In vivo pretargeted dual-modality imaging of HER2 and TAG-72 expression using the HaloTag enzyme

Objectives The covalent bond-forming reaction that occurs between the dehalogenase enzyme HaloTag and a chloroalkane HaloTag ligand (HTL) has been shown to proceed rapidly and selectively in an in vivo setting (1). We propose that this reaction can form the basis of a novel pretargeting strategy for both imaging and therapeutic applications. We have previously demonstrated in a series of in vitro experiments that HaloTag-mediated pretargeting is capable of detecting elevated expression of the cancer biomarker HER2 in a dual-modality (SPECT/optical) approach (2). Here, we report on the first successful in vivo pretargeting experiments using HaloTag, detecting HER2 and TAG-72 in xenograft tumors in mice.

Methods BALB/c nude mice were inoculated subcutaneously with either MDA-MB-231/H2N cells (HER2-transfected human breast cancer) or LS174T cells (TAG-72-expressing human colorectal cancer). Trastuzumab-HaloTag and CC49-HaloTag conjugates were employed as the primary agents targeting HER2 and TAG-72, respectively. Two ¹¹¹In-radiolabeled and fluorescent (TMR; λ_ex/em=552/578 nm) HaloTag ligands (¹¹¹In-HTL-2 and ¹¹¹In-HTL-3) were evaluated as secondary agents and were administered at 24 h after injection of the primary agent (100 µg). SPECT/CT imaging and ex vivo biodistribution experiments were then performed at 4 h and 24 h post-injection.

Results Ex vivo biodistribution experiments at 4 h p.i. revealed higher uptake of ¹¹¹In-HTL-3 in MDA-MB-231/H2N tumors pretargeted with Trastuzumab-HaloTag conjugated versus a non-pretargeted control (2.83±0.95 and 1.17±0.37 %ID/g, respectively; P<0.05). Uptake of ¹¹¹In-HTL-3 was also higher in LS174T tumors which had been pretargeted with CC49-HTP (3.15±0.29 %ID/g) compared with those pretargeted with a non-specific rabbit-IgG-HaloTag conjugate (2.15±0.33 %ID/g) or without pretargeting (1.48±0.43 %ID/g) (P<0.01). ¹¹¹In-HTL-2 had higher uptake in LS174T tumors which were pretargeted with CC49-HTP compared to non-pretargeted tumors (2.31±0.64 and 1.19±0.19, respectively; P<0.05). Intratumoural distribution was confirmed by autoradiography and fluorescence microscopy, utilizing the TMR-moiety on the HaloTag ligand.

Conclusions The HaloTag-HTL reaction has strong potential to form the basis of a novel pretargeting strategy for the rapid and selective delivery of reporter groups and/or therapeutic agents. REFERENCES: (1) Hong et al., Am. J. Trans. Res., 2013, 5, 291-302. (2) Knight et al., Chem. Commun., 2015, 51, 4055-4058. RESEARCH SUPPORT: This work received financial support from Cancer Research UK.