[(64) Cu]-labelled trastuzumab: optimisation of labelling by DOTA and NODAGA conjugation and initial evaluation in mice

Abstract

The human epidermal growth factor receptor-2 (HER2) is overexpressed in 20-30% of all breast cancer cases, leading to increased cell proliferation, growth and migration. The monoclonal antibody, trastuzumab, binds to HER2 and is used for treatment of HER2-positive breast cancer. Trastuzumab has previously been labelled with copper-64 by conjugation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. The aim of this study was to optimise the (64) Cu-labelling of DOTA-trastuzumab and as the first to produce and compare with its 1,4,7-triazacyclononane, 1-glutaric acid-5,7 acetic acid (NODAGA) analogue in a preliminary HER2 tumour mouse model. The chelators were conjugated to trastuzumab using the activated esters DOTA mono-N-hydroxysuccinimide (NHS) and NODAGA-NHS. (64) Cu-labelling of DOTA-trastuzumab was studied by varying the amount of DOTA-trastuzumab used, reaction temperature and time. Full (64) Cu incorporation could be achieved using a minimum of 10-µg DOTA-trastuzumab, but the fastest labelling was obtained after 15 min at room temperature using 25 µg of DOTA-trastuzumab. In comparison, 80% incorporation was achieved for (64) Cu-labelling of NODAGA-trastuzumab. Both [(64) Cu]DOTA-trastuzumab and [(64) Cu]NODAGA-trastuzumab were produced after purification with radiochemical purities of >97%. The tracers were injected into mice with HER2 expressing tumours. The mice were imaged by positron emission tomography and showed high tumour uptake of 3-9% ID/g for both tracers.

Keywords: DOTA; NODAGA; PET; trastuzumab.

Figure 1

Structure of DOTA‐trastuzumab and NODAGA‐trastuzumab.

Figure 2

⁶⁴Cu‐incorporation of DOTA‐trastuzumab in antibody containing fractions collected from PD‐10 purification after DOTA conjugation with 5, 20 and 100 equivalents of DOTA‐NHS ester. The bars show the antibody concentration in the fractions. Twenty‐five microgram DOTA‐trastuzumab and a total volume of 300 µl were used for all experiments. Reactions were performed at room temperature and analysed after 15 min.

Figure 3

HPLC chromatogram of DOTA‐trastuzumab (blue) with overlay of [⁶⁴Cu]DOTA‐trastuzumab (red) (A) and a crude mixture of [⁶⁴Cu]DOTA‐trastuzumab with [⁶⁴Cu]EDTA (B).

Figure 4

Incorporation curves for the ⁶⁴Cu‐labelling of DOTA‐trastuzumab using different amounts of DOTA‐trastuzumab. Reactions were performed at room temperature (blue) and at 40°C (green) and analysed at different times: 15 (A) and 90 min (B) are shown. The volume was held constant in all experiments.

Figure 5

⁶⁴Cu‐incorporation of NODAGA‐trastuzumab in antibody containing fractions collected from PD‐10 purification after NODAGA conjugation with 5, 20 and 100 equivalents NODAGA‐NHS ester. Conjugation was also tested with 5 equivalents NODAGA‐NHS ester at room temperature (5 equivalents—RT, blue). The bars show the antibody concentration in the fractions. Twenty‐five microgram NODAGA‐trastuzumab and a total volume of 300 µl were used for all experiments. Reactions were performed at room temperature and analysed after 15 min.

Figure 6

Saturation binding of [⁶⁴Cu]DOTA‐trastuzumab and [⁶⁴Cu]NODAGA‐trastuzumab to SK‐BR‐3 cells. The K_D values were estimated to 14.1 and 12.0 nM respectively.

Figure 7

Coronal and axial PET/CT images of [⁶⁴Cu]DOTA‐trastuzumab (DOTA) and [⁶⁴Cu]NODAGA‐trastuzumab (NODAGA) 13, 26 and 43‐h post injection (left), and maximal projection images 43‐h post injection (right). H, heart; L, liver; T, tumour; MFP, mammary fad pad.