Over the past two decades, targeted therapy has actively developed and, demonstrating impressive clinical results, has gained an increasingly important role in the treatment of cancer. This was facilitated to a large extent by an in-depth understanding of the mechanisms of cancer development, and mainly, the discovery of molecular targets. Despite the fact that targeted therapy can radically change the results of treatment and the prognosis of the disease course in some cancer cases, its effectiveness is sometimes replaced by drug resistance, in others. The authors of the lecture analyzed and systematized therapeutic approaches to addressing a number of important molecular targets that are key for implementing a specific stage in human tumor pathogenesis. These include maintaining chronic proliferative signaling, promoting evasion of cell growth suppressors, inducing angiogenesis, forming immune surveillance, and activating invasion and metastasis. The lecture presented targeted therapy drugs used in the Russian Federation, including antibody-based drugs and small molecule tyrosine kinase inhibitors. It also analyzed mechanisms of molecular interaction between these drugs and their targets, as well as possible factors for developing resistance and ways to overcome these resistance mechanisms.

Aim. To study in vitro and in vivo the functional suitability of ^99mTc-labeled lyophilized formulation containing designed ankyrin repeat protein (DARPin) G3-(GGGS)₃Cys for radionuclide imaging of HER2/neu overexpression in malignant tumors.Materials and methods. To create a targeted protein, a modified genetic construct with the sequence encoding DARPin G3-(GGGS)₃Cys was used. To generate the experimental probe, we used a lyophilized formulation containing DARPin G3-(GGGS)₃Cys with auxiliary substances and ^99mTc sodium pertechnetate (500 MBq) incubated at 60 °C for 30 min. Radiochemical purity of ^99mTc-G3-(GGGS)₃Cys was analyzed by thin-layer radiochromatography. SKOV-3, BT-474, and DU-145 cell lines were used to test binding specificity in vitro. The dissociation constant was determined via a saturation binding assay on SKOV-3 cells with a range of protein concentrations from 0.2 to 40 nM. Nu/j mice bearing HER2-positive SKOV-3 xenografts and HER2-negative Ramos xenografts were used to evaluate the targeting properties and biodistribution.Results. A radiocomplex based on ^99mTc and a lyophilized formulation with DARPin G3-(GGGS)₃Cys was obtained with the radiochemical purity of more than 96%. Binding of ^99mTc-G3-(GGGS)₃Cys to the cells was specific (K_D 3.9 ± 0.5 nM) and proportional to the level of HER2/neu expression in the cells. The uptake of ^99mTc-G3-(GGGS)₃Cys in SKOV-3 xenografts was significantly higher than in Ramos xenografts. ^99mTc-G3-(GGGS)₃Cys demonstrated rapid blood and renal clearance and had low activity in the salivary glands and stomach. Liver uptake was about 5–7%ID/g. In addition, ^99mTc-G3-(GGGS)₃Cys exhibited very low uptakes in the lungs, muscles, small intestine, and bones.Conclusion. The ^99mTc-labeled lyophilized formulation with DARPin G3-(GGGS)₃Cys is functionally suitable for imaging HER2/neu overexpression in tumors, as it binds specifically to the receptor, is stable in vivo, and has favorable biodistribution in organs and tissues. The radiocomplex based on ^99mTc-G3-(GGGS)₃Cys was obtained by a simple method with high radiochemical purity.

Introduction. Overexpression of epidermal growth factor receptor (EGFR) is characteristic of a number of malignant neoplasms, making it an important molecular target for targeted radionuclide imaging. The designed ankyrin repeat protein DARPin (HE)₃-E01 with subnanomolar affinity to EGFR is considered as a promising chemical precursor for the preparation of radiopharmaceuticals labeled with technetium-99m or iodine-123. To be introduced into pharmaceutical practice, a set of validated quality control methods for this protein is required. Objective. To develop and validate methods for identification, purity assessment, and quantification of the DARPin (HE)₃-E01 protein. Materials and Methods. Identity was confirmed by liquid chromatography–electrospray ionization mass spectrometry (LC-ESI/MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Quantification was performed using direct UV spectrophotometry at 280 nm and the bicinchoninic acid (BCA) colorimetric assay. Validation was conducted according to the criteria of specificity, linearity, accuracy (standard addition method), and precision (repeatability, intermediate precision). The two quantitative methods were compared using the F-test and t-test. Results. Mass spectrometric analysis showed complete agreement between the experimental molecular mass (18,260.29 Da) and the theoretically expected value (18,260.25 Da). Electrophoretic determination of molecular weight gave a value of 18.1 kDa, and the protein purity was at least 99%, with impurity content not exceeding 1%. Both quantitative methods demonstrated linearity over the studied concentration range (correlation coefficients of 0.9979 and 0.9997), high accuracy (CV not more than 2% for both methods), and precision (RSD 0.05). Conclusion. The developed methods for identification and quantification of DARPin (HE)₃-E01 have been successfully validated and can be included in the draft regulatory documentation for quality control of the chemical precursor intended for the preparation of EGFR-targeted radiopharmaceuticals.

Non-invasive radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression can guide patient stratification for EGFR-targeted therapies. The designed ankyrin repeat protein (DARPin) E01, which binds EGFR ectodomain III with sub-nanomolar affinity, is a promising scaffold for single-photon emission computed tomography (SPECT) imaging probes. In the present study, we compared site-specific radioiodination of DARPin E01 using the bifunctional prosthetic group (4-hydroxyphenyl)ethyl maleimide (HPEM) with site-unspecific radioiodination via [¹²³I]I-para-iodobenzoate (PIB). [¹²³I]I-HPEM was conjugated to the C-terminus of DARPin E01 via Glu-Glu-Glu-Cys ([¹²³I]I-E01-E₃C-HPEM) or Gly-Gly-Gly-Cys ([¹²³I]I-E01-G₃C-HPEM) linkers. Radiolabelling yields were 6 ± 2% and 13 ± 5%, respectively. Size-exclusion purification provided radiochemical purity > 98%. Both HPEM conjugates retained nanomolar EGFR-binding affinity (K_D: 3.2 ± 0.6 and 4.8 ± 0.9 nM) and demonstrated EGFR-specific tumour accumulation in A-431 xenografts. Cellular processing was characterised by rapid binding, slow internalisation, and non-residualising behaviour of all variants. Kidney uptake was lower for the site-specifically labelled variants. However, site-specific labelling evidently elevated hepatobiliary excretion and uptake in Na/I-symporter-expressing organs compared to [¹²³I]I-(HE)₃-E01-PIB, while linker composition (E₃C vs. G₃C) did not significantly alter biodistribution. Site-unspecific radioiodination with [¹²³I]I-PIB remains the preferred approach for clinical SPECT imaging of EGFR expression with DARPin E01.

Radionuclide-based molecular imaging modalities are active and developing areas of functional and molecular diagnosis. Among the radionuclides used for SPECT imaging in oncology, ^99mTc is a leading candidate for radiolabeling. At present, a sufficient number of complexons for ^99mTc have been described; however, the development of effective delivery systems for this isotope to the area of interest is a complex research task. The use of tumor-targeting molecules as carriers for radioactive tracers is an effective strategy that has enabled the development of many novel radiopharmaceuticals for cancer imaging. Background: To date, a number of studies have shown tumorotropicity of tetrapyrrole compounds to tumor tissues, in particular derivatives of natural chlorophyll A. Methods: Purification was performed using solid-phase extraction. Assessment of radiochemical yield and purity was performed via radio-ITLC. The in vitro tumor cell accumulation was assessed using SKOV-3 and A-431 cell lines. Dose-dependent biodistribution was evaluated in Nu/J mice bearing epidermoid carcinoma (A-431) xenografts. Results: In this work, we obtained complexes with ^99mTc based on water-soluble carboxylate chlorin e₆ derivatives in order to evaluate their potential for use as SPECT radiopharmaceuticals. We performed radiolabelling optimization of a series of the novel chlorins and primary preclinical studies, including an assessment of the effect of their lipophilicity and charge on tumor uptake. Conclusions: Modification of the periphery of the chlorin macrocycle with chelating groups allows for complexing a wide range of metals, including ^99mTc, which can be used for targeted delivery of the radionuclide to the area of interest.