Radiolabeling of Anti-HER2 scFv with 99m-Tc tricarbonyl through His-tag

Radiolabeling of Anti-HER2 scFv with 99m-Tc tricarbonyl through His-tag
Negar Bozorgchami,¹ Maryam Ahmadzadeh,² Elham Mohit,^3,* Soraya Shahhosseini,⁴
1. Department of Pharmaceutical Chemistry and Radiopharmacy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4. Department of Pharmaceutical Chemistry and Radiopharmacy, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Introduction: Breast cancer is the most common cancer and the second cause of cancer death in women, worldwide. Recently, invasive biopsy method is used for detection of benign from malignant tissues. Correlation between breast cancer malignancy and human epidermal growth factor 2 (HER2) overexpression has led to the application of monoclonal antibodies targeting HER2 receptors in treatment and diagnosis of HER2-overexpressing breast cancer. Single chain variable fragment (scFv) contains variable regions of heavy and light chains of an antibody and is the smallest functional region of an antibody for antigen binding. The penetrability of scFvs into tumors is improved while its specific affinity is retained and its immunogenicity is lowed as compared to full length antibody. scFv against HER2 can be conjugated to radioisotopes for staging and diagnosis of HER2-positive breast cancers and their metastasis. 99mTc is used in over 98% of radiopharmaceuticals. Its optimal nuclear properties including short half-life (6 h), decomposition through isomeric transition (IT), gamma photon emission of 140 keV, availability via 99Mo/99mTc generator and known chemical properties make 99mTc a suitable radionuclide for diagnostic applications in nuclear medicine. Technetium-99m forms very stable complexes in its inert stable +1 oxidation state in the form of aqueous 99m-Tc-tricarbonyl with His-tag which is used as purification tag for immobilized metal affinity chromatography (IMAC). 99mTc(I) tricarbonyl provides high labeling yield and the least damage to biological activity of scFv. The aim of this study is to label anti-HER2 scFv with 99m-Tc-tricarbonyl and evaluate the effect of radiolabeling on biological activity of anti-HER2 scFv.
Methods: Anti-HER2 scFv gene was expressed in Escherichia coli BL21 (DE3) host. Anti-HER2 scFv protein was then purified by IMAC under native condition. Anti-HER2 scFv was directly labeled by techneyium-99m tricarbonyl. Radiochemical purity (RCP¬%) was determined using thin layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). Biologic activity of anti-HER2 scFv and 99m-Tc-anti-HER2 scFv was evaluated by HER2-based ELISA.
Results: Highly pure anti-HER2 scFv with approximately 28 kDa molecular weight was obtained by IMAC using Ni-NTA resin under native condition. RCP% of radiolabeled anti-HER2 scFv (99m-Tc-anti-HER2 scFv) was approximately around 98%. HER2-based ELISA demonstrated no significant difference between the biologic activity of anti-HER2 scFv and 99m-Tc-anti-HER2 scFv.
Conclusion: Anti-HER2 scFv was directly radiolabeled by 99m-Tc- tricarbonyl. Based on the results, 99m-Tc-anti-HER2 scFv is a proper candidate for specific targeting of HER2 receptor and can be used for diagnosis of breast cancer type, detecting metastasis and evaluation of treatment efficacy in HER2 overexpressing-breast cancer.
Keywords: Breast cancer, HER2, scFv, Radiolabeling, Technetium-99m tricarbonyl