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Purpose: In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. Methods: A synthetic variant of the anti-HER2 Z Affibody molecule, Z, was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with In, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. Results: The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z, NOTA-Z and NODAGA-Z, respectively. A comparative study of In-labelled DOTA-Z, NOTA-Z and NODAGA-Z in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. In-NODAGA-Z had the most rapid clearance from blood and healthy tissues. In-NOTA-Z showed high hepatic uptake and was excluded from further evaluation. In-DOTA-Z and In-NODAGA-Z demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of In-NODAGA-Z, 5.6 ± 0.4%ID/g, was significantly lower than the uptake of In-DOTA-Z, 7.4 ± 0.5%ID/g, presumably because of lower bioavailability due to more rapid clearance. In-NODAGA-Z provided higher tumour-to-blood ratio, but somewhat lower tumour-to-liver, tumour-to-spleen and tumour-to-bone ratios. Conclusion: Since distant prostate cancer metastases are situated in bone or bone marrow, the higher tumour-to-bone ratio is the most important. This renders In-DOTA-Z a preferable agent for imaging of HER2 expression in disseminated prostate cancer. [ABSTRACT FROM AUTHOR]

Affibody-mediated PNA-based pretargeting shows promise for HER2-expressing tumor radiotherapy. In our recent study, a 15-mer Z HER2:342 -HP15 affibody-PNA conjugate, in combination with a shorter 9-mer [ 177 Lu]Lu-HP16 effector probe, emerged as the most effective pretargeting strategy. It offered a superior tumor-to-kidney uptake ratio and more efficient tumor targeting compared to longer radiolabeled effector probes containing 12 or 15 complementary PNA bases. To enhance the production efficiency of our pretargeting system, we here introduce even shorter 6-, 7-, and 8-mer secondary probes, designated as HP19, HP21, and HP20, respectively. We also explore the replacement of the original 15-mer Z-HP15 primary probe with shorter 12-mer Z-HP12 and 9-mer Z-HP9 alternatives. This extended panel of shorter PNA-based probes was synthesized using automated microwave-assisted methods and biophysically screened in vitro to identify shorter probe combinations with the most effective binding properties. In a mouse xenograft model, we evaluated the biodistribution of these probes, comparing them to the Z-HP15:[ 177 Lu]Lu-HP16 combination. Tumor-to-kidney ratios at 4 and 144 h postinjection of the secondary probe showed no significant differences among the Z-HP9:[ 177 Lu]Lu-HP16, Z-HP9:[ 177 Lu]Lu-HP20, and the Z-HP15:[ 177 Lu]Lu-HP16 pairs. Importantly, tumor uptake significantly exceeded, by several hundred-fold, that of most normal tissues, with kidney uptake being the critical organ for radiation therapy. This suggests that using a shorter 9-mer primary probe, Z-HP9, in combination with 9-mer HP16 or 8-mer HP20 secondary probes effectively targets tumors while minimizing the dose-limiting kidney uptake of radionuclide. In conclusion, the Z-HP9:HP16 and Z-HP9:HP20 probe combinations offer good prospects for both cost-effective production and efficient in vivo pretargeting of HER2-expressing tumors., Competing Interests: The authors declare the following competing financial interest(s): KW, AV, AO, AEK and VT own shares in Zytox Therapeutics AB. MO is employed by Affibody AB. KW, HT and AEK are co-inventors of a patent application on PNA pretargeting (PCT/EP2021/078854). The other co-authors declare no competing interest., (© 2024 The Authors. Published by American Chemical Society.)

Affibody molecules are a class of affinity agents for molecular imaging based on a non-immunoglobulin protein scaffold. Previous studies have demonstrated high contrast for in vivo imaging of cancer-associated molecular abnormalities using Affibody molecules. Using the radionuclide (18)F for labeling and PET as the imaging modality, the sensitivity of molecular imaging using Affibody molecules can be further increased. The use of oxime formation between an aminooxy-functionalized peptide and (18)F-fluorobenzaldehyde ((18)F-FBA) is a promising way of radiolabeling of targeting peptides. However, previous studies demonstrated that application of this method to Affibody molecules is associated with high liver uptake. We hypothesized that incorporation of a triglutamyl spacer between the aminooxy moiety and the N-terminus of a synthetic Affibody molecule would decrease the hepatic uptake of the (18)F-N-(4-fluorobenzylidine)oxime) ((18)F-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting ZHER2:342 Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E3-PEP4313, where a triglutamyl spacer was introduced between the aminooxy moiety and the N-terminus. We have found that the use of the spacer is associated with a minor decrease of affinity, from KD = 49 pM to KD = 180 pM. Radiolabeled (18)F-FBO-E3-PEP4313 demonstrated specific binding to HER2-expressing ovarian carcinoma SKOV-3 cells and slow internalization. Biodistribution studies in mice demonstrated that the use of a triglutamyl linker decreased uptake of radioactivity in liver 2.7-fold at 2 h after injection. Interestingly, radioactivity uptake in kidneys was also reduced (2.4-fold). Experiments in BALB/C nu/nu mice bearing SKOV-3 xenografts demonstrated HER2-specific uptake of (18)F-FBO-E3-PEP4313 in tumors. At 2 h pi, the tumor uptake (20 ± 2% ID/g) exceeded uptake in liver 5-fold and uptake in kidneys 3.6-fold. The tumor-to-blood ratio was 21 ± 3. The microPET/CT imaging experiment confirmed the biodistribution data. In conclusion, the use of a triglutamyl spacer is a convenient way to improve the biodistribution profile of Affibody molecules labeled at the N-terminus using (18)F-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.

Affibody molecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the C-terminus. The biodistribution of 68Ga- and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.

Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide (68)Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with (68)Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with (68)Ga and (111)In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for (68)Ga than for (111)In. The tumor uptake of (68)Ga-NODAGA-ZHER2:2395 and (68)Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for (68)Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

The effects of polar (mercaptoacetyl-triseryl) and negatively charged (mercaptoacetyl-triglumatyl) chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules were previously investigated. With glycine, serine, and glutamate, we demonstrated that substitution with a single amino acid in the chelator can significantly influence the biodistribution properties and the excretion pathways. Here, we have taken this investigation further, by analyzing the effects of introduction of a positive amino acid residue on the in vivo properties of the 99mTc-labeled Affibody molecule. The Affibody molecules with mercaptoacetyl-seryl-lysyl-seryl (maSKS) and mercaptoacetyl-trilysyl (maKKK) extensions were produced by peptide synthesis and labeled with 99mTc in alkaline conditions. A comparative biodistribution was performed in normal mice to evaluate the excretion pathway. A shift toward renal excretion was obtained when serine was substituted with lysine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced 3-fold for the 99mTc-maSKS-Z(HER2:342) and 99mTc-maKKK-Z(HER2: 342) in comparison with the 99mTc-maSSS-Z(HER2:342) conjugate 4 h post injection (p.i.). The radioactivity in the liver was elevated when a triple substitution of positively charged lysine was used. The tumor targeting properties of 99mTc-maSKS-Z(HER2:342) were further investigated in SKOV-3 xenografts. The tumor uptake of 99mTc-maSKS-Z(HER2: 342) was 17+/-7% IA/g 4 h p.i. Tumor xenografts were well-visualized by gamma scintigraphy. In conclusion, the substitution with one single lysine in the chelator results in better tumor imaging properties of the Affibody molecule Z(HER2:342) and is favorable for imaging of tumors and metastases in the abdominal area. Multiple lysine residues in the chelator are, however, undesirable due to elevated uptake both in the liver and kidneys.