Comparison of approaches for increasing affinity of affibody molecules for imaging of B7-H3: dimerization and affinity maturation

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 ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=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. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-024-00261-3.


Introduction
Non-invasive detection of overexpression of cell-surface proteins in malignant tumours can provide diagnostic information influencing patient stratification for specific therapies.Analysis of biopsy samples, a commonly used method to assess the expression level of molecular targets in solid tumours, is associated with several limitations caused by its invasiveness and heterogeneity of targets' expression.Alternatively, radionuclide molecular imaging modalities such as single photon emission computed tomography (SPECT) and/or positron emission tomography (PET) could be used as non-invasive techniques to estimate expression levels of the cell-surface proteins enabling selection of targeted therapies and personalized anti-cancer treatment (Wu et al. 2023).
An emerging molecular target for cancer treatment is the immune checkpoint protein B7-H3 (Sun et al. 2002).This protein is involved in oncogenic signalling, tumour cell plasticity, and drug resistance.The protumourigenic activity of B7-H3 includes promotion of endothelial-to-mesenchymal transition, invasion, and angiogenesis (Zhou and Jin 2021).It should be noted that B7-H3 is overexpressed on differentiated malignant and cancer-initiating cells in many different cancer types (Roth et al. 2007;Zang et al. 2007Zang et al. , 2010;;Crispen et al. 2008;Boorjian et al. 2008;Lemke et al. 2012;Ingebrigtsen et al. 2014;Sun et al. 2014;Bachawal et al. 2015;Inamura et al. 2018;Pulido and Nunes-Xavier 2023), while its expression level is low in most normal organs and tissues (Modak et al. 2001) making it an attractive molecular target for diagnostic and/or treatment applications.
Preclinical studies (Burvenich et al. 2018;Kasten et al. 2018) and phase I clinical trials (Modak et al. 2020;Kramer et al. 2022) using radiolabelled antibodies have demonstrated successful radionuclide targeting of B7-H3-expressing tumours.In spite of a specific tumour localisation and acceptable safety in patients, the bulkiness of such agents causes a relatively slow accumulation in tumours and clearance of activity from blood circulation.It means that several days are needed to obtain a reasonable imaging contrast.An alternative would be to reduce the size of the imaging agent, which is beneficial in term of better extravasation and faster clearance from blood and non-targeted sites (Garousi et al. 2020).The Affibody molecule is a small (6-7 kDa) engineered affinity protein based on a robust triple helical scaffold (Ståhl et al. 2017).Robustness, straightforward production and site-specific labelling are advantages of the use of Affibody molecules.The reduction of the size resulted in a high extravasation rate and fast clearance from blood providing high-contrast imaging a few hours after injection.In addition, small proteins are not accumulated in tumours due to the enhanced permeability and retention (EPR) effect (Wester and Kessler 2005), which increases specificity of imaging in comparison with imaging using monoclonal antibodies.These are essential advantages of Affibody molecules compared with monoclonal antibodies.Affibody molecules have been successfully evaluated as promising targeting agents for both preclinical (Orlova et al. 2006;Strand et al. 2014;Rosestedt et al. 2015;Garousi et al. 2016;Andersson et al. 2016;Oroujeni et al. 2018Oroujeni et al. , 2021;;Liu et al. 2022) and clinical (Sörensen et al. 2014(Sörensen et al. , 2016;;Alhuseinalkhudhur et al. 2023;Bragina et al. 2023) imaging of different molecular targets.Safety of this drug class has furthermore been demonstrated clinically with up to three years chronic patient dosing, suggesting viability for repeated administrations (Klint et al. 2023).
Proof-of principle of radionuclide visualization of expression levels of B7-H3 in vivo 2-4 h after injection was demonstrated using the [ 99m Tc]Tc-AC12-GGGC Affibody molecule (Oroujeni et al. 2022).It should be noted that the high sensitivity provided by the imaging agent is an essential precondition for successful translation of a radionuclide-based imaging agent into the clinic.The sensitivity depends on imaging contrast (tumour-to-organ ratios).These ratios are influenced by several characteristics of the imaging agent such as cellular retention of the label during cellular processing, tumour localization rate, clearance rate from normal organs, and biologic properties of the target.For development of new successful imaging agents, the focus should be on factors increasing the imaging contrast.This could be possible by both increasing tumour uptake and/or by decreasing uptake in non-targeted organs and tissues.affinity, i.e. a strength of binding to the desirable molecular target, is one of the crucial factors defining the efficacy of tumour targeting.Since the internalization of Affibody molecules is relatively slow (around 20-30% of cell-bound conjugate per day), the cellular retention of the radionuclide is strongly influenced by the binding strength of imaging agents to its target on a cancer cells surface (Wållberg and Orlova 2008;Tran et al. 2009).Cellular retention thus depends on the rate of dissociation of the tracer from target and on cellular processing of the tracer-target complex.Several studies have demonstrated that optimization of the binding strength of protein-based targeting probes to their tumour targets is critical for efficient tumour localization (Adams et al. 1998a;Adams et al. 1998b;Adams et al. 2001;Adams et al. 2006;Tolmachev et al. 2012).Affinity maturation can be used as an approach to improve affinity of Affibody molecules binding to different molecular targets, such as EGFR/HER1 (Friedman et al. 2008), HER2 (Orlova et al. 2006), HER3 (Malm et al. 2013) and PDGFRβ (Lindborg et al. 2011).Di-or multimerization is another approach for improving tumour-targeting properties of targeting probes, e.g.single-chain Fv fragments (Wu et al. 1996;Nielsen et al. 2000;Goel et al. 2001).This approach usually decreases the dissociation rate.This increases an apparent affinity of binding to a molecular target and might increase tumour localization of a number of targeting proteins (Wu et al. 1996;Adams et al. 1998b;Nielsen et al. 2000;Batra et al. 2002).However, there is a risk that an increase in size of the targeting molecule might outweigh the benefit of an increased affinity (Orlova et al. 2006;Tolmachev et al. 2009;Garousi et al. 2019).
The results of a recent study (Oroujeni et al. 2023) have shown that affinity maturation of anti-B7-H3 Affibody molecules resulted in an appreciably increased affinity compared to parental AC12 Affibody molecule.The use of these affinity matured Affibody molecules has shown an improved imaging contrast for imaging of B7-H3-expressing tumours in a murine model.However, once a promising targeting protein is found, further optimization of a tracer is often required.
The aim of this study was to find out, which approaches for increasing apparent affinity of B7-H3 Affibody molecules, dimerization or affinity maturation, would could improve the tumour-targeting properties of Affibody molecules for imaging of B7-H3-expressing tumours.In this study, according to previous studies (Altai et al. 2012;Oroujeni et al. 2021), peptide-based cysteine containing chelator -GGGC was incorporated at the C-terminus at Affibody molecules for labelling with 99m Tc.In this complex (Ahlgren et al. 2009;Altai et al. 2012), the amide nitrogens of amino acids from glycine and the thiol group of cysteine at peptide-based cysteine containing chelator form together a N 3 S chelator, providing a stable complex with 99m Tc (Fig. 1B).In order to compare different affinities at the same size, two dimeric variants of anti-B7-H3 Affibody molecules, Z AC12* -GGGC and Z AC12* -Z Taq_3 -GGGC, were produced, were produced, characterized and labelled with 99m Tc as a commonly used radionuclide for SPECT imaging in clinics.The first variant, Z AC12* -Z AC12* -GGGC contained two units of the parental Z AC12* Affibody molecule binding to B7-H3.To evaluate impact of size, the second variant contained only one B7-H3-binding unit and the second unit was Z Taq_3 Affibody molecule, which is a nonbinding Affibody.Z Taq is an Affibody originally selected as a binder to Thermus aquaticus (Taq) DNA polymerase, which doesn't interact with any mammalian proteins.Thus, Z AC12* -Z Taq_3 -GGGC is a monovalent bi-Affibody.In vitro evaluation of new variants (testing specificity and affinity) was performed using B7-H3-expressing cell lines and compared with the affinity matured monomeric Affibody molecule SYNT-179 that was recently generated (Oroujeni et al. 2023).

General 99m
Tc as pertechnetate was obtained by elution of an Ultra TechneKow generator (Mallinckrodt, Petten, The Netherlands) with sterile 0.9% sodium chloride (Mallinckrodt, Petten, the Netherlands).For quantitative measurement of radioactivity distribution in instant thin-layer chromatography strips, a Cyclone Storage Phosphor system (Perkin-Elmer, Wellesley, MA, USA) was used.An automated gamma-spectrometer with a 3-inch NaI (TI) well detector (2480 Wizard, Wallac, Turku, Finland) was used to measure activity from cell and animal samples.A dose calibrator VDC-405 (Veenstra Instruments BV, Joure, The Netherlands) equipped with an ionization chamber was utilised for measuring the radioactivity for labelling and injection formulation.
B7-H3-expressing ovarian cancer SKOV-3 and breast cancer BT-474 cell lines, obtained from the American Type Culture Collection (ATCC), was used for in vitro cell studies.Ramos lymphoma cells (ATCC) were used to establish B7-H3-negative xenografts.Cells were cultured in RPMI medium (Flow Laboratories, Irvine, UK) supplemented with 10% of fetal bovine serum (20% of fetal bovine serum for BT-474), 2 mM of L-glutamine, 100 IU/mL of penicillin, and 100 mg/mL of streptomycin.
To determine significant differences (p < 0.05), in vitro studies and biodistribution data were analysed by unpaired 2-tailed t-test and ANOVA using GraphPad Prism (version 6 for Windows; GraphPad Software).

Production, purification, and characterization of Novel Anti-B7-H3 Affibody Molecules
Genes encoding dimeric versions of a Z AC−12 were ordered from GeneArt (Thermo Fisher Scientific, MA, USA) as codon optimized and synthesized genes cloned in a custom plasmid to form fusion proteins with a His 6 tag and a TEV protease cleavage site.After TEV protease treatment, resulting sequences were in the format G-Z AC12* -Z AC12 -GGGC and G-Z AC12* -Z Taq_3 -GGGC respectively.Z AC−12 variants were expressed in autoinducing medium (Overnight Express TB, Novagen) inoculated with precultures of E. coli T7E2 clones carrying plasmids with sequence verified gene fragments of each B7-H3 binding Z AC−12 variant.Cells were disrupted, centrifuged and supernatants were used immediately for purification.
The lysates were purified using 1 mL His GraviTrap IMAC column (Cytiva).Contaminants were removed by washing with wash buffer and the Z AC−12 variants were subsequently eluted with elution buffer, with 1 mM dithiothreitol (DTT) added to all buffers.The Z AC−12 variants were buffer exchanged to 1 × DPBS with 1 mM DTT added and incubated over night at 4 °C with His-tagged TEV protease in a 20:1 molar ratio of Z AC−12 variant: TEV protease, and DTT was added to 2 mM final concentration.The incubated samples, supplemented with 20 mM imidazole, were applied on a 1 mL His GraviTrap IMAC column (Cytiva) equilibrated with binding buffer.TEV protease cleaved Z variants were collected in the flow through whereas His-tagged material bound to the IMAC resin.The non-tagged Z variants were further purified by reverse phase chromatography (RPC).and the buffer was exchanged to 1 × DPBS + 2 mM EDTA using PD-10 desalting columns (Cytiva).
RP-HPLC-MS analysis: Samples were analyzed using the Agilent 1290 Infinity UHPLC-system, equipped with single quadrupole and AP-ESI and the analytical column Zorbax 300SB-C8 RRHD (Agilent).

Cell assays
The Affibody molecules SYNT-179, Z AC12* -Z AC12* -GGGC and Z AC12* -Z Taq_3 -GGGC were tested in terms of binding to B7-H3 expressing SKOV-3 cells.The cells were placed in a V-bottom 96-well plate (0.2 × 10 6 cells/well) and incubated at 4 °C for 1 h with Affibody molecules at decreasing concentrations from 555 nM to 28 pM.After 1× washing in PBS with 1% fetal bovine serum (FBS), binding of Affibody molecules was identified by an anti-Affibody polyclonal antibody (4 µg/mL) and incubated at 4 °C for 1 h followed by an Alexa488 conjugated goat-anti-rabbit IgG diluted 1:2000 and incubated at 4 °C for 1 h.After 2× washing in PBS with 1% FBS, fluorescence intensity was measured by a multimode plate reader (Enspire).Each sample was test-ed in quadruplicates.Binding curves were plotted and EC50 values were determined using software GraphPad Prism.

Labelling with 99m Tc and in vitro stability of radioconjugates
Labelling of Affibody molecules with 99m Tc was performed using a lyophilized kit, as described earlier (Ahlgren et al. 2010).Briefly, a freeze-dried labelling kit containing 75 µg of tin (II) chloride dihydrate (Fluka Chemika, Buchs, Switzerland), 5 mg of gluconic acid sodium salt (Celsus Laboratories, Geel, Belgium), and 100 µg of ethylenediaminetetraacetic acid tetra sodium salt (EDTANa 4 ) (Sigma-Aldrich, Munich, Germany) was prepared for labelling of Affibody molecules with 99m Tc.Radiolabelling of Affibody molecules was performed by adding the contents of the freeze-dried kit, dissolved in 120-µL degassed PBS, to 100 µg of the Affibody molecule.80 µL (200-300 MBq) of 99m Tc-pertechnetate was added to the reaction mixture and degassed to protect the mixture from oxidation.The reaction vial was thoroughly vortexed and incubated at 90 °C for 1 h.Radiochemical yield of Affibody molecules was analysed using instant thin layer chromatography (ITLC-SG) (Agilent Technologies, Santa Clara, CA, USA) developed with PBS (Affibody: R f = 0.0, other forms of 99m Tc: R f = 1.0).The reduced hydrolysed technetium colloid (RHT) level in the labelling mixture was analysed using pyridine: acetic acid: water (10:6:3) as the mobile phase ( 99m Tc colloid: Rf = 0.0, other forms of 99m Tc and radio-labelled affibody molecule: Rf = 1.0).Since the radiochemical yield was more than 95% for all radioconjugates, no further purification was performed for in vitro and in vivo experiments.
To test in vitro stability, a fraction of a fresh radioconjugate (10 µL, 4 µg) was incubated with excess amount of PBS (40 µL) at 37 °C for 4 h.The test was performed in triplicates.To evaluate stability in blood serum, two samples of each radioconjugate (0.4 nmol) were mixed with the murine blood serum (100 µL) to mimic a concentration of the tracer in murine blood immediately after injection.Two control samples were mixed with the same amount of PBS.All samples were incubated at 37 ℃ for 4 h, and thereafter they were passed through NAP5 columns to separate a high molecular weight compounds (over 5 kDa) and low molecular weight compounds (less than 5 kDa).Activities in both high-and low-molecular weight fractions were measured to calculate percentage of 99m Tc associated with the high molecular weight fraction (i.e., bound to Affibody molecules).

In vitro studies
Ovarian carcinoma SKOV-3 and breast carcinoma BT-474 cell lines as B7-H3-positive cells were used for in vitro studies.Ramos lymphoma cell line was used as a B7-H3-negative control for in vitro specificity and in vivo studies.B7-H3 expression levels were estimated to be 68000, 45000 and 250 receptors per cell for SKOV-3, BT-474 and Ramos, respectively (Oroujeni et al. 2022).Cells were seeded in cell-culture dishes (35 mm in diameter) with a density of 10 6 cells/dish for in vitro study.A set of three dishes was used for in vitro binding specificity test.
To test in vitro binding specificity of each radioconjugate, cells in three control dishes were pre-saturated with 200-fold excess of a non-labelled Affibody molecule 15 min before addition of a radiolabelled conjugate.Cells in both blocked and nonblocked dishes were incubated with a radiolabelled conjugate (10 nM) in a humidified incubator (5% CO 2 , 37 °C) for 1 h.The medium was discarded, the cells were washed with cold serum-free medium before trypsin-EDTA solution (0.5 mL per dish) was added, and cells were additionally incubated for 10 min.Detached cells were diluted with 0.5 mL of complete medium, re-suspended, and transferred to fraction tubes.The radioactivity associated with cells was measured using an automated gamma counter and the cellbound radioactivity was calculated.
Affinity of binding of each radiolabelled Affibody molecule to B7-H3 target was measured using a LigandTracer Yellow instrument (Ridgeview Instruments AB, Vänge, Sweden), as previously described (Björke and Andersson 2006).SKOV-3 cells (2 × 10 6 cells/ dish) were seeded on a local area of a cell culture dish (89 mm in diameter, NunclonTM, NUNC A/S, Roskilde, Denmark).The measurements were performed at room temperature to prevent internalization.Uptake curves were recorded for concentrations 1, 3 and 9 nM of 99m Tc-labelled conjugates.After incubation with labelled conjugate, the radioactive medium was replaced with fresh non-radioactive medium, and the dissociation curve was recorded for several hours.Analysis was performed in duplicates.The data were analysed using the Interaction Map software (Ridgeview Diagnostics AB, Uppsala, Sweden) to calculate the association rate, the dissociation rate, and the dissociation constant at equilibrium (K D ).The basics of the InteractionMap analysis is described by Altschuh et al. (Altschuh et al. 2012).

In vivo studies
Animal experiments were performed in accordance with the national legislation for work with laboratory animals.All animal studies were approved by the Ethics Committee for Animal Research in Uppsala (Sweden), following the national legislation on the protection of laboratory animals (permit 5.8.18-00473/2021, approved 26 February 2021).Four mice per data point were used in biodistribution experiments.
Biodistribution and targeting properties of 99m Tc-labelled Affibody conjugates were evaluated in BALB/C nu/nu mice bearing B7-H3-positive SKOV-3 xenografts.To establish xenografts, SKOV-3 cells (10 7 cells/mouse) were subcutaneously injected on the right hind leg of female BALB/c nu/nu mice.For in vivo specificity, B7-H3-negative Ramos cells (5 × 10 6 cells/mouse) were subcutaneously implanted on the left hind leg of female BALB/c nu/nu in mice.Biodistribution measurement was performed two weeks after cell implantation.Average animal weight was 17.9 ± 1.5 g.Average tumour weight was 0.27 ± 0.18 g and 0.8 ± 0.4 g for SKOV-3 and Ramos xenografts, respectively.The biodistribution was measured 4 h after injection in mice bearing SKOV-3 xenografts.Groups of 4 mice bearing tumour were injected with 99m Tc-labelled Affibody molecules (0.4 nmol, 7.8 µg, 60 kBq, 100 µL in PBS) into the tail vein.To test B7-H3-specific accumulation, one group of animals bearing B7-H3-negative Ramos xenografts was injected with the same peptide and activity doses for each conjugate and the biodistribution was measured 4 h after injection.After 4 h, mice were euthanized by overdosing of anaesthetic solution (20 µL of solution per gram of body weight: ketamine, 10 mg/mL; Xylazine, 1 mg/mL).This was followed by a heart puncture, and blood samples were collected.Organs and tissue samples were collected and weighed.Organ radioactivity was measured using a gamma spectrometer along with three standards of injected activity and empty syringes for each animal.Uptake values for organs were calculated as the percentage of injected dose per gram tissue (%ID/g).For comparison, one group of mice was injected with monomeric Affibody molecule SYNT-179 labelled with 99m Tc (0.4 nmol, 3 µg, 60 kBq, 100 µL in PBS) into the tail and biodistribution was measured 4 h after injection.To show in vivo binding specificity, two groups of mice bearing SKOV-3 xenografts were intraperitoneally injected with 600 µg of Affibody molecules 40 min before injection of the best binders, [ 99m Tc]Tc-Z AC12*− Z AC12*− GGGC and [ 99m Tc]Tc-SYNT-179.The biodistribution was measured 4 h after injection as described above.
To confirm biodistribution results, a small animal SPECT/CT imaging was performed.One SKOV-3 bearing mouse was intravenously injected with 14 MBq/0.4nmol of 99m Tc-labelled Affibody molecule.To confirm in vivo specificity, one mouse bearing Ramos xenograft was intravenously injected with the same mass and activity of 99m Tc-labelled Affibody molecule.The mice were imaged 4 h after injection using a nanoSPECT/CT scanner (Mediso Medical Imaging Systems, Budapest, Hungary).The mice were euthanized by CO 2 asphyxiation immediately before being placed in the camera.The computed tomography (CT) acquisition was carried out at the following parameters: energy peak of 50 kV, 670 µA, 480 projections, and 2.29-min acquisition time.SPECT acquisition was performed at the following parameters: 99m Tc energy peak of 140 keV, window width of 20%, matrix of 256 × 256, and acquisition time of 1 h.CT images were reconstructed in real-time using Nucline 2.03 Software (Mediso Medical Imaging Systems, Budapest, Hungary).SPECT raw data were reconstructed using Tera-Tomo™ 3D SPECT reconstruction technology.

Production, purification, and characterization of novel Anti-B7-H3 affibody molecule
To evaluate targeting properties of Affibody molecule labelled with 99m Tc, the peptidebased cysteine containing -GGGC chelator was incorporated at the C-terminus of Affibody molecules.Figure 1B shows the general structure of the N 3 S chelator formed by a C-terminal cysteine and three adjacent amino acids (triglycine-cysteine, ̶ GGGC, chelator).The sequences of Affibody molecules, which were evaluated in this study, as well as the sequence of the parental Z AC−12 -GGGC Affibody molecules are shown in Fig. 1A.

Labelling with 99m Tc and in vitro stability of radioconjugates
Labelling of dimeric anti-B7-H3 Affibody molecules with 99m Tc was successfully performed providing the radiochemical yield exceeding 95% and radiocolloid concentration below 5% (Table 1).The specific activity of 99m Tc-labelled conjugates was 3 MBq/µg.Because of the high radiochemical purity of the labelled constructs, no additional purification was performed for in vitro and in vivo studies.99m Tc-labelled Affibody molecules were stable during incubation at 37 °C for 4 h in the presence of excess of PBS (Table 1).
After 4-h incubation of in murine serum at 37 °C, 94.3 ± 3.6, 96.9 ± 0.3 and 91.3 ± 0.5% of technetium was associated with the high molecular weight fraction (molecular weight over 5 kDa) for [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC, [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC and [ 99m Tc]Tc-SYNT-179, respectively.In control samples, which were diluted in PBS to the same concentration and incubated in the same condition, 96.4 ± 0.9, 98.8 ± 1.7 and 92.1 ± 0.7% of technetium was associated with the high molecular Fig. 1 (A) Amino acid sequences of the evaluated Affibody molecules and (B) general structure of the N 3 S chelators formed by a C-terminal cysteine and three adjacent amino acids.X 1 , X 2 and X 3 are denoting the side chains of the amino acids (in this case X 1 ,X 2 , X 3 = hydrogen atom for glycine).The variable amino acids are marked in red and the chelator composition in green bold fraction for [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC, [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC and [ 99m Tc]Tc-SYNT-179, respectively.
According to radiochromatogram, both 99m Tc-labelled conjugates were eluted as single peaks with the retention time of between 12 and 13 min (Fig. S1) with a good match to the retention time of non-labelled standards (ultraviolet detector).

In vitro studies
In vitro binding specificity of [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC and [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC to B7-H3-expressing cells was measured by a saturation experiment.The binding was significantly (p < 0.005) decreased when the cells were presaturated with the excess amount of non-labelled anti-B7-H3 Affibody molecule (Fig. 3).The binding of both variants was significantly (p < 0.005) lower on Ramos as a negative-B7-H3 cell line compared with B7-H3-positive cell lines (SKOV-3 and BT-474) confirming the binding level is proportional to the expression level of the target.

Discussion
The small size and engineered excellent binding specificity of scaffold proteins make them promising targeting vectors for radionuclide molecular imaging (Garousi et al. 2020;Tolmachev et al. 2022).The favourable imaging properties of Affibody molecules were confirmed by development of the B7-H3 imaging probe [ 99m Tc]Tc-Z AC12− GGGC (Oroujeni et al. 2022), which in a mouse model provided better tumour-to-blood ratio (11.0 ± 0.5) already 4 h after injection than the conventional antibody-based tracer 89 Zr-DS-5573a (Burvenich et al. 2018) provided 7 days after injection (5.03 ± 0.73).Still, scaffold proteins are much less studied for imaging than antibodies, and influence of different parameters on their properties should be further elucidated.
Affinity is an essential feature of tracers (Eckelman et al. 2006) and how affinity is optimized is important.To improve the imaging properties of the Affibody-molecule-mediated imaging of B7-H3, an affinity maturation was performed.The affinity maturation resulted in improved monomeric affinity and a two-fold increase of tumour-to-blood ratio as compared to the parental Z AC12 construct (Oroujeni et al. 2023).Dimeric formatting of a targeting molecule is another strategy to increase the apparent affinity and may be easier and faster to accomplish.In this study, we compared the effects of affinity maturation and a dimeric format on imaging properties of B7-H3-targeting Affibody molecules.As control we used the parental Z AC12 molecule as monomer binding but in dimeric format with a Taq polymerase binding Affibody molecule to create a construct of the same size as the high affinity dimer but with parental affinity.The two monomeric constructs, Z AC12 and SYNT-179, have been compared previously (Oroujeni et al. 2023).
Despite a seeming simplicity, the dimeric formatting might be associated with some pitfalls.For example, selection of inappropriate linkers might hamper a proper folding of the target-binding domains and reduce the affinity of a dimer (Garousi et al. 2019).However, the use of a trimeric-(G 3 S) 3 -linker in this study permitted to obtain a binding-competent dimer.Another risk is that one or both of the binding domains become sterically hindered from interacting with the epitope on the target molecule.In this case, [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC preserved the affinity of the strongest interaction of parental [ 99m Tc]Tc-Z AC12 -GGGC (1.9 nM) with positive B7-H3 cells (Table 2).Moreover, the abundance of the strongest interaction increased from 23 to 100% for [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC.The reason for this unexpected positive effect is not quite clear, but it is thinkable that the non-binding ZTaq_3 domain could help stabilize the binding interaction.On the other hand, the detection efficiency of secondary reagents to the heterodimeric Z-scaffold may have been impacted, which can be suggested from cell binding experiments in Fig. 2 where the absolute signal seems to be lower than for the other constructs.With a successful design of the architecture of the construct, the homodimeric formatting had a clear positive effect on the binding strength of Z AC12* -Z AC12* -GGGC as its half-inhibition concentration (EC 50 ) decreased nearly 50-fold compared with the Z Taq -containing variant.The EC 50 of Z AC12* -Z AC12* -GGGC was also 5.4-fold better than for the monomeric affinity matured SYNT-179.The EC 50 data were concordant with the measurements of kinetics of binding to the living cells (Table 2), where the apparent dissociation constant for the strongest interaction of [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC was 7-fold lower compared with the strongest interaction of monomer [ 99m Tc]Tc-Z AC12 -GGGC.
The affinity maturation enabled to obtain lower K D values for the strongest interaction of [ 99m Tc]Tc-SYNT-179 compared with both parental [ 99m Tc]Tc-Z AC12− GGGC and dimeric [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC, but this high-affinity interaction was relatively less abundant (K D1 =0.028 ± 0.001nM, %weight 1 = 12).The most abundant interaction of [ 99m Tc]Tc-SYNT-179 (K D2 =8.2 ± 0.5 nM, %weight 2 = 88) was 8-fold stronger that the most abundant interaction of [ 99m Tc]Tc-Z AC12 -GGGC (K D2 =68.8 ± 7.4, %weight 2 = 67).Still, a direct interpretation of kinetic measurement might be complicated, because the human B7-H3 has two identical domains and, accordingly, two potential epitopes.It could also not be excluded that binding to one epitope causes conformational changes, which modify affinity to another epitope.Overall, the EC 50 might be a better measure for comparison of binding strength, and the results of EC 50 -measurements were more in favour of the dimeric format.
Still, in vivo data are crucial for selection of the optimal format of an imaging probe.The biodistribution data from this study (Figs. 5 and 6) were in a good agreement (with overlapping of errors) with similar data obtained in earlier experiments.For example, the tumour uptake of [ 99m Tc]Tc-SYNT-179 in this study was 2.17 ± 0.54%ID/g, while Oroujeni et al. (Oroujeni et al. 2023) reported the uptake 1.54 ± 0.19%ID/g in SKOV-3 xenografts at the same time point.The tumour-to-blood ratios were 20.6 ± 5.0 in this study and 25.7 ± 2.5 in the earlier investigation.
The uptake of [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC in B7-H3-positive SKOV-3 xenografts was 12-fold higher than in B7-H3-negative Ramos xenografts (Fig. 8A), which clearly demonstrated that the tumour uptake in vivo was B7-H3-mediated.These data were confirmed by imaging results (Fig. 10A and B).Despite stronger binding of [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC in vitro, no significant difference in tumour uptake for [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC and the affinity maturated monomer [ 99m Tc]Tc-SYNT-179 was observed (p > 0.05 in ANOVA test with Bonferroni's correction for multiple comparisons).Moreover, there was a tendency to lower tumour uptake of the dimeric variant [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC.Furthermore, the tumour uptake of [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC (1.25 ± 0.3%ID/g) was not better than the tumour uptake of historical data for [ 99m Tc]Tc-Z AC12− GGGC (1.04 ± 0.08%ID/g at the same time point) reported earlier (Oroujeni et al. 2022) despite higher binding strength of the [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC.Some insight into this phenomenon might be obtained by comparison with targeting properties of [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC.The affinity of this construct was (at least) not worse than the affinity of [ 99m Tc]Tc-Z AC12 -GGGC, but its tumour uptake was lower (0.15 ± 0.05%ID/g) than the uptake of [ 99m Tc]Tc-Z AC12 -GGGC (Oroujeni et al. 2022).This suggests that the small size of an Affibody-based construct is critical for maximizing a tumour uptake.A possible explanation can be that the extravasation rate is essential for scaffold proteins, which are cleared rapidly from circulation.This rapid clearance results in a disappearance of the concentration gradient between blood and tumour.It is quite likely, that the increase of the size of a dimer slows down the extravasation during the critical time when the tracer's concentration in blood is still high (Schmidt and Wittrup 2009).This decrease cannot be compensated by improvement of the affinityImportantly, previous studies using Affibody molecules for targeting HER2 have also demonstrated that keeping the size small might be more important than gaining functional affinity by dimerization (Orlova et al. 2006;Tolmachev et al. 2009).The influence of dimerization on targeting properties of a series of HER2-targeting ADAPT molecules has similarly showed that despite higher affinity, dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer (Garousi et al. 2019).If the size penalty observed in this study is of general value, it suggests that even relatively small affinity molecules of 14 kDa size e.g.nanobodies and DARPins, may have similar extravasation penalty and therefore lower tumour uptake.

Conclusion
To conclude, increase in affinity by dimeric formatting in order to improve targeting properties of an anti-B7-H3 Affibody molecule was not efficient.It appears that increase of affinity should be performed by affinity maturation in order to keep the molecular format as small as possible.The affinity matured SYNT-179 Affibody molecule is hence a more favourable B7-H3-specific Affibody molecule than the dimeric variants tested herein for imaging of B7-H3-expressing tumours in vivo.

Fig. 5
Fig. 5 Comparative biodistribution of 99m Tc-labelled Affibody molecules in BALB/C nu/nu mice bearing SKOV-3 xenografts 4 h after injection.Data are expressed as the percentage of administered activity (injected probe) per gram of tissue (% ID/g).The data are presented as the average (n = 4) and SD

Fig. 6
Fig. 6 Tumour-to-organ ratios of 99m Tc-labelled Affibody molecules in BALB/C nu/nu mice bearing SKOV-3 xenografts 4 h after injection.The data are presented as the average (n = 4) and SD