Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD₀₃₅) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.

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.

HER3 (human epidermal growth factor receptor type 3) is a challenging target for diagnostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the ⁸⁹Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab)₂-fragment labeled with ⁸⁹Zr and ⁶⁸Ga, and the ⁶⁸Ga-labeled affibody molecule [⁶⁸Ga]Ga-Z_HER3. The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribution was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [⁶⁸Ga]Ga-DFO-seribantumab-F(ab’)₂ was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [⁸⁹Zr]Zr-DFO-seribantumab and [⁸⁹Zr]Zr-DFO-seribantumab-F(ab’)₂ was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [⁶⁸Ga]Ga-Z_HER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt–chelator complex on the biodistribution of anti-HER3 affibody molecule (HE)₃-Z_HER3 and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE)₃-Z_HER3-X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [⁵⁷Co]Co (surrogate for ⁵⁵Co). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [⁵⁷Co]Co-(HE)₃-Z_HER3-DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA 3 h pi, [⁵⁷Co]Co-(HE)₃-Z_HER3-DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [⁵⁷Co]Co–chelator complex influenced the uptake in tumors and normal tissue. [⁵⁷Co]Co-(HE)₃-Z_HER3-DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [⁵⁷Co]Co-(HE)₃-Z_HER3-DOTA improved imaging contrast compared to early-time-point imaging with [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA.

Human epidermal growth factor receptor 3 (HER3) has been increasingly scrutinized as a potential drug target since the elucidation of its role in mediating tumor growth and acquired therapy resistance. Affibody molecules are so-called scaffold proteins with favorable biophysical properties, such as a small size for improved tissue penetration and extravasation, thermal and chemical stability, and a high tolerance to modifications. Additionally, affibody molecules are efficiently produced in prokaryotic hosts or by chemical peptide synthesis. We have previously evaluated the biodistribution profiles of five mono- and bivalent anti-HER3 affibody molecules (designated as 3) fused to an albumin-binding domain (designated as A), 3A, 33A, 3A3, A33, and A3, that inhibit ligand-dependent phosphorylation. In the present study, we examined the therapeutic efficacy of the three most promising variants, 3A, 33A, and 3A3, in a direct comparison with the HER3-targeting monoclonal antibody seribantumab (MM-121) in a preclinical BxPC-3 pancreatic cancer model. Xenografted mice were treated with either an affibody construct or MM-121 and the tumor growth was compared to a vehicle group. Receptor occupancy was estimated by positron emission tomography/computed tomography (PET/CT) imaging using a HER3-targeting affibody imaging agent [⁶⁸Ga]Ga-(HE)₃-Z₀₈₆₉₈-NODAGA. The affibody molecules could inhibit ligand-dependent phosphorylation and cell proliferation in vitro and demonstrated tumor growth inhibition in vivo comparable to that of MM-121. PET/CT imaging showed full receptor occupancy for all tested drug candidates. Treatment with 3A and 3A3 affibody constructs was more efficient than with 33A and similar to the anti-HER3 antibody seribantumab, showing that the molecular design of affibody-based therapeutics targeting HER3 in terms of the relative position of functional domains and valency has an impact on therapeutic effect.

Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)₃-Z_HER3:08698-DOTAGA affibody molecule with non-residualizing [¹²⁵I]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [¹²⁵I]I-PIB-(HE)₃-Z_HER3:08698-DOTAGA was compared side-by-side with [¹¹¹In]In-(HE)₃-Z_HER3:08698-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24 h pi showed faster clearance of the [¹²⁵I]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [¹²⁵I]I-PIB-(HE)₃-Z_HER3:08698-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 ± 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [¹²⁵I]I-PIB label. In conclusion, the use of non-residualizing [¹²⁵I]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.