Gastric cancer (GC) remains one of the deadliest forms of cancer worldwide, largely due to its often late-stage diagnosis as well as high inter- and intratumoral heterogeneity. While HER2-targeted treatments like trastuzumab have improved outcomes, they are often limited by resistance mechanisms and limited tumour penetration.

This project aimed to develop and optimize a pretargeted radiotherapy system for HER2-positive gastric cancer using biorthogonal click chemistry. First, a TCO-conjugated antibody is injected, followed by a small radiolabelled tetrazine probe. The goal is to utilize the high specificity from the antibody without the high background radiation caused by its long circulation half-life.

Trastuzumab was conjugated to TCO at four different ratios; 10, 25, 50 and 75 molar equivalents. The conjugates were evaluated for TCO reactivity, stability and functionality both in vitro, using NCI-N87 and SNU-216 cell lines, and in vivo on NCI-N87 xenograft-bearing mice.

Quantification of reactive TCOs showed successful conjugation of 1.1, 4.8, 4.9 and 7.1 TCOs per antibody and the reactivity was maintained through storage at -80°C for up to 8 weeks. 

In vitro studies confirmed the functionality of the pretargeting system and LigandTracer assays confirmed the preserved binding kinetics of the antibody conjugate. However, 50Eq and 75Eq proved unreliable throughout the in vitro experiments and in the end, 25Eq was chosen as the lead candidate.

In vivo studies demonstrated successful tumour accumulation of both the antibody conjugate and the 111In labelled tetrazine probe although high blood background activity remains a challenge.

Overall, this study confirmed the feasibility of HER2-targeted pretargeted radiotherapy for gastric cancer. Optimisation of antibody dose and pretargeting interval slightly improved tumour-to-blood ratios but additional strategies, including the use of masking agents, may help reduce background activity and improve the therapeutic potential of the pretargeting approach.