Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers

Abstract

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules

“…The biodistribution of [ 68 Ga]-Ga-(HE) 3 -Z HER3 -NODAGA 3 h pi was directly compared with [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA 24 h pi in the same batch of Balb/c nu/nu mice bearing BxPC-3 xenografts (Table S1, Figure 5). Data for [ 68 Ga]-Ga-(HE) 3 -Z HER3 -NODAGA was in agreement with previously published data [31]. Comparing [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA at 3 h pi with [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA at 24 h pi, the cobalt-labeled variant had significantly lower activity concentration in blood, liver and stomach, and slightly higher uptake in tumor.…”

“…The different levels of uptake observed in the in vitro specificity test correlated to the different levels of HER3 expression, but no differences were observed in the cellular processing of the different conjugates. While low internalization rate seems to be characteristic for anti-HER3 affibody molecules, the observed rate is considerably lower than for the gallium-68 and indium-111-labeled analogs [26,31,43]. It could be speculated that cobalt-efflux mechanisms are triggered, which in addition to the already slow internalization of (HE) 3 -Z HER3 , partly affect the level of the internalized fraction [27].…”

“…It was hypothesized that an increased negative charge can reduce unspecific hepatic uptake and, therefore, enhance the tumorto-liver contrast [27]. This hypothesis was further supported by comparing the effect of differently charged radiometal-chelator complexes on the biodistribution of ZHER3 labeled with 68 Ga and 111 In and HER2-targeting affibody molecules [26,31,36].…”

“…The production and purification of the different affibody molecules (HE) 3 -Z HER3 -NOTA, (HE) 3 -Z HER3 -NODAGA, (HE) 3 -Z HER3 -DOTA, and (HE) 3 -Z HER3 -DOTAGA are described in Dahlsson Leitao et al [31]. An overview of the different chelators is depicted in Figure 1.…”

“…For PET imaging of HER3 expression, 18 F-and 68 Ga-labeled Z HER3 affibody molecules were able to visualize tumor uptake already 1 and 3 h pi in rodents. However, the tumor-to-liver contrast did not exceed 1 [21,31]. Unfortunately, these two commonly used PET isotopes have short half-lives (t 1/2 ( 18 F) = 110 min, t 1/2 ( 68 Ga) = 68 min) and are not suitable for next-day imaging.…”