Development of quality control techniques of chemical precursor – recombinant protein with ankyrin repeats for radionuclide imaging of HER2 overexpression in malignant tumors

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 ^99mTc[Tc]-G3-(G₃S)₃C 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-(G₃S)₃C protein included in the developed composition of chemical precursor lyophilizate lyophilizate for preparation of ^99mTc-containing preparation for radionuclide imaging of HER2 overexpression in malignant tumors

Materials and methods. A lyophilizate containing DARPin G3-(G₃S)₃C 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-(G₃S)₃C 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-(G₃S)₃C 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-(G₃S)₃C protein in the lyophilizer ±10 % of the nominal content.

Conclusion. To determine the "Genuineness" of a new chemical precursor of DARPin G3-(G₃S)₃C protein in the lyophilizate for the preparation of ^99mTc-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.

1. Bartley A. N., Washington M. K., Ventura C. B., Ismaila N., Colasacco C., Benson A. B., Carrato A., Gulley M. L., Jain D., Kakar S., Mackay H. J., Streutker C., Tang L., Troxell M., Ajani J. A. HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline From the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology. Archives of Pathology & Laboratory Medicine. 2016;140(12):1345–1363. DOI: 10.5858/arpa.2016-0331-CP.

2. Gebhart G., Lamberts L. E., Wimana Z., Garcia C, Emonts P., Ameye L., Stroobants S., Huizing M., Aftimos P., Tol J., Oyen W. J. G., Vugts D. J., Hoekstra O. S., Schröder C. P., Menke-van der Houven van Oordt C. W., Guiot T., Brouwers A. H., Awada A., de Vries E. G. E., Flamen P. Molecular imaging as a tool to investigate heterogeneity of advanced HER2-positive breast cancer and to predict patient outcome under trastuzumab emtansine (T-DM1): the ZEPHIR trial. Annals of oncology. 2016;27(4):619–624. DOI: 10.1093/annonc/mdv577.

3. Tolmachev V. Imaging of HER-2 overexpression in tumors for guiding therapy. Current Pharmaceutical Design. 2008;14(28):2999–3019. DOI: 10.2174/138161208786404290.

4. Wolff A. C., Somerfield M. R., Dowsett M., Hammond M. E. H., Hayes D. F., McShane L. M., Saphner T. J., Spears P. A., Allison K. H. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: ASCO-College of American Pathologists Guideline Update. Journal of Clinical Oncology. 2023;41(22):3867–3872. DOI: 10.1200/jco.22.02864.

5. Azhar A., Ahmad E., Zia Q., Rauf M. A., Owais M., Ashraf G. M. Recent advances in the development of novel protein scaffolds based therapeutics. International Journal of Biological Macromolecules. 2017;102:630–641. DOI: 10.1016/j.ijbiomac.2017.04.045.

6. Sörensen J., Velikyan I., Sandberg D., Wennborg A., Feldwisch J., Tolmachev V., Orlova A., Sandström M., Lubberink M., Olofsson H. Measuring HER2-receptor expression in metastatic breast cancer using [68Ga] ABY-025 Affibody PET/CT. Theranostics. 2016;6(2):262.

7. Skuridin V. S., Stasyuk E. S., Bragina O. D., Yusubov M. S., Chernov V. I., Larkina M. S., Zelchan R. V., Rogov A., Sinilkin I., Larionova L. Development of radiopharmaceutical based on mini-antibody for early cancer detection. Annual Congress of the European-Association-of-Nuclear-Medicine. 2016;43:465–465.

8. Bragina O. D., Larkina M. S., Stasyuk E. S., Chernov V. I., Yusubov M. S., Skuridin V. S., Deyev S. M., Zel’chan R. V., Buldakov M. A., Podrezova E. V., Belousov M. V. The development of a highly specific radiochemical compound based on labeled ^99mТс recombinant molecules for targeted imaging of cells with the overexpression of Her-2/neu. Bulletin of Siberian Medicine. 2017;16(3):25–33. (In Russ.) DOI: 10.20538/1682-0363-2017-3-25-33.

9. Bragina O. D., Chernov V. I., Zeltchan R. V., Sinilkin I. G., Medvedeva A. A., Larkina M. S. Alternative scaffolds in radionuclide diagnosis of malignancies. Bulletin of Siberian Medicine. 2019;18(3):125–133. (In Russ.) DOI: 10.20538/1682-0363-2019-3-125-133

10. Plückthun A. Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annual review of pharmacology and toxicology. 2015;55:489–511.

11. Deyev S. M., Xu T., Liu Y., Schulga A., Konovalova E., Garousi J., Rinne S. S., Larkina M., Ding H., Gräslund T., Orlova A., Tolmachev V., Vorobyeva A. Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec1. Cancers. 2021;13(14):3589. DOI: 10.3390/cancers13143589.

12. Zelchan R., Chernov V., Medvedeva A., Rybina A., Bragina O., Mishina E., Larkina M., Varvashenya R., Fominykh A., Schulga A., Konovalova E., Vorobyeva A., Orlova A., Tashireva L., Deyev S. M., Tolmachev V. Phase I Clinical Evaluation of Designed Ankyrin Repeat Protein [^99mTc]Tc(CO₃-(HE)₃-Ec1 for Visualization of EpCAM-Expressing Lung Cancer. Cancers. 2024;16(16):2815. DOI: 10.3390/cancers16162815.

13. Kobe B, Kajava A.V. When protein folding is simplified to protein coiling: the continuum of solenoid protein structures. Trends in Biochemical Sciences. 2000;25(10):509–515. DOI: 10.1016/s0968-0004(00)01667-4.

14. Binz H. K., Stumpp M. T., Forrer P., Amstutz P., Plückthun A. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. Journal of Molecular Biology. 2003;332(2):489–503. DOI: 10.1016/s0022-2836(03)00896-9.

15. Goldstein R., Sosabowski J., Livanos M., Leyton J., Vigor K., Bhavsar G., Nagy-Davidescu G., Rashid M., Miranda E., Yeung J., Tolner B., Plückthun A., Mather S., Meyer T., Chester K. Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging. European Journal of Nuclear Medicine and Molecular Imaging. 2015;42(2):288–301. DOI: 10.1007/s00259-014-2940-2.

16. Vorobyeva A., Schulga A., Konovalova E., Güler R., Löfblom J., Sandström M., Garousi J., Chernov V., Bragina O., Orlova A. Optimal composition and position of histidine-containing tags improves biodistribution of 99mTc-labeled DARPin G3. Scientific Reports. 2019;9(1):9405.

17. Bragina O., Chernov V., Schulga A., Konovalova E., Garbukov E., Vorobyeva A., Orlova A., Tashireva L., Sörensen J., Zelchan R., Medvedeva A., Deyev S., Tolmachev V. Phase I Trial of ^99mTc-(HE)₃-G3, a DARPin-Based Probe for Imaging of HER2 Expression in Breast Cancer. Journal of Nuclear Medicine. 2022;63(4):528–535. DOI: 10.2967/jnumed.121.262542.

18. Larkina M., Plotnikov E., Bezverkhniaia E., Shabanova Y., Tretyakova M., Yuldasheva F., Zelchan R., Schulga A., Konovalova E., Vorobyeva A. Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with ^99mTc for Radionuclide Imaging of HER2 Expression in Cancer. International Journal of Molecular Sciences. 2022;23(21):13443.

19. Larkina M., Varvashenya R., Yuldasheva F., Plotnikov E., Bezverkhniaia E., Tretyakova M., Zelchan R., Schulga A., Konovalova E., Vorobyeva A., Belousov M., Orlova A., Tolmachev V., Deyev S. Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the ^99mTc-Based Imaging of HER2-Expressing Malignant Tumors. Molecular Pharmaceutics. 2024;21(4):1919–1932. DOI: 10.1021/acs.molpharmaceut.3c01173.

20. Varvashenya R. N. Prach A. A., Plotnikov E. B., Deev S. M., Belousov M. V., Larkina M. S., Chernov V. I. Assessing functional suitability of a lyophilized formulation containing designed ankyrin repeat proteins for radionuclide imaging of HER2/neu overexpression in malignant tumors. Bulletin of Siberian Medicine. 2024;23(3):16–24. (In Russ.) DOI: 10.20538/1682-0363-2024-3-16-24.

21. Malakhov M. P., Mattern M. R., Malakhova O. A., Drinker M., Weeks S. D., Butt T. R. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. Journal of Structural and Functional Genomics. 2004;5(1–2):75–86.

22. Vorobyeva A., Sсhulga A., Konovalova E., Güler R., Mitran B., Garousi J., Rinne S., Löfblom J., Orlova A., Deyev S. Comparison of tumor-targeting properties of directly and indirectly radioiodinated designed ankyrin repeat protein (DARPin) G3 variants for molecular imaging of HER2. International Journal of Oncology. 2019;54(4):1209–1220.

23. Kiseleva D. G., Zigashin R. H., Fotin D. P., Markin A. M. Proatherogenic proteomic profile of LDL isolated from plasma of patients with diabetes mellitus: immunological aspects. Russian Journal of Immunology. 2024;27(2):253–258. (In Russ.) DOI: 10.46235//1028-7221-16674-PPP.

24. Ma B., Zhang K., Hendrie C., Liang C., Li M., Doherty-Kirby A., Lajoie G. PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Communications in Mass Spectrometry. 2003;17(20):2337–2342. DOI: 10.1002/rcm.1196.

25. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry. 1989;182(2):319–326. DOI: 10.1016/0003-2697(89)90602-7.