Affibody molecules are small proteins engineered using a non-antibody scaffold. Radiolabeled affibody molecules are excellent imaging probes, but their application for radionuclide therapy was so far prevented by high renal reabsorption. The aim of this study was to test the hypothesis that affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of HER2-expressing cancer extends survival without accompanying renal toxicity. Methods: Human epidermal growth factor (HER2)-targeting affibody molecule ligated with AGTCGTGATGTAGTC PNA hybridization probe (ZHER2:342-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide ¹⁷⁷Lu (¹⁷⁷Lu-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in six cycles separated by 7 days. Results: Optimal tumor targeting was achieved when 16 MBq/3.5 µg (0.65 nmol) ¹⁷⁷Lu-HP2 was injected 16 h after injection of 100 µg (7.7 nmol) ZHER2:342-SR-HP1. The calculated absorbed dose to tumor was 1076 mGy/MBq, while the absorbed dose to the kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice in the treatment group (66 d) was significantly (p<0.05) longer than survival of mice in control groups treated with the same amount of ZHER2:342-SR-HP1 only (37 d), the same amount and activity of ¹⁷⁷Lu-HP2 only (32 d), or PBS (37 d). Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.

Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for ⁸⁹Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both ⁸⁹Zr-FSC-ZEGFR:2377 and ⁸⁹Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison ⁸⁹Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of ⁸⁹Zr-FSC-ZEGFR:2377 as well as ⁸⁹Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that ⁸⁹Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast.

The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide 99mTc should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with 99mTc using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, 99mTc-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that 99mTc-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6±1 and 2.5±0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8±0.4 and 8±3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of 99mTc-labeled ZEGFR:2377 for imaging of EGFR in vivo.

Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide ⁶⁸Ga. Stability, specificity of binding to EGFR-expressing cells, and processing of [⁶⁸Ga]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [⁶⁸Ga]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [⁸⁹Zr]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 ± 3%) and stably labeled with ⁶⁸Ga. Binding of [⁶⁸Ga]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [⁶⁸Ga]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [⁸⁹Zr]Zr-DFO-ZEGFR:2377, [⁶⁸Ga]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [⁶⁸Ga]Ga-DFO-ZEGFR:2377. In conclusion, [⁶⁸Ga]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.

The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, Z_HER2:2891, fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, Z_HER2:2891-ABD-E₃-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. Z_HER2:2891-ABD-E₃-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC₅₀ values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcDM1, was counteracted by incorporation of hydrophilic and negatively charged glutamate residues near the site of mcDM1 conjugation. A dose-escalation experiment showed that increasing doses up to 15.1 mg/kg gave a proportional increase in uptake in xenografted HER2-overexpressing SKOV3 tumors, after which the tumors became saturated. Experimental therapy with four once-weekly injection of 10.3 or 15.1 mg/kg led to efficient regression of tumors in all animals and complete regression in some. Weight loss was detected for some animals in the group receiving the highest dose, suggesting that it was close to the maximum tolerated dose. In conclusion, the monovalent HER2-targeting affibody drug conjugate presented herein have potent anti-tumor activity in vivo.