The use of radiopharmaceuticals for diagnostics in oncology allows for the detection of the disease at an early stage. Among diagnostic radionuclides, ^99mTc is a promising isotope that has been used to create several drugs for clinical use. One of the most effective ^99mTc chelators is 6-hydrazinylnicotinic acid (HYNIC), which, when combined with various vector molecules, can be used for targeted delivery of radionuclides to tumor tissues. At the same time, it is known that tetrapyrrole macrocycles are capable of selective accumulation in tumors, and thus can be used to target radiopharmaceuticals with ^99mTc. In this work, the conjugate of natural chlorin and HYNIC was obtained, and preliminary preclinical studies were carried out on its radiocomplex with ^99mTc.

Radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression might permit the selection of patients for EGFR-targeting therapies. Designed ankyrin repeat protein (DARPin) E01 with a high affinity to the ectodomain III of the EGFR is a possible EGFR imaging probe. The goal of this study was to evaluate the potential of radiolabeled DARPin E01 for in vivo imaging of EGFR. DARPin E01 containing the (HE)₃-tag was site-specifically labeled with a residualizing ^99mTc (using ^99mTc]Tc(CO)₃). Two methods providing non-residualizing ¹²³I labels, direct electrophilic radioiodination and indirect radioiodination using [¹²³I]I-para-iodobenzoate (PIB), were tested. [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB preserved specific binding to EGFR-expressing cells and affinity in the single-digit nanomolar range. Direct labeling with ¹²³I resulted in a substantial loss of binding. In vitro cellular processing studies showed that both [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB had rapid binding and relatively slow internalization. Evaluation of [^99mTc]Tc-(HE)₃-E01 biodistribution in normal CD1 mice showed that its hepatic uptake was non-saturable, suggesting that this tracer does not bind to murine EGFR. A side-by-side comparison of biodistribution and tumor targeting of [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB was performed in Nu/j mice bearing EGFR-positive A-431 and EGFR-negative Ramos human cancer xenografts. Both radiolabeled DARPins demonstrated EGFR-specific tumor uptake. However, [¹²³I]I-(HE)₃-E01-PIB had appreciably lower uptake in normal organs compared to [^99mTc]Tc-(HE)₃-E01, which provided significantly (p < 0.05) higher tumor-to-organ ratios. Gamma-camera imaging confirmed that [¹²³I]I-(HE)₃-E01-PIB demonstrated a higher imaging contrast in preclinical models than [^99mTc]Tc-(HE)₃-E01. In conclusion, DARPin (HE)₃-E01 labeled using a non-residualizing [¹²³I]I-para-iodobenzoate (PIB) label is the preferred radiotracer for in vivo imaging of EGFR expression in cancer.

Introduction. Overexpression of the HER2 receptor is associated with aggressive cancer progression and poor prognosis. Traditional immunohistochemical diagnostics have a number of limitations, including invasiveness, inability to assess tumor heterogeneity, and total spread of the process. A promising alternative is radionuclide imaging using targeted scaffold DARPin G3 proteins. Based on the results of preclinical studies, the drug ^99m Tc[Tc]-G3-(G ₃ S) ₃ C has been proposed for Phase I clinical trials. The experimental drug is a sterile lyophilizate of a chemical precursor in a single vial and must comply with the requirements of OFS 1.11.0005 "Chemical Precursors for Radiopharmaceuticals" and OFS 1.7.1.0007.15 "Medicinal Products Obtained by Recombinant DNA Methods." Aim. The aim of the present study was to develop approaches and techniques for quality control of DARPin G3-(G ₃ S) ₃ C protein included in the developed composition of chemical precursor lyophilizate lyophilizate for preparation of ^99m Tc-containing preparation for radionuclide imaging of HER2 overexpression in malignant tumors Materials and methods. A lyophilizate containing DARPin G3-(G ₃ S) ₃ C with excipients was prepared for quality control. HPLC with a tandem mass spectrometer equipped with an electrospray ionization source was used to record mass spectra. Vertical electrophoresis with SDS in a heterogeneous buffer system was carried out in a polyacrylamide gel with an acrylamide concentration of 15 % at 110 V for 2 hours. Coomassie B-250 solution was used as the dye solution. Peptide mapping was performed using an HPLC system with a mass spectrometer via a nano-electrospray source. When correlating the amino acid sequence, carbamidomethylation of Cys, deamidation of Asn/Gln and oxidation of Met were counted as variable modifications. UV spectrophotometry was performed to determine the Quantification index at a wavelength of 280 nm. Validation evaluation of the developed method was carried out in accordance with the requirements of OFS.1.1.0012 "Validation of analytical methods". Results and discussion. The authenticity and purity of DARPin G3-(G ₃ S) ₃ C was confirmed using HPLC-MS. The purity of the protein was close to 100 %. Application of 2 μg of protein for electrophoresis in polyacrylamide gel showed a distinct stain allowing the authenticity of DARPin G3-(G ₃ S) ₃ C to be established. To determine the homodimer impurity, 10 μg of protein should be applied. The purity of the protein established by the peptide mapping method is 98 %. More than 700 variations of peptide fragments were identified by protein hydrolysate analysis. The value of –10LogP was more than 20. The developed UV-visible spectrophotometry technique meets the validation requirements and allows the quantification of DARPin G3-(G ₃ S) ₃ C protein in the lyophilizer ±10 % of the nominal content. Conclusion. To determine the "Genuineness" of a new chemical precursor of DARPin G3-(G ₃ S) ₃ C protein in the lyophilizate for the preparation of ^99m Tc-containing preparation for radionuclide imaging of HER2 overexpression in malignant tumors the following methods were proposed: mass spectrometry, peptide mapping, electrophoresis in polyacrylamide gel. For the indicator "Related impurities" – the technique of electrophoresis in polyacrylamide gel. For the indicator "Quantitative determination" the method of UV-spectrophotometry.

INTRODUCTION. Over the past two decades, targeted therapy and diagnostics have been developing rapidly and have acquired an increasingly important role in the treatment of cancer. This progress is largely driven by a deeper understanding of the mechanisms of cancer development and by the discovery of molecular targets, among which the human epidermal growth factor receptor (EGFR) occupies one of the key positions. Despite the clinical success of tyrosine kinase inhibitors, the development of resistance and the limitations of biopsy-based diagnostic methods necessitate the search for new solutions using radionuclide technologies. AIM. To systematize and analyze current approaches to targeted radionuclide the­rapy and to the diagnostics of EGFR-expressing tumors. DISCUSSION. The evolution of tyrosine kinase inhibitors (TKIs) targeting EGFR-­expressing tumors, from reversible first-generation agents to irreversible fourth-­generation inhibitors (JIN-A02), has enabled the consistent overcoming of resis­tance mutations, including T790M and C797S. For JIN-A02, partial responses with up to 77% regression and intracranial metastasis control in C797S-mediated resistance have already been documented. New classes of agents, including antibody–drug conjugates (MRG003, approved in China in 2025) and photoimmunoconjugates (cetuximab–IR700, approved in Japan in 2020), have expanded therapeutic capabi­lities through the targeted delivery of cytotoxic agents. In the field of radionuclide molecular imaging, full-length radiolabeled antibodies, such as ⁸⁹ Zr-cetuximab, are characterized by slow clearance and a high background signal. Small vector molecules currently under preclinical development exhibit markedly more favorable diagnostic characteristics: [ ⁸⁹ Zr]Zr-DFO-ZEGFR:2377 (affibody) exhibits a tumor-to-blood ratio of 3.7 at 24 h after injection, which is 2-fold higher than that of ⁸⁹ Zr-cetuximab; [ ⁶⁸ Ga]Ga-Df-Bz-NCS-7D12 (nanobody) accumulates in tumors (A431 xenografts) at a level of 6% injected dose per gram of tissue as early as 1 h after injection, while [ ¹²³ I]I-(HE) ₃ -E01-PIB (DARPin, designed ankyrin repeat protein) provides tumor-to-blood ratios of up to 13. CONCLUSIONS. The integration of innovative therapeutic agents with highly sensitive molecular imaging creates a basis for personalized treatment of EGFR-­expressing tumors and for overcoming resistance mechanisms. A promising direction is the development of theranostic approaches based on single vector molecules labeled with diagnostic ( ⁸⁹ Zr, ⁶⁸ Ga) and therapeutic ( ¹⁷⁷ Lu, ⁹⁰ Y) radionuclides. Howe­ver, no EGFR-targeted theranostic platform has yet been approved for clinical use, which defines the direction of further research.

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.