Development and Validation of the ELISA Method for Neutralizing Anti-trastuzumab Antibodies Detection in Human Blood Serum

Introduction. Trastuzumab is the first known anti-HER2 agent, which revolutionized the treatment of one of the most common cancer types – breast cancer. Despite trastuzumab being approved long time ago, further improvement of related analytical methods remains relevant primarily due to the emergence of new biosimilars. For instance, immunogenicity – adverse reaction which is usually associated with biological drugs, can still be relevant for trastuzumab. Anti-drug antibodies, including neutralizing antibodies, caused by trastuzumab therapy, can affect drug effectiveness and safety profile.

Aim. The aim of this study was to develop and validate the analytical method for neutralizing anti-trastuzumab antibodies determination in human blood serum.

Materials and methods. The neutralizing anti-trastuzumab antibody determination was carried out by the competitive ELISA method, using spectrophotometric detection in the visible range of the spectrum.

Results and discussion. The developed method was validated for cut-point, selectivity, sensitivity, specificity, precision and stability (short-term and long-term). To decrease the background noise from non-specific binding of sera components, the minimum required dilution value was determined at 0.5 % serum. The calculated value for cut-point was 14.62 %. The sensitivity of the developed method was estimated at 1985.2 ng/mL of neutralizing anti-trastuzumab antibodies.

Conclusion. The obtained results allowed us to apply the developed ELISA method for the neutralizing anti-trastuzumab antibodies determination in human blood serum during trastuzumab immunogenicity assessment in bioequivalence clinical trials.

1. Kaprin A. D., Starinskij V. V., Shahzadova A. O. Malignant neoplasms in Russia in 2020 (morbidity and mortality). Moscow: MNIOI named after P. A. Herzen – branch of the Federal State Budgetary Institution "NMITs Radiology" of the Ministry of Health of Russia; 2021. 252 p. (In Russ.)

2. Kaprin A. D., Starinskij V. V., Shahzadova A. O. The State of Oncological Care for the Population of Russia in 2020. Moscow: MNIOI named after P. A. Herzen – branch of the Federal State Budgetary Institution "NMITs Radiology" of the Ministry of Health of Russia; 2021. 239 p. (In Russ.)

3. Duan S., Buxton I. L. O. Evolution of Medical Approaches and Prominent Therapies in Breast Cancer. Cancers. 2022;14(10):2450. DOI: 10.3390/cancers14102450.

4. Tarantino P., Morganti S., Curigliano G. Biologic therapy for advanced breast cancer: recent advances and future directions. Expert Opinion on Biological Therapy. 2020;20(9):1009–1024. DOI: 10.1080/14712598.2020.1752176.

5. Semiglazova T. Yu., Sharashenidze S. M., Kerimova S. N., Klimenko V. V., Malygin A. Yu., Dashyan G. A., Paltuev R. M., Semiglazov V. V., Krivorotko P. V., Novikov S. N., Semiglazov V. F. Current approaches to the treatment of HER2-positive breast cancer with brain metastases. Tumors of female reproductive system. 2021;17(1):27–34. (In Russ.) DOI: 10.17650/1994-4098-2021-17-1-27-34.

6. Liu X., Fang Y., Li Y., Li Y., Qi L., Wang X. Pertuzumab combined with trastuzumab compared to trastuzumab in the treatment of HER2-positive breast cancer: A systematic review and meta-analysis of randomized controlled trials. Frontiers in Oncology. 2022;12:894861. DOI: 10.3389/fonc.2022.894861.

7. Mohammadi M., Jeddi-Tehrani M., Golsaz-Shirazi F., Arjmand M., Bahadori T., Judaki M. A., Shiravi F., Zare H. A., Haghighat F. N., Mobini M., Amiri M. M., Shokri F. A Novel Anti-HER2 Bispecific Antibody with Potent Tumor Inhibitory Effects In Vitro and In Vivo. Frontiers in Immunology. 2021;11:600883. DOI: 10.3389/fimmu.2020.600883.

8. Li B., Meng Y., Zheng L., Zhang X., Tong Q., Tan W., Hu S., Li H., Chen Y., Song J., Zhang G., Zhao L., Zhang D., Hou S., Qian W., Guo Y. Bispecific antibody to ErbB2 overcomes trastuzumab resistance through comprehensive blockade of ErbB2 heterodimerization. Cancer Research. 2013;73(21):6471–6483. DOI: 10.1158/0008-5472.CAN-13-0657.

9. Komarova, T. V., Sheshukova, E. V., Kosobokova, E. N. Kosorukov V. S., Shindyapina A. V., Lipskerov F. A., Shpudeiko P. S., Byalik T. E., Dorokhovet Y. L. The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells. Scientific Reports. 2019;9:16168. DOI: 10.1038/s41598-019-52507-9.

10. Volk A. L., Mebrahtu A., Ko B. K., Lundqvist M., Karlander M., Lee H. J., Frejd F. Y., Kim K. T., Lee J. S., Rockberg J. Bispecific Antibody Molecule Inhibits Tumor Cell Proliferation More Efficiently Than the Two-Molecule Combination. Drugs in R&D. 2021;21(2):157–168. DOI: 10.1007/s40268-021-00339-2.

11. Wang Z. H., Zheng Z. Q., Jia S. C., Liu S. N., Xiao X. F., Chen G. Y., Liang W. Q., Lu X. F. Trastuzumab resistance in HER2-positive breast cancer: Mechanisms, emerging biomarkers and targeting agents. Frontiers in Oncology. 2022;12:1006429. DOI: 10.3389/fonc.2022.1006429.

12. Kilany L. A. A., Gaber A. A. S., Aboulwafa M. M., Zedan H. H. Trastuzumab immunogenicity development in patients’ sera and in laboratory animals. BMC Immunology. 2021;22(1):15. DOI: 10.1186/s12865-021-00405-z.

13. da Luz F.A.C., da Costa Marinho E., Nascimento C. P., de Andrade Marques L., Delfino P. F. R., Antonioli R. M., Silva M. J. B., de Araújo R. A. The benefits of trastuzumab in the treatment of HER2⁺ breast cancer as a function of exposure time. Ecancermedicalscience. 2022;16:1347. DOI: 10.3332/ecancer.2022.1347.

14. Barbier, L., Declerck, P., Simoens, S., Neven P., Vulto A., Huys I. The arrival of biosimilar monoclonal antibodies in oncology: clinical studies for trastuzumab biosimilars. British Journal of Cancer 2019;121:199–210. DOI: 10.1038/s41416-019-0480-z.