Targeting Tumor Cells Overexpressing the Human Epidermal Growth Factor Receptor 3 with Potent Drug Conjugates Based on Affibody Molecules.

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

HER3 PET Imaging: 68Ga-Labeled Affibody Molecules Provide Superior HER3 Contrast to 89Zr-Labeled Antibody and Antibody-Fragment-Based Tracers.

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-ZHER3. 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-ZHER3 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.

Evaluating the Therapeutic Efficacy of Mono- and Bivalent Affibody-Based Fusion Proteins Targeting HER3 in a Pancreatic Cancer Xenograft Model.

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.

Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts.

Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with 111In and characterized in vitro. Tumor-targeting properties of [111In]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [99mTc]Tc(CO)3-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [111In]In-DTPA-G250(Fab')2, in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the 99mTc-labeled parental variant, [111In]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [111In]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [111In]In-G250(Fab')2. In conclusion, [111In]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

Improved contrast of affibody-mediated imaging of HER3 expression in mouse xenograft model through co-injection of a trivalent affibody for in vivo blocking of hepatic uptake.

Human epidermal growth factor receptor type 3 (HER3) plays a crucial role in the progression of many cancer types. In vivo radionuclide imaging could be a reliable method for repetitive detection of HER3-expression in tumors. The main challenge of HER3-imaging is the low expression in tumors together with endogenous receptor expression in normal tissues, particularly the liver. A HER3-targeting affibody molecule labeled with radiocobalt via a NOTA chelator [57Co]Co-NOTA-Z08699 has demonstrated the most favorable biodistribution profile with the lowest unspecific hepatic uptake and high activity uptake in tumors. We hypothesized that specific uptake of labeled affibody monomer might be selectively blocked in the liver but not in tumors by a co-injection of non-labeled corresponding trivalent affibody (Z08699)3. Biodistribution of [57Co]Co-NOTA-Z08699 and [111In]In-DOTA-(Z08699)3 was studied in BxPC-3 xenografted mice. [57Co]Co-NOTA-Z08699 was co-injected with unlabeled trivalent affibody DOTA-(Z08699)3 at different monomer:trimer molar ratios. HER3-expression in xenografts was imaged using [57Co]Co-NOTA-Z08699 and [57Co]Co-NOTA-Z08699: DOTA-(Z08699)3. Hepatic activity uptake of [57Co]Co-NOTA-Z08699: DOTA-(Z08699)3 decreased with increasing monomer:trimer molar ratio. The tumor activity uptake and tumor-to-liver ratios were the highest for the 1:3 ratio. SPECT/CT images confirmed the biodistribution data. Imaging of HER3 expression can be improved by co-injection of a radiolabeled monomeric affibody-based imaging probe together with a trivalent affibody.

Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-111.

Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTAGA (1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z08698 and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged 111In-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs.

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.

Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab.

Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER-targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.