Ga-68 labeled Trastuzumab Fab for imaging HER2 positive lesions: formulation and characterization.

Introduction: A monoclonal antibody Trastuzumab is FDA approved for the treatment HER2 expressing breast cancer. The HER2 have specific binding domain IV for the Trastuzumab. The binding of Fab region of Trastuzumab binds to HER2 receptor, stop the dimerization of the receptor and inhibits the tumour growth. Fab being a smaller peptide (45 kDa) is explored for imaging and therapeutic use. Methodology The Fab was generated from the whole monoclonal antibody Trastuzumab by papain digestion in phosphate buffer (1M) at 37°C and 850 rpm for the 22 hours. The purification of Fab from digested trastuzumab was carried out with Amicon ultrafiltration (30 kDa) tube. The purified Fab molecular weight 45 kDa was analyzed by the MALDI. The concentration of Fab after the digestion was measured by the UV-VIS spectrophotometry and nanodrop at 280 nm wavelength. After the Fab generation, the Fab was conjugated with NOTA, a bifunctional chelating agent. The molar ratios of NOTA and Fab were optimized by the repetition of experiments at different molar ratios. The incubation time and temperature conditions were also optimized. The purification of NOTA conjugated Fab from unconjugated chelating agents was performed with a PD-10 column. The number of chelating molecules in conjugated Fab was calculated by the mass of conjugated and unconjugated Fab obtained by mass spectra. The radiolabeling of NOTA- Fab was standardized with the Ga-68, purified and passed through 0.22 micron filter. The quality control was performed to be used for intravenous injection. For patient study, 3- 5 mCi dose was used. The patients with biopsy-proven HER2 positive tumour were recruited for the study. The biodistribution of Ga-68 Fab and localization in lesions were observed.

Results: The Fab of 45 kDa was obtained papain digestion and the same was characterized by mass spectra. The NOTA chelator conjugation conditions were standardized as 22 h incubation at 4 °C. The average number NOTA conjugated per Fab was 1.5. The radiolabeling efficiency of Ga-68 Fab was 48% and after purification of radiolabeled product, the radiochemical purity was more than 80%. The Ga-68 Fab was found sterile and the endotoxin concentration was below 175 EU/V. The whole body PET/CT demonstrated high blood pool, kidney and bladder activity. The high liver uptake was also observed in the Ga-68 Fab image and the metastatic lesions were visualized at similar locations as observed in FDG PET/CT image. However not all the lesions were visualized in Ga-68 Fab PET/CT. Additionally, the uptake of Ga-68 Fab in lesions was improved in 2 h image.

Conclusions: The Ga-68 Fab may have the potential to image HER 2 lesions and be used to select the patients for Trastuzumab therapy and also to evaluate response to Trastuzumab therapy.