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The Insulin-like growth factor-1 receptor (IGF-1R) is a molecular target for several monoclonal antibodies undergoing clinical evaluation as anticancer therapeutics. The non-invasive detection of IGF-1R expression in tumors might enable stratification of patients for specific treatment and improve the outcome of both clinical trials and routine treatment. The affibody molecule Z(IGF-1R:4551) binds specifically to IGF-1R with subnanomolar affinity. The goal of this study was to evaluate the Ga-68 and In-111-labeled affibody construct NODAGA-(HE)(3)-Z(IGF-1R:4551) for the imaging of IGF-1R expression, using PET and SPECT. The labeling was efficient and provided stable coupling of both radionuclides. The two imaging probes, [Ga-68]Ga-NODAGA-(HE)(3)-Z(IGF-1R:4551) and [In-111]In-NODAGA-(HE)(3)-Z(IGF-1R:4551), demonstrated specific binding to IGF-1R-expressing human cancer cell lines in vitro and to IGF-1R-expressing xenografts in mice. Preclinical PET and SPECT/CT imaging demonstrated visualization of IGF-1R-expressing xenografts already one hour after injection. The tumor-to-blood ratios at 3 h after injection were 7.8 +/- 0.2 and 8.0 +/- 0.6 for [Ga-68]Ga-NODAGA-(HE)(3)-Z(IGF-1R:4551) and [In-111]In-NODAGA-(HE)(3)-Z(IGF-1R:4551), respectively. In conclusion, a molecular design of the Z(IGF-1R:4551) affibody molecule, including placement of a (HE)(3)-tag on the N-terminus and site-specific coupling of a NODAGA chelator on the C-terminus, provides a tracer with improved imaging properties for visualization of IGF-1R in malignant tumors, using PET and SPECT.

Affibody molecules are small (58 amino acids) engineered scaffold proteins that can be selected to bind to a large variety of proteins with a high affinity. Their small size and high affinity make them attractive as targeting vectors for molecular imaging. High-affinity affibody binders have been selected for several cancer-associated molecular targets. Preclinical studies have shown that radiolabeled affibody molecules can provide highly specific and sensitive imaging on the day of injection; however, for a few targets, imaging on the next day further increased the imaging sensitivity. A phase I/II clinical trial showed that 68Ga-labeled affibody molecules permit an accurate and specific measurement of HER2 expression in breast cancer metastases. This paper provides an overview of the factors influencing the biodistribution and targeting properties of affibody molecules and the chemistry of their labeling using positron emitters.

Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699NOTA HER3-specific affibody molecules were labeled with indium-111 ([sup.111]In) and assessed in vitro and in vivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with [sup.111]In provided stable conjugates. In vitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT-474 breast carcinoma cells. In mice bearing BT-474 xenografts, the tumor uptake of the two conjugates was receptor-specific. Direct in vivo comparison of [sup.111]In-HEHEHE-Z08698-NOTA and [sup.111]In-HEHEHE-Z08699-NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for [sup.111]In-HEHEHE-Z08698-NOTA [12 ± 3 vs. 8 ± 1, 4 h post injection (p.i.)] due to more efficient blood clearance. [sup.111]In-HEHEHEZ0869-8-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy. Key words: NOTA, indium-111, affibody molecules, HER3, molecular imaging

Radionuclide imaging of HER2 expression in tumours may enable stratification of patients with breast, ovarian, and gastroesophageal cancers for HER2-targeting therapies. A first-generation HER2-binding affibody molecule [Tc-99m]Tc-ZHER2:V2 demonstrated favorable imaging properties in preclinical studies. Thereafter, the affibody scaffold has been extensively modified, which increased its melting point, improved storage stability, and increased hydrophilicity of the surface. In this study, a second-generation affibody molecule (designated ZHER2:41071) with a new improved scaffold has been prepared and characterized. HER2-binding, biodistribution, and tumour-targeting properties of [Tc-99m]Tc-labelled ZHER2:41071 were investigated. These properties were compared with properties of the first-generation affibody molecules, [Tc-99m]Tc-ZHER2:V2 and [Tc-99m]Tc-ZHER2:2395. [Tc-99m]Tc-ZHER2:41071 bound specifically to HER2 expressing cells with an affinity of 58 +/- 2 pM. The renal uptake for [Tc-99m]Tc-ZHER2:41071 and [Tc-99m]Tc-ZHER2:V2 was 25-30 fold lower when compared with [Tc-99m]Tc-ZHER2:2395. The uptake in tumour and kidney for [Tc-99m]Tc-ZHER2:41071 and [Tc-99m]Tc-ZHER2:V2 in SKOV-3 xenografts was similar. In conclusion, an extensive re-engineering of the scaffold did not compromise imaging properties of the affibody molecule labelled with Tc-99m using a GGGC chelator. The new probe, [Tc-99m]Tc-ZHER2:41071 provided the best tumour-to-blood ratio compared to HER2-imaging probes for single photon emission computed tomography (SPECT) described in the literature so far. [Tc-99m]Tc-ZHER2:41071 is a promising candidate for further clinical translation studies.

Vectors based on Adenovirus type 5 (Ad5) are among the most common vectors in cancer gene therapy trials to date. However, for increased efficiency and safety, Ad5 should be de-targeted from its native receptors and re-targeted to a tumor antigen. We have described earlier an Ad5 vector genetically re-targeted to the tumor antigen HER2/neu by a dimeric version of the Affibody molecule ZH inserted in the HI-loop of the fiber knob of a coxsackie and adenovirus receptor-binding ablated fiber. This virus showed almost wild-type growth characteristics and infected cells through HER2/neu. Here we generate vectors with double specificity by incorporating two different Affibody molecules, ZH (HER2/neu-binding) and ZT (Taq polymerase-binding), at different positions relative to one another in the HI-loop. Receptor-binding studies together with viral production and gene transfer assays showed that the recombinant fiber with ZT in the first position and ZH in the second position (ZT ZH ) bound to both its targets, whereas surprisingly, the fiber with ZH ZT was devoid of binding to HER2/neu. Hence, it is possible to construct a recombinant adenovirus with dual specificity after evaluating the best position for each ligand in the fiber knob.

Simple Summary Affibody molecules are small, engineered affinity proteins based on a nonimmunoglobulin scaffold. Affibody-based radionuclide imaging probes have demonstrated excellent tumor targeting. However, the renal clearance of affibody molecules is accompanied by high reabsorption and retention of activity in the kidney, which prevents their use for radionuclide therapy. We have previously shown the feasibility of overcoming the high renal uptake using a pretargeting approach for affibody-mediated therapy based on peptide nucleic acid (PNA) hybridization. In this study, we test the hypothesis that shortening the PNA pretargeting probes would further increase the difference between the accumulation of radiometals in tumor xenografts and in kidneys. A series of novel PNA probes has been designed and evaluated in vitro and in vivo. We have found that a variant containing 9 nucleobases enables a two-fold increase of the tumor-to-kidney dose ratio compared with a variant containing 15 nucleobases. This creates preconditions for more efficient therapy of cancer. Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe Z(HER2:342)-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with Lu-177. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [Lu-177]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all Lu-177-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe's size and decreased with an increased number of nucleobases. The shortest PNA probe, [Lu-177]Lu-HP16, showed the highest tumor-to-kidney ratio. [Lu-177]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a radionuclide Zr-89. The Zr-89-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160 +/- 60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, Zr-89-DFO-ZEGFR: 2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6 +/- 0.5% ID/g and tumour-to-blood ratio of 3.7 +/- 0.6 at 24 h after injection. Zr-89-DFO-ZEGFR: 2377 provides higher tumour-to-organ ratios than anti-EGFR antibody Zr-89-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using Zr-89-DFO-ZEGFR: 2377 at both 3 and 24 h after injection. In conclusion, Zr-89-DFO-ZEGFR: 2377 is a potential probe for PET imaging of EGFR-expression in vivo.

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 Tc-99m 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 Tc-99m 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, Tc-99m-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 Tc-99m-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 Tc-99m-labeled ZEGFR:2377 for imaging of EGFR in vivo.

Fusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required. The HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with Lu. Affinity, specificity, and in vivo targeting properties of [ Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated. The maximum molar activity of 52 GBq/µmol [ Lu]Lu-PEP49989 was obtained. [ Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [ Lu]Lu-PEP49989 was similar to the affinity of [ Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [ Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [ Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [ Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [ Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild. In preclinical studies, [ Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

HER2-targeted radionuclide therapy might be helpful for the treatment of breast, gastric, and ovarian cancers which have developed resistance to antibody and antibody-drug conjugate-based therapies despite preserved high HER2-expression. Affibody molecules are small targeting proteins based on a non-immunoglobulin scaffold. The goal of this study was to test in an animal model a hypothesis that the second-generation HER2-targeting Affibody molecule 188Re-ZHER2:41071 might be useful for treatment of HER2-expressing malignant tumors. ZHER2:41071 was efficiently labeled with a beta-emitting radionuclide rhenium-188 (188Re). 188Re-ZHER2:41071 demonstrated preserved specificity and high affinity (KD = 5 ± 3 pM) of binding to HER2-expressing cells. In vivo studies demonstrated rapid washout of 188Re from kidneys. The uptake in HER2-expressing SKOV-3 xenografts was HER2-specific and significantly exceeded the renal uptake 4 h after injection and later. The median survival of mice, which were treated by three injections of 16 MBq 188Re-ZHER2:41071 was 68 days, which was significantly longer (<0.0001 in the log-rank Mantel-Cox test) than survival of mice in the control groups treated with vehicle (29 days) or unlabeled ZHER2:41071 (27.5 days). In conclusion, the experimental radionuclide therapy using 188Re-ZHER2:41071 enabled enhancement of survival of mice with human tumors without toxicity to the kidneys, which is the critical organ.