Analysis of Transferrin in the Urine of Patients with Bladder Cancer Using Nanobodies

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Anastasiya M. Sachko¹, Oksana S. Goryainova¹, Tatiana I. Ivanova^1,2, Irina Yu. Nikolaeva³, Mariya E. Tarnopolskaia³, Andrew Y. Bychkov³, Margarita Ya. Gaas⁴, Nikolay V. Vorob’ev^5,6, Andrey D. Kaprin^4,7, Petr V. Shegay^4,7, and Sergei V. Tillib^1,a*

¹Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

²Tsyb Medical Radiological Research Centre, Branch of the National Medical Research Radiological Centre, Ministry of Health of the Russian Federation, 249036 Obninsk, Russia

³Faculty of Geology, Lomonosov Moscow State University, 119234 Moscow, Russia

⁴Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia

⁵Hertsen Moscow Oncology Research Institute, Branch of the National Medical Research Radiological Centre, Ministry of Health of the Russian Federation, 125284 Moscow, Russia

⁶Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation, 119991 Moscow, Russia

⁷National Medical Research Radiological Centre, Ministry of Health of the Russian Federation, 125284 Moscow, Russia

^* To whom correspondence should be addressed.

Received November 30, 2022; Revised June 14, 2023; Accepted July 3, 2023

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It is known that the saturation ratio of transferrin (Tf) with iron in human blood is an important clinical parameter. Specific antibodies can be used to analyze subtle changes in the relative abundance of different forms of transferrin potentially associated with a pathological process. Recently, the authors of this study were able to obtain and characterize highly specific single-domain antibodies (nanobodies) that predominantly recognize the iron-saturated (holo-Tf) or iron-unsaturated (apo-Tf) form of transferrin. In this work, under conditions closer to physiological than in the previous experiments, we further demonstrated that these unique nanobodies have extremely high differential binding specificity for different forms of Tf in different human biological fluids. Using these nanobodies, we were able to analyze for the first time relative abundance of the transferrin forms in urine samples from the patients with bladder cancer (BC). We have shown that increase in the concentration of total Tf in the urine samples normalized for creatinine is associated with the degree of progress and growth of malignancy of BC. In the samples of healthy donors and in the early stages of BC (G1), Tf is detected in much smaller amounts (compared to the later stages) and only with additional concentration of the studied samples. For most of the studied urine samples from the BC patients, it is expected (as previously shown in the case of Tf in the blood of terminal ovarian cancer patients) that the concentration of apo-Tf is clearly higher than holo-Tf, especially in the case of the most advanced muscle-invasive BC. It was a surprise for us that approximately equal amounts of apo-Tf and holo-Tf were found in the urine samples of some patients with BC. We hypothesized that the holo-Tf fraction in this case could be largely represented by the “secondary complexes” formed by apo-Tf in combination with ions other than Fe³⁺, which accumulate in the urine of some cancer patients and are able to bind to apo-Tf, changing its conformation towards holo-Tf. By using inductively coupled plasma mass spectroscopy (ICP-MS), we obtained first results confirming our hypothesis. Preparation of the holo-Tf in these urine samples was found to be highly enriched in zinc and nickel. Also, relative enrichment in cadmium has been observed in this preparation, but at much lower concentrations. The obtained data indicate that the used nanobody, while recognizing predominantly the iron-saturated form of transferrin (holo-Tf), is also capable of binding transferrin in association with other metal ions that are different from iron. This ability could potentially open up new possibilities for investigation of relative abundance of various metal ions in association with transferrin in human biological fluids in normal and pathological conditions.

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KEY WORDS: single-domain antibody, nanobody, apo- and holo-transferrin, affinity chromatography, bladder cancer, ICP-MS

DOI: 10.1134/S0006297923080059

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