28P - Development 68Ga trastuzumab Fab and bioevaluation by PET imaging in HER2/neu-expressing breast cancer patients

Background

A target-specific mAb for the HER2 receptor is trastuzumab, which is used for treatment of breast cancer. This mAb coupled with radionuclides β or α particle emitters are used for radioimmunotherapy. However, radioimmunotherapy with the whole mAb is still limited due to slow pharmacokinetics. This leads to the slow elimination of trastuzumab from the blood and low tumor to blood and tumor to background ratios. To limit this, a small molecule Fab was introduced but the role in HER2 detection by immuno-PET imaging is unknown.

Methods

Fab has been generated by papain digestion and conjugation with NOTA. The characterization of Fab and NOTA Fab were performed by the SDS-PAGE and mass spectrometry technique MALDI-TOF. In vitro stability and target specificity for HER2/neu receptor were performed in plasma and receptor binding with bio-layer interferometry technique. After standardization of radiolabelling with 68GaCl3 the in vivo target efficiency was performed in HER2.positive breast cancer patients, which compared with 18F-FDG and corelated with immunohistochemistry.

Results

Fab generation was standardized at molar ratio 140:1 wt/wt of trastuzumab and papain, temp 37°C, stirrer at 850 rpm, time 20-24h.The purity were found >99% on SDS-PAGE (band 43 and 29 kDa) and MALDI-TOF (45 kDa). The conjugation was done at molar ratio of 1:25 and 1:30. The radiolabelling yield 48-70% were standardized at parameters noted pH 4.5-5, temp 37-40°C, incubation time 15 min for Rxn Vol 2.5mL. The trastuzumab affinity for HER2 were found in the picomolar range (Kon and Koff 3.79 E+05M-1s-1 and <1.0E-7s-1), Fab 1.9nM (Kon and Koff 3.9 E+05M-1s-1 and 4.77E-4s-1) and NOTA Fab 20 nM (Kon Koff 7.4 E+04M-1s-1 and 1.6E-3s-1). The stability of the radiopharmaceutical was found to be more than 4h and sterile up to 14 days. The endotoxin limit was found 5-10 EU/mL. In vivo targeting potential was checked in HER2-positive patients with uptake at primary lesion (SUV)max 3-5.8 and at lymph node 2.0-3.1, while liver and kidney also showed high uptake.

Conclusions

The 68Ga trastuzumab Fab can be formulated with high radiochemical purity and demonstrates high receptor affinity for HER2/neu receptor. The 68Ga trastuzumab Fab can be used as a diagnostic tool in immunoPET imaging for the HER2/neu receptor.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Board of Research in Nuclear Sciences (BRNS) and University Grants Commission (UGC).

Disclosure

All authors have declared no conflicts of interest.