic iron-dependent form of regulated cell death. Ferroportin is the only exporter of iron from cells, however, the involvement of ferroportin in cell ferroptosisis poorly understood. Activation or blocking of ferroptosis pathways may provide a therapeutic strategy for the treatment of a number of diseases. Control of ferroportin expression will make it possible to select a more effective tumor therapy. Obtaining monoclonal antibodies to human ferroportin will help to further study its role in ferroptosis of tumor cells.Aim. Obtaining and characterization of monoclonal antibodies to human ferroportin.Materials and methods. Mouse monoclonal antibodies against a synthetic peptide corresponding to the ferroportin protein region were obtained using hybridomic technology. The peptide was synthesized by the method of solid-phase automatic synthesis.Results. A 15-membered peptide with the sequence ELKQLNLHKDTEPKP (ELK) was selected and synthesized. Conjugates of the synthetic peptide with carrier proteins were obtained. 4 stable hybridomic clones were obtained, and samples of monoclonal antibodies were obtained. The samples were successfully tested using methods of flow cytometry, enzyme immunoassay, and polyacrylamide gel electrophoresis.Conclusion. Four antibodies against ferroportin binding the ELK peptide were obtained and characterized. The antibodies can be used in further studies of iron transport in tumor cells.

The evolutionary conserved DNA-sensing cGAS-STING innate immunity pathway represents one of the most important cytosolic DNA-sensing systems that is activated in response to viral invasion and/or damage to the integrity of the nuclear envelope. The key outcome of this pathway is the production of interferon, which subsequently stimulates the transcription of hundreds of genes. In oncology, the situation is complex because this pathway may serve either anti- or pro-oncogenic roles, depending on context. The prevailing understanding is that when the innate immune response is activated by sensing cytosolic DNA, such as DNA released from ruptured micronuclei, it results in the production of interferon, which attracts cytotoxic cells to destroy tumors. However, in tumor cells that have adjusted to significant chromosomal instability, particularly in relapsed, treatment-resistant cancers, the cGAS-STING pathway often supports cancer progression, fostering the epithelial-to-mesenchymal transition (EMT). Here, we review this intricate pathway in terms of its association with cancer progression, giving special attention to pancreatic ductal adenocarcinoma and gliomas. As the development of new cGAS-STING-modulating small molecules and immunotherapies such as oncolytic viruses involves serious challenges, we highlight several recent fundamental discoveries, such as the proton-channeling function of STING. These discoveries may serve as guiding lights for potential pharmacological advancements.

Aquatic toxins are a group of toxic compounds produced by several types of freshwater and marine algae and cyanobacteria and transported through the food chains of water bodies. Potential contamination of aquaculture products (raw and processed fish and seafood) with aquatic toxins requires the use of efficient screening methods for their control. In this study, a multiplex immunochromatographic test system for the simultaneous detection of three aquatic toxins—phycotoxins domoic acid (DA) and okadaic acid (OA), and cyanotoxin microcystin-LR (MC-LR)—is for the first time developed. For this, a competitive indirect immunochromatographic analysis (ICA) based on gold-labeled secondary antibodies was carried out. The LODs/cutoffs/working ranges of the ICA were 0.05/0.3/0.07–0.29, 1.3/100/3.2–58.2, and 0.1/2.0/0.2–1.1 ng/mL for MC-LR, DA, and OA, respectively. The assay duration was 18 min. The developed test system was used to analyze water samples from natural sources (salt and fresh water) and fish samples. For sample preparation of water, simple dilution with a buffer was proposed; for fish samples, methanol–water extraction was utilized. It was demonstrated that the triple LFIA specifically detected target aquatic toxins with recoveries of 85.0–121.5

A lateral flow immunoassay (LFIA) of phycotoxin domoic acid (DA) contaminating seawater and marine organisms was developed in this investigation. Nine clones of monoclonal antibodies against DA were produced and characterized. The test system was implemented in the indirect competitive format, where gold nanoparticles as a marker were conjugated with secondary antibodies. The developed test system allows for the detection of DA with a cutoff of 60 ng mL-1 and an instrumental detection limit of 1.4 ng mL-1 within 15 min. The LFIA was applied to detect DA in seawater, mussels, shrimps, and octopuses. A simple method of seafood sample preparation was proposed. The entire analytical cycle, from obtaining a sample to the estimation of final results, takes only 30 min. The assay recoveries ranged from 88.5% to 124%. The developed analytical method is a promising solution for rapid on-site monitoring of marine toxicants in water and food throughout the farm-to-fork chain.

In this study, a lateral flow immunoassay (LFIA) was developed to detect okadaic acid (OA) belonging to the diarrheic shellfish poisoning group of aquatic toxins. Newly obtained anti-OA monoclonal antibodies and bimetallic core@shell Au@Pt nanoparticles were used in the indirect format of the LFIA. Peroxidase-mimicking nanozyme properties of Au@Pt enabled using them to enhance band coloration on the test strips and, consequently, for increasing the LFIA sensitivity. The instrumental limit of detection (LOD), the working range of detectable concentrations, and the visual cutoff of the assay were 0.5, 0.8–6.8, and 10 ng/mL, respectively. The assay duration was 20 min. The rapid and simple sample preparation procedure was applied for seawater, river water, and fish samples. The total duration of the sample pretreatment and LFIA was 25/40 min for water/fish samples, ensuring testing rapidity. The developed test system provides sensitive control of raw materials and food products and can be used to detect OA at all stages of the food industry «from sea to fork» chains.

The PRAME antigen, which is a significant target for monoclonal antibodies, is a tumor-specific marker that is active at all stages of tumor cell differentiation; it induces a spontaneous T-cell response. In this work, a humanized 5D3Hu antibody was constructed on the basis of the mouse monoclonal antibody 5D3 to the PRAME protein and produced in CHO (Chinese hamster ovary tumor) cells. The 5D3Hu antibody demonstrated high affinity for the antigen (1.4 nM), binding to both recombinant and native PRAME protein; it also had an inhibitory effect on the proliferation of PRAME-positive cell lines. These results allow considering the humanized 5D3Hu antibody as a promising therapeutic agent for the treatment of oncological diseases in which overexpression of the PRAME protein is observed.

In this investigation, a double immunochromatographic analysis (ICA) of two relevant phycotoxins, domoic acid (DA) and okadaic acid (OA), was developed for the first time. The ICA was performed in the indirect competitive format using gold nanoparticles conjugated with anti-species antibodies. Under optimal conditions, the instrumental detection limits/cutoffs for simultaneous detection of DA and OA were 1.2/100 and 0.1/2.5 ng/mL, respectively. The time of the assay was 18 min. The ICA was applied to test seawater and a large panel of seafood, including mussels, tiger shrimps, octopuses, whelks, crabs, and scallops. The proposed simple sample preparation method for seafood takes only 20 min. For seawater, a dilution by buffer was implemented. The assay recoveries varied from 80.8% to 124.5%. The competitive potential of the proposed technique as a tool to control natural water and seafood samples is determined by its simplicity, rapidity, and sensitivity.

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells.

Two monoclonal antibodies recognizing non-overlapping epitopes of the PRAME protein were injected into immunocompetent mice to study their influence on the growth of subcutaneous tumor nodes. The B16F10 murine melanoma line, either expressing human PRAME protein or bearing only a vector without PRAME gene, were used as transplants. Each of the antibodies showed the ability to suppress tumor growth of a PRAME-expressing tumour, but not a tumor without PRAME.

We investigated the epitope specificity of different monoclonal antibodies recognizing the cancer testis antigen PRAME. Antibody 5D3 binds to the fragment of PRAME corresponding to 160–180 amino acid residues. Antibodies 6H8 and F11 bind to the fragment corresponding to 180–200 amino acid residues of PRAME. These antibodies retained the ability to recognize these PRAME fragments after chimerization.

The development of and research into new therapies that can selectively and effectively destroy tumor cells that overexpress the ErbB2 receptor is apressing task. Recently, research into the use of type I interferons in the treatment of cancer has intensified. Cytokine therapy is aimed at activating the cells of the immune system to fight tumors, but it has drawbacks that limit its use because of a number of side effectsthe severity of which varies depending on the dosage and type of used cytokine. At the moment, a number of studies are being conducted regarding the use of IFNin oncology. The studies areaimed at mitigating the systemic action of this cytokine. The immunocytokine complex made of a bispecific antibody against the ErbB2 receptor and recombinant IFNdeveloped in this study underlies themechanism meant to avoid the systemic action of this cytokine. Part of this study focuses on the development of full-length antibodies that bind to the ErbB2 receptor on the one hand, and bind and neutralize IFN, on the other hand, which allows us to consider the antibodies as a means of cytokine delivery to tumor cells.

— The extracellular part of the ErbB2 receptor (ecdErbB2), an oncological marker and a target for therapeutic antibodies, has been expressed in eukaryotic CHO cells. The extracellular part of the ErbB2 receptor has been isolated from a culture medium by metal affinity chromatography and characterized by biochemical and immunochemical methods. It has been shown that the receptor is produced in monomeric, dimeric, and trimeric forms. The oligomeric forms have been separated by gel filtration, and their ability to bind to Herceptin, one of the main drugs in the treatment of breast cancer, has been characterized by ELISA. The receptor produced can be used for in vitro immunochemical experiments.