Maryam Oroujeni

This study investigates how the structural design of secondary PNA probes affects their ability to carry the cytotoxic agent DM1 in a novel affibody-PNA-based pretargeting approach directed at Human Epidermal growth factor Receptor 2 (HER2)-expressing cells. Six different DM1-conjugated secondary 8-mer PNA probes with strategic hydrophilic modifications were designed and evaluated. Surface plasmon resonance analysis confirmed that the modifications preserved hybridization to the HER2-targeting affibody-PNA primary probe, with all secondary probes showing similar binding kinetics (KD = 310–660 pM). Biodistribution studies in nontumor-bearing NMRI mice with lutetium-177 (177Lu)-labeled secondary probes revealed that probe modifications dramatically influenced organ distribution patterns. The secondary probes SP2, SP5, and SPc demonstrated favorable biodistribution profiles with reduced accumulation in critical organs, i.e., liver and kidneys. Receptor-mediated endocytosis was also affected by secondary probe design, showing an increase in cellular internalization for SP5 (20.4% after 24 h) compared to SP2 (12.1%). Our findings highlight how rational design can optimize in vivo pharmacokinetics while preserving binding properties essential for effective pretargeting, providing a foundation for developing novel targeted cytotoxic delivery systems with potentially improved therapeutic indexes.

B7-H3 (CD276), an immune checkpoint protein, is overexpressed in malignant tumors, while its expression in normal tissues is low, and several B7-H3-targeting therapies are under clinical evaluation. Radionuclide molecular imaging offers a non-invasive method for determining B7-H3 expression levels and may aid in improved patient selection. The feasibility of the use of Affibody molecules for the visualization of B7-H3 was demonstrated earlier. The selection of an approach for routine labeling providing high radiochemical yields and reproducibility is, however, critical for successful clinical translation. The optimal combination of a targeting protein, chelator/linker, and radionuclide should provide high-contrast visualization. In this study, we evaluated an acyclic chelator, tris(3,4-hydroxypyridinone) (THP), for labeling of the Affibody molecule Z_{B7-H3_2} with ⁶⁸Ga and compared its impact on radiolabeling efficiency and targeting properties with the impact of the cyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Maleimide derivatives of THP and NOTA were site-specifically coupled to the C-terminal cysteine of Z_{B7-H3_2}. THP-Z_{B7-H3_2} was successfully labeled with ⁶⁸Ga within 5 min of incubation at room temperature, achieving a 100% radiochemical yield. NOTA-Z_B7-H3 required heating at 60 °C for 10 min, and the radiochemical yield was lower. Both radioconjugates exhibited specific binding to B7-H3-expressing cells with similar binding strength, and both tracers demonstrated similar tumor uptake (p > 0.05) in a murine model. The biodistribution was similar, although [⁶⁸Ga]Ga-NOTA-Z_{B7-H3_2} provided slightly but significantly higher tumor-to-liver and tumor-to-spleen ratios. Nonetheless, the advantages of THP include rapid and mild radiolabeling with high efficiency, eliminating the need for heating and a post-purification step, which suggests a potential for streamlined clinical translation of Z_{B7-H3_2}.

Affibody molecules, nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging of different molecular targets. One of the molecular targets for radionuclide diagnosis and therapy is B7-H3 (known as CD276), which is overexpressed in various cancers, whereas its expression is low in most normal organs and tissues. The visualization of expression levels of B7-H3 has been performed using Affibody molecules labeled with Tc-99m. However, radionuclide molecular imaging using PET offers several advantages such as superior sensitivity, quantitation accuracy, and better spatial resolution compared to SPECT. In this study, we aimed to introduce a radiotracer for PET imaging of B7-H3. To design imaging agents for labeling with the generator-produced positron-emitting radionuclide ⁶⁸Ga, the macrocyclic triaza chelator (2-[4,7-bis-(carboxymethyl)-1,4,7-triazonan-1-yl]-acetic acid) (NOTA) was site-specifically coupled to the C-terminal cysteine of the anti-B7-H3 Affibody molecules. Four different variants of Affibody molecules, Z_B7-H3_₂, Z_B7-H3_₃, Z_B7-H3_₄, and Z_AC12 (as control), were produced, characterized, and successfully labeled with ⁶⁸Ga. ⁶⁸Ga-labeled conjugates bound specifically to B7-H3-expressing cells in vitro and in vivo. Biodistribution showed that [⁶⁸Ga]-Ga-Z_B7-H3_₂ had the highest tumor accumulation only 2 h after administration, which was 2.8-fold higher than that for the control Z_AC12. There was a tendency for higher tumor-to-organ ratios compared to the other variants, resulting in higher imaging contrast using [⁶⁸Ga]-Ga-Z_B7-H3_₂ for preclinical PET imaging of B7-H3-expressing tumors. Thus, [⁶⁸Ga]-Ga-Z_B7-H3_₂ could be a promising candidate for further development aimed at clinical PET in the future.

Background: Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is overexpressed in several types of malignancies. Designed ankyrin repeat protein (DARPin) Ec1 is a 19 kDa engineered scaffold protein that binds with high affinity to EpCAM. Radiolabelled Ec1 might be used as a companion diagnostic for the selection of patients for personalized therapy. This study aimed to investigate the influence of different radiometal-chelator complexes on the biodistribution and imaging contrast of ⁶⁸Ga-labelled Ec1. To investigate this, two macrocyclic chelators, 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the C-terminus of the Ec1. The previously developed DARPin Ec1 conjugated to 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA) was used as a comparator. Results: All Ec1 variants were successfully labelled with ⁶⁸Ga. The use of NOTA and NODAGA provided twice higher radiochemical yield and improved label stability compared to DOTA. All labelled Ec1 variants bound to the EpCAM-expressing cells with nanomolar affinity and preserved targeting specificity in vitro and in vivo. Biodistribution studies in mice bearing EpCAM-expressing SKOV-3 xenografts showed that [⁶⁸Ga]Ga-Ec1-NOTA had lower uptake in most normal organs while maintaining tumor uptake. Among all variants, [⁶⁸Ga]Ga-Ec1-NOTA showed the lowest liver uptake, with no significant differences in tumor uptake. Additionally, [⁶⁸Ga]Ga-Ec1-NOTA provided the highest tumor-to-blood ratio compared to [⁶⁸Ga]Ga-Ec1-DOTA and [⁶⁸Ga]Ga-Ec1-NODAGA. Conclusion: [⁶⁸Ga]Ga-Ec1-NOTA is the preferred radioconjugate for PET imaging of EpCAM expression.

Designed ankyrin repeat protein (DARPin) Ec1, a small scaffold protein (18 kDa), binds with high affinity the epithelial cell adhesion molecule (EpCAM) that is overexpressed in several carcinomas. To enhance the targeted delivery of cytotoxic drugs using Ec1, we investigated the potential of fusing Ec1 with an albumin-binding domain (ABD) to improve its circulation time and decrease renal uptake. Two fusion proteins were created, Ec1-ABD, with the ABD at the C-terminus, and ABD-Ec1, with the ABD at the N-terminus. Both variants were labeled with ¹¹¹In. ABD-fused variants bound specifically to EpCAM-expressing cells with picomolar affinity. Adding human albumin reduced the affinity. This effect was more pronounced for Ec1-ABD; however, the affinity remained in the subnanomolar range. The position of the ABD did not influence the internalization rate of both variants by human cancer cells. In mouse models with human cancer xenografts, both variants demonstrated over 10-fold lower renal uptake compared to the Ec1. Tumor uptake of the ABD-fused variants was higher than the uptake of Ec1. ABD-Ec1 provided two-fold higher tumor uptake, indicating fusion with an ABD as a promising way to modulate the targeting properties of an Ec1-based construct. However, the effect of fusion depends on the order of the domains.

The immune checkpoint protein B7-H3 (CD276) is overexpressed in various cancers and is an attractive target for the treatment of malignant tumors. Radionuclide molecular imaging of B7-H3 expression using engineered scaffold proteins such as Affibody molecules is a promising strategy for the selection of potential responders to B7-H3-targeted therapy. Feasibility of B7-H3 imaging was demonstrated using two ^99mTc-labeled probes, AC12 and an affinity-matured SYNT179 using a [^99mTc]Tc-GGGC label. This study aimed to evaluate whether the use of a residualizing ¹¹¹In-based label provides better imaging contrast compared with a nonresidualizing label. To do that, SYNT179 and AC12-GGGC Affibody molecules were labeled with ¹¹¹In using (4,10-bis-carboxymethyl-7-{[2-(2,5-dioxo-3-thioxo-pyrrolidin-1-yl)-ethylcarbamoyl]-methyl}-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid (maleimide-DOTA) chelator, site-specifically coupled to the C-terminus of Affibody molecules. The binding affinities of the ¹¹¹In-labeled conjugates to B7-H3-expressing living cells were higher compared with the affinities of the ^99mTc-labeled variants. In mice with B7-H3-expressing xenografts, the tumor uptake of ¹¹¹In-labeled proteins (3.6 ± 0.3 and 1.8 ± 0.5%ID/g for [¹¹¹In]In-SYNT179-DOTA and [¹¹¹In]In-AC12-DOTA, respectively) was significantly (p < 0.05, ANOVA) higher than those for ^99mTc-labeled counterparts (1.6 ± 0.2%ID/g and 0.8 ± 0.2%ID/g for [^99mTc]Tc-SYNT179 and [^99mTc]Tc-AC12-GGGC, respectively). The best variant, [¹¹¹In]In-SYNT179-DOTA, provided a tumor-to-blood ratio of 31.1 ± 2.9, which was twice higher than that for [^99mTc]Tc-SYNT179 and 7-fold higher than that for [^99mTc]Tc-AC12-GGGC. Both ¹¹¹In-labeled Affibody molecules had higher renal retention compared with ^99mTc-labeled ones, but the hepatobiliary excretion of ¹¹¹In-labeled proteins was appreciably lower, potentially improving the imaging of abdominal metastases. Overall, [¹¹¹In]In-SYNT179-DOTA is the most promising tracer for visualization of B7-H3 expression.

Purpose: 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. Methods: 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 ¹⁷⁷Lu. Affinity, specificity, and in vivo targeting properties of [¹⁷⁷Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated. Results: The maximum molar activity of 52 GBq/µmol [¹⁷⁷Lu]Lu-PEP49989 was obtained. [¹⁷⁷Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [¹⁷⁷Lu]Lu-PEP49989 was similar to the affinity of [¹⁷⁷Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [¹⁷⁷Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [¹⁷⁷Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [¹⁷⁷Lu]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 [¹⁷⁷Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild. Conclusion: In preclinical studies, [¹⁷⁷Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

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 [¹⁷⁷Lu]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:[¹⁷⁷Lu]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:[¹⁷⁷Lu]Lu-HP16, Z-HP9:[¹⁷⁷Lu]Lu-HP20, and the Z-HP15:[¹⁷⁷Lu]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.

Background: Radionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast. Results: Two dimeric variants of anti-B7-H3 Affibody molecules were produced (designated Z_AC12*-Z_AC12*-GGGC and Z_AC12*-Z_{Taq_3}-GGGC). Both variants were labelled with Tc-99m (^99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [^99mTc]Tc-Z_AC12*-Z_AC12*-GGGC showed subnanomolar affinity (K_D1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [^99mTc]Tc-Z_AC12*-Z_{Taq_3}-GGGC (K_D=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with ^99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule. Conclusion: The improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.