Abstract B043: Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer
Authors: Shukla Jaya , Rathore Yogesh , Kaur Komalpreet , Irrinky Santosh , Kumar Rajender , Bal Amanjit , Mittal Bhagwant R 
Published: February 2024

Breast cancer is the most common cause of mortality among women worldwide. Among all subtypes, HER2-positive breast cancers are highly aggressive and have poor prognosis. Surgical resection is choice of treatment. However, surgery may not be possible if disease is widespread. Trastuzumab, a monoclonal antibody, is globally used for immunotherapy. It is a large sized molecule (141kDa) and show slow pharmacokinetics and high liver uptake. We have explored trastuzumab fragment as a radio-theranostic agent. Trastuzumab Fab was generated using Papain enzyme tagged beads, incubation time 18 to 20 h at 850 rpm in phosphate buffer. Fab were collected with ultrafiltration device, cut off 30 kDa, in phosphate buffer pH 8. Quantification of Fab was done at 280 nm. Fab characterization was done using MALDI-TOF and SDS-PAGE. To enable radiolabelling of Fab with Ga-68 and Lu-177, Fab was conjugated with bifunctional chelator, DTPA. Fab-DTPA was separated by micro bio-spin P-6 chromatography columns and the average number of DTPA/Fab were calculated. Affinity of Fab conjugated Fab-DTPA for HER2 receptors was studied using the biolayer interferometry technique (BLI). Radiolabelling of Fab with Ga-68 for PET imaging and Lu-177 for therapy was optimized. Quality control included radiochemical purity using TLC in sodium citrate (pH 5) mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. Receptor affinity and apoptosis were studied with HER2 positive SKBR-3 cells using flow cytometry assay. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a known amount of Lu-177-DTPA-Fab (0- 18.5 MBq). First in human Ga-68 Fab PET/CT and Lu-177-Fab gamma camera images were acquired after obtaining clearance from Institutional Ethics Committee and inform signed consent. Patients were recruited with histopathological proven HER2 positive breast cancer and uptake on F-18 FDG PET/CT. Ga-68-DTPA-Fab (3.5-5 mCi) and 10-15 mCi Lu-177 DTPA-Fab was injected and images were acquired at 2/3 h and up to 7 days respectively. Dominant Fab peak at 45kDa on MALD-TOF, single band on SDS-PAGE (Non-reduced or reduced) have been obtained. Two DTPA/Fab were conjugated and confirmed by MALDI-TOF. BLI data showed Kd value of trastuzumab, Fab, and conjugated DTPA-Fab as 0.2 nM, 0.57 nM, and 20 nM for HER2. High labelling efficiency (≥98%) of Lu-177-DTPA-Fab and Ga-68-DTPA-Fab was achieved. Apoptosis (%) was 1.5%- 42.5% in SKBR-3 cells treated with Lu-177-DTPA-Fab. Both formulations were found sterile and pyrogen-free. Kidney and bladder activity of Ga-68/Lu-177-DTPA-Fab demonstrate renal clearance. Blood pool activity of both tracers were high. However, it was decreased, and lesion uptake was increased with time. All the lesions on Ga-68-DTPA-Fab, same as in F-18 FDG PET/CT, were picked up accept those near heart. Good lesion retention of Lu-177-DTPA-Fab was observed showing suitability for therapy. Fab-DTPA has potential to target HER2 receptor. It could be radiolabelled with Ga-68 for PET imaging and Lu-177 for targeted therapy.

Citation Format: Jaya Shukla, Yogesh Rathore, Komalpreet Kaur, Santosh Irrinky, Rajender Kumar, Amanjit Bal, Bhagwant R Mittal. Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B043.