Radionuclide molecular imaging of human epidermal growth factor type 2 (HER2) expression may enable a non-invasive discrimination between HER2-positive and HER2-negative breast cancers for stratification of patients for HER2-targeted treatments. DARPin G3 is a small (molecular weigh 14 kDa) scaffold protein with picomolar affinity to HER2. The aim of this first-in-human study was to evaluate the safety, biodistribution and dosimetry of ^99mTc-(HE)₃-G3. Methods: Three cohorts of patients with primary breast cancer (each including at least 4 patients with HER2-negative and 5 patients with HER2-positive tumors) were injected with either 1000, 2000 or 3000 µg of ^99mTc-(HE)₃-G3 (287±170 MBq). Whole-body planar imaging followed by SPECT was performed at 2, 4, 6 and 24 h after injection. Vital signs and possible side effects were monitored during imaging and up to 7 days after injection. Results: All injections were well tolerated. No side effects were observed. The results of blood and urine analyses did not differ before and after studies. ^99mTc-(HE)₃-G3 cleared rapidly from the blood. The highest uptake was detected in the kidneys and liver followed by the lungs, breasts and small intestinal content. The hepatic uptake after injecting with 2000 or 3000 µg was significantly (p<0.05) lower than the uptake after injecting with 1000 µg. Effective doses did not differ significantly between cohorts (average 0.011± 0.004 mSv/MBq). Tumor-to-contralateral site ratios for HER-positive tumors were significantly (p< 0.05) higher than for HER2-negative at 2 and 4 h after injection. Conclusion: Imaging of HER2 expression using ^99mTc-(HE)₃-G3 is safe, well-tolerated and provides a low absorbed dose burden on patients. This imaging enables discerning HER2-positive and HER2-negative breast cancer. Phase I study data justifies further clinical development of ^99mTc-(HE)₃-G3.

We report here a combined anti-cancer therapy directed toward HER2 and EpCAM, common tumor-associated antigens of breast cancer cells. The combined therapeutic effect is achieved owing to two highly toxic proteins—a low immunogenic variant of Pseudomonas aeruginosa exotoxin A and ribonuclease Barnase from Bacillus amyloliquefaciens. The delivery of toxins to cancer cells was carried out by targeting designed ankyrin repeat proteins (DARPins). We have shown that both target agents efficiently accumulate in the tumor. Simultaneous treatment of breast carcinoma-bearing mice with anti-EpCAM fusion toxin based on LoPE and HER2-specific liposomes loaded with Barnase leads to concurrent elimination of primary tumor and metastases. Monotherapy with anti-HER2- or anti-EpCAM-toxins did not produce a comparable effect on metastases. The proposed approach can be considered as a promising strategy for significant improvement of cancer therapy.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%–75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with ¹²⁵I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [¹²⁵I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [¹²⁵I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [¹²⁵I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [¹²⁵I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [¹²⁵I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [¹²⁵I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with ¹⁷⁷Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [¹⁷⁷Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

Theranostic approach is currently among the fastest growing trends in cancer treatment. It implies the creation of multifunctional agents for simultaneous precise diagnosis and targeted impact on tumor cells. A new type of theranostic complexes was created based on NaYF₄: Yb,Tm upconversion nanoparticles coated with polyethylene glycol and functionalized with the HER2-specific recombinant targeted toxin DARPin-LoPE. The obtained agents bind to HER2-overexpressing human breast adenocarcinoma cells and demonstrate selective cytotoxicity against this type of cancer cells. Using fluorescent human breast adenocarcinoma xenograft models, the possibility of intravital visualization of the UCNP-based complexes biodistribution and accumulation in tumor was demonstrated.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [¹²⁵I]I-para-iodobenzoate (PIB) label and a residualizing [^99mTc]Tc(CO)₃ label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [¹²⁵I]I-PIB-Ec1 and [^99mTc]Tc(CO)₃-Ec1 in TNBC tumors. [¹²⁵I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [^99mTc]Tc(CO)₃-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.