Annotation of the results obtained in 2022
The project is dedicated to the use of an efficient technology for generating recombinant single-domain antibodies (nanobodies) and their use in order to search for the most informative and reliable early prognostic biomarkers of bladder cancer (BC) and to develop a non-invasive diagnostic method for the quantitative analysis of these markers in the patient's urine. In the past reporting year of the project, additional urine samples from patients at risk, different stages and forms of bladder cancer, as well as control samples from healthy donors, were accumulated in the library being created. To date, a collection of morning urine samples has been collected from 135 patients with benign and malignant diseases of the bladder undergoing treatment at the P.A. Herzen - a branch of the "NMITs Radiology" of the Ministry of Health of Russia and signed a voluntary consent to the study, as well as from 10 healthy volunteer colleagues. With the help of the obtained combined immunosorbents based on nanobodies, along with the subsequent separation of eluates or breakthroughs from the column using traditional 1D SDS-PAGE, an increased content of a number of marker proteins in the urine samples of patients with bladder cancer was revealed, which was especially pronounced when the tumor progressed from non-muscle-invasive to muscle-invasive stages. In addition to serum albumin, transferrin, IgA and IgG, it was also found to be enriched in fibrinogen (moreover, its degradation was observed in BCC of a high degree of malignancy), lactoferrin, α2-macroglobulin, apolipoprotein A1, fibronectin. A variant of the combined immunosorbent containing nanobodies to the marker proteins listed above, which have a diagnostic potential for bladder cancer, can also be used to develop a non-invasive diagnostic method in the near future for the purpose of primary screening of patients at risk. It has been shown that IgM is practically not detected in the urine samples of patients with bladder cancer, while IgG and IgA are detected as one of the major protein components. All 4 subclasses of IgG are present in the urine of BC patients, but the relative proportions of IgG1 and IgG4 are increased compared to the representation of IgG subclasses in the blood plasma of a healthy donor. Using immobilized nanobodies against human immunoglobulins IgG or IgA, a parallel analysis of protein components isolated from urine samples of patients with bladder cancer together with immunoglobulins was carried out. Shortened variants of immunoglobulins with abnormal mobility during electrophoresis have been identified. Their number increases in more advanced stages of muscle-invasive bladder cancer. In association with immunoglobulins, lactoferrin, the alpha subunit of hemoglobin, beta-lactoglobulin, and a number of other as yet unidentified proteins were also detected in various urine samples of patients with bladder cancer. New nanobodies were selected that bind the transferrin receptor TfR1. Potentially, these nanobodies can recognize TfR1 on the surface of exosomes, which can help to effectively immunoaffinity isolate the corresponding exosomes from urine for further studies. Changes in transferrin (Tf) in urine samples of patients with bladder cancer were studied using previously obtained nanobodies recognizing predominantly holo-Tf (aTf1) or apo-Tf (aTf2). A direct quantitative association of Tf with bladder cancer progression was shown. However, in a number of samples, an unexpected relative increase in Tf released by anti-holo-Tf aTf1 nanobodies was found. We assume that these may be largely secondary complexes formed by apo-Tf in combination with ions other than Fe3 +, but accumulating in the urine of a cancer patient and able to bind to apo-Tf, changing its conformation and making available epitopes that are recognizable aTf1 nanobody. A noticeable decrease in the proportion of holo-Tf relative to apo-Tf in the blood of patients with ovarian cancer was found. It has been shown that a decrease in holo-Tf is associated with functional iron deficiency associated with malignant ovarian diseases. The use of immunoaffinity chromatography to separate the two forms of transferrin and determine their ratio (holo-Tf / apo-Tf) seems to be a valuable addition in the formation of a risk group among patients with benign ovarian diseases and may be a predictor for the effectiveness of therapy in cancer patients. The use of the polarization fluorescence immunoassay (FPIA) method, which is based on measuring the change in the polarization of the fluorescence of a labeled nanobody upon its specific binding to a noticeably larger target antigen, has been demonstrated to be promising for the quantitative detection of major high-molecular-weight proteins in a biological fluid. As a model system, we used nanobodies previously obtained for different eptopes of lactoferrin (LF) protein, which were fluorescently labeled and detected by LF in diluted milk. The FPIA method takes very little time, does not require mandatory separation of bound and free forms of proteins or lengthy sample preparation. It has been shown that this method works better in diluted urine than with the same dilution in milk in detecting high-molecular-weight urine proteins in a similar way (the same LF or apo-transferrin using the corresponding labeled nanobodies). Definitely need to keep this method in mind for rapid detection of relatively major high molecular weight proteins in urine samples.

 

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Annotation of the results obtained in 2020
The project is dedicated to the use of an effective technology for the generation and use of new recombinant single-domain antibodies (nanobodies) in order to search for the most informative and reliable early predictive biomarkers of bladder cancer (BC) and the development of a non-invasive diagnostic method for quantitative analysis of these markers in the patient's urine. In the past first reporting year, the creation of a Bank of annotated urine samples from patients with different stages and forms of bladder cancer, as well as control samples from healthy donors, was initiated. To date, the Bank that is being created already contains about 40 urine samples from various patients (both primary and undergoing treatment) with bladder cancer, as well as from several control samples from healthy donors. A full cycle of work on the creation of nanobodies for conservative surface proteins common to exosomes of various types of cells and biological fluids has been carried out. For this, a fraction of membrane proteins was isolated from extracellular vesicles (exosomes) of the blood and used as an antigenic material for immunizing an experimental animal (Camelus bactrianus) for the purpose of generating, cloning and subsequent selection by the phage display method of the desired nanobodies. A new immune library of nanobody-coding DNAs was obtained, enriched with variants of nanobodies that bind membrane proteins of extracellular vesicles (exosomes). The last stages of selection were carried out on the material of extracellular vesicles that bind to immobilized antibodies against conserved exosome proteins - CD9, CD81, or EpCam. A total of about 24 different variants of nanobodies, potentially recognizing exosome surface proteins, were selected. Work has begun on the identification of target antigens for selected nanobodies. A full cycle of work was carried out to create an immune library of cDNA sequences encoding single-domain antibodies, including immunization of a camel with a preparation of extracellular vesicles (exosomes) from the urine of patients with bladder cancer and subsequent cloning of the entire repertoire of genes of nanobodies of the immunized animal. Three preparations of urine exosomes are prepared for the forthcoming selection of new nanobodies: a) exosomes from the urine of healthy donors, b) exosomes from the urine of primary patients with non-muscle invasive bladder cancer, and c) exosomes from the urine of patients with muscle invasive aggressive bladder cancer. A comparison was made of major urine proteins electrophoretically separated in a 5-19% gradient polyacrylamide 1-D gel (in reducing and non-reducing conditions): a) in healthy people, b) patients with muscle-non-invasive bladder cancer, and c) patients with muscle-invasive bladder cancer. There were revealed clear quantitative differences in the major proteins between samples from healthy donors and samples from patients with bladder cancer. In all studied patients with bladder cancer, there is a significant increase in serum albumin, IgA, IgG, transferrin, as well as two more proteins of about 150 and 13 kDa (with electrophoresis under reducing conditions). The performed electrophoretic analysis of major urine proteins seems to be a very informative method for a preliminary assessment of the probability of bladder cancer.

 

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Annotation of the results obtained in 2021
The project is dedicated to the use of an efficient technology for the generation and use of new recombinant single-domain antibodies (nanobodies) in order to search for the most informative and reliable early predictive biomarkers of bladder cancer (BC) and the development of a non-invasive diagnostic method for the quantitative analysis of these markers in the patient's urine. In the last second reporting year of the project, the number of samples in the bank of urine samples from patients at risk, different stages and forms of bladder cancer, as well as control samples from healthy donors was significantly increased. To date, a collection of morning urine samples has been collected from 105 patients with benign and malignant diseases of the bladder. The group of primary patients consisted of 48 people. The group of people under observation during treatment and after - 57. The vast majority of patients with malignant tumors of the bladder were diagnosed with urothelial carcinoma (92%). Non-muscle-invasive urothelial carcinoma was found in 54 patients (17 primary patients and 37 treated); 29 were diagnosed with muscle-invasive cancer (9 primary and 20 treated). Most of them are high-grade tumors (72%). The promising methods of using new immunosorbents based on the nanobodies obtained by us for the preprocessing of human urine proteomes for the analysis of oncobiomarkers have been demonstrated. We used several previously obtained high-affinity nanobodies that specifically bind a specific major protein in blood or urine (serum albumin, immunoglobulin A, immunoglobulin G, fibrinogen, transferrin, alpha-2-macroglobulin, apolipoprotein A1, apolipoprotein B, fibronectin), lactoferrinectin. We used a combined approach to study urine supernatant samples obtained from patients with different stages of bladder cancer. This approach consisted in the use of immunoaffinity columns with immobilized nanobodies for specific pretreatment of urine samples and subsequent electrophoretic fractionation of proteins in the original or pretreated urine using the traditional SDS polyacrylamide gel (PAGE) gradient electrophoresis method. It was demonstrated that the development of bladder cancer in almost all 22 studied samples is accompanied by an increase in the content of major blood proteins in the urine, some of which have already been proposed as biomarkers of bladder cancer (fibrinogen, alpha-2-macroglobulin, apolipoprotein A1, fibronectin, lactoferrin). The isolation of immune complexes from the urine of a particular patient is of particular interest. During the initial study of these complexes, along with the increased content of IgA and IgG at the invasive stages of the disease, many other components can be seen in their composition, which can potentially be biomarkers of the pathological process in a particular patient. By using immunosorbents created on the basis of previously obtained nanobodies, which differently bind iron-free transferrin (apo-Tf) and iron-containing transferrin (holo-Tf), it was demonstrated that such immunosorbents allow detecting changes in the relative abundance of different forms of transferrin. in biological fluids of a person (in blood and urine). Works on cloning, creation of producers for the development in the bacterial system of the expression of a number of extracellular domains and proteins, which can be useful for early diagnosis of bladder cancer and with the aim of using them to obtain new nanobodies for them. Producers of the selected fragments of 21 proteins were obtained. A new library of clones of nanobodies sequences was created, enriched with nanobodies to exosomes of patients with bladder cancer. It has been shown that the method of polarization fluorescence immunoassay, based on measuring the change in the polarization of the fluorescence of a labeled nanobody upon its specific binding to the target antigen, according to our new data (on the quantitative detection of lactoferrin in urine) is very promising for the detection of major proteins in urine.

 

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