Dual-isotope imaging allows in vivo immunohistochemistry using radiolabelled antibodies in tumours

Abstract

While radiolabelled antibodies have found great utility as PET and SPECT imaging agents in oncological investigations, a notable shortcoming of these agents is their propensity to accumulate non-specifically within tumour tissue. The degree of this non-specific contribution to overall tumour uptake is highly variable and can ultimately lead to false conclusions. Therefore, in an effort to obtain a reliable measure of inter-individual differences in non-specific tumour uptake of radiolabelled antibodies, we demonstrate that the use of dual-isotope imaging overcomes this issue, enables true quantification of epitope expression levels, and allows non-invasive in vivo immunohistochemistry. The approach involves co-administration of (i) an antigen-targeting antibody labelled with zirconium-89 (⁸⁹Zr), and (ii) an isotype-matched non-specific control IgG antibody labelled with indium-111 (¹¹¹In). As an example, the anti-HER2 antibody trastuzumab was radiolabelled with ⁸⁹Zr, and co-administered intravenously together with its ¹¹¹In-labelled non-specific counterpart to mice bearing human breast cancer xenografts with differing HER2 expression levels (MDA-MB-468 [HER2-negative], MDA-MB-231 [low-HER2], MDA-MB-231/H2N [medium-HER2], and SKBR3 [high-HER2]). Simultaneous PET/SPECT imaging using a MILabs Vector4 small animal scanner revealed stark differences in the intratumoural distribution of [⁸⁹Zr]Zr-trastuzumab and [¹¹¹In]In-IgG, highlighting regions of HER2-mediated uptake and non-specific uptake, respectively. Normalisation of the tumour uptake values and tumour-to-blood ratios obtained with [⁸⁹Zr]Zr-trastuzumab against those obtained with [¹¹¹In]In-IgG yielded values which were most strongly correlated (R = 0.94; P = 0.02) with HER2 expression levels for each breast cancer type determined by Western blot and in vitro saturation binding assays, but not non-normalised uptake values. Normalised intratumoural distribution of [⁸⁹Zr]Zr-trastuzumab correlated well with intratumoural heterogeneity HER2 expression.

Keywords: Antibody; Dual-isotope; HER2; Molecular imaging; PET; SPECT.

Fig. 1

(A) Western blot of tumour xenograft lysates showing the different levels of HER2 expression. (B) Saturation binding plot of [¹¹¹In]In-trastuzumab on each of the cell lines used in the study.

Fig. 2

(A) PET/SPECT images of mice bearing MDA-MB-468 tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab and [¹¹¹In]In-IgG. (B) Biodistribution data showing uptake (%ID/g) of each radiolabelled agent in tumours and selected organs. (C) Tumour-to-blood ratios and (D) tumour-to-muscle ratios for each radiolabelled agent. (ns: no statistical significance).

Fig. 3

(A) PET/SPECT images of mice bearing MDA-MB-231 tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab and [¹¹¹In]In-IgG. (B) PET/SPECT images of mice bearing MDA-MB-231 tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab, [¹¹¹In]In-IgG, and a blocking dose (0.5 mg) of unlabelled trastuzumab. (C) Biodistribution data showing uptake (%ID/g) of each radiolabelled agent in tumours and selected organs. (D) Tumour-to-blood ratios and (E) tumour-to-muscle ratios for each radiolabelled agent. (ns: no statistical significance; *P < 0.05).

Fig. 4

(A) PET/SPECT images of mice bearing MDA-MB-231/H2N tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab and [¹¹¹In]In-IgG. (B) PET/SPECT images of mice bearing MDA-MB-231/H2N tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab, [¹¹¹In]In-IgG, and a blocking dose (0.5 mg) of unlabelled trastuzumab. (C) Biodistribution data showing uptake (%ID/g) of each radiolabelled agent in tumours and selected organs. (D) Tumour-to-blood ratios and (E) tumour-to-muscle ratios for each radiolabelled agent. (*P < 0.05; ****P < 0.0001).

Fig. 5

(A) PET/SPECT images of mice bearing SKBR3 tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab and [¹¹¹In]In-IgG. (B) PET/SPECT images of mice bearing SKBR3 tumours at 3 days post injection of [⁸⁹Zr]Zr-trastuzumab, [¹¹¹In]In-IgG, and a blocking dose (0.5 mg) of unlabelled trastuzumab. (C) Biodistribution data showing uptake (%ID/g) of each radiolabelled agent in tumours and selected organs. (D) Tumour-to-blood ratios and (E) tumour-to-muscle ratios for each radiolabelled agent. (***P < 0.001; ****P < 0.0001).

Fig. 6

(A) The [⁸⁹Zr]Zr-trastuzumab/[¹¹¹In]In-IgG uptake ratios and (B) the [⁸⁹Zr]Zr-trastuzumab T/B: [¹¹¹In]In-IgG T/B ratios for each of the tumour models used in the study. (C) Correlation between overall uptake of [⁸⁹Zr]Zr-trastuzumab/[¹¹¹In]In-IgG and expression levels of HER2 determined by saturation binding assay. (D) Correlation between [⁸⁹Zr]Zr-trastuzumab T/B: [¹¹¹In]In-IgG T/B ratios and expression levels of HER2 determined by saturation binding assay. Error bars for (A) and (B) represent standard error of the mean.

Fig. 7

(A) Magnified sections of a representative tumour (MDA-MB-231) showing differences in the intratumoural distribution of [⁸⁹Zr]Zr-trastuzumab (A) and [¹¹¹In]In-IgG (B). Images overlaid in (C). (D) Image resulting from subtraction of the [¹¹¹In]In-IgG signal from the overlaid image showing the HER2-specific uptake of [⁸⁹Zr]Zr-trastuzumab. (E–F) H&E staining and immunohistochemistry staining HER2 in adjacent sections (blue: DAPI; green: HER2).