*2.3. [18F]AlF Radiolabeling and In Vitro Stability*

The affibody conjugates were radiolabeled with [18F]AlF in a one-pot reaction at pH 4, 100 ◦C for 15 min using ethanol as organic co-solvent (50% *v*/*v*) (Scheme 2). Consistent with previously reported results for maleimide-derived affibody conjugates, some thermal degradation products were formed during the reaction (Figures S8,S9) [17]. Therefore, a RP–HPLC purification procedure, followed by HLB-SPE, was required to produce [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA -PODS-ZEGFR:03115 with a RCP > 98%, verified by RP–HPLC (Figure 2). As shown in Table 1, the radiochemical yields and the apparent specific/molar activities (SA/MA) achieved for [18F]AlF-NODAGA -PODS-ZEGFR:03115 were lower than for [18F]AlF-NOTA-PODS-ZEGFR:03115. Similar findings have been described by other research groups and attributed to the chelator structure and chelation capacity towards the [18F]AlF complex [19,20].



**Figure 2.** Radiochromatograms of pure [18F]AlF-NOTA-PODS-ZEGFR:03115 (**A**), and [18F]AlF-NODAGA-PODS-ZEGFR:03115 (**B**). The retention time (Rt) is indicated as min:sec.

To investigate the impact of the PODS linker on the radioconjugate properties, the distribution coefficient (logD7.4) values of the two products were determined (Table S1). The logD7.4 for [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 was found to be hydrophilic, with values of −1.73 ± 0.07 and −3.62 ± 0.06, respectively. Including the maleimide-bearing product [ 18F]AlF-NOTA-ZEGFR:03115 logD7.4 value of <sup>−</sup>1.13 <sup>±</sup> 0.1, the NODAGA-radioconjugate proved to be the most hydrophilic of the tested compounds.

The stability of the PODS-bearing radioconjugates, with respect to change in radiochemical purity (RCP) and loss of radioactivity from the affibody molecule, was determined in mouse serum at 37 ◦C for 1 h (Table S2). As affibody molecules have short blood half-life, and the in vivo studies (imaging and biodistribution) are typically performed 1 h post-injection, a longer incubation time was not investigated [21]. According to RP–HPLC analysis, both conjugates exhibited good stability as 92.7 ± 2.6% of [18F]AlF-NOTA-PODS-ZEGFR:03115 and 97.2 <sup>±</sup> 1.2% of [18F]AlF-NODAGA-PODS-ZEGFR:03115 remained intact after 1 h (Figure S10, Table S2). As previously observed for [18F]AlF-NOTA-ZEGFR:03115, a residual activity associated with the pelleted protein indicates some non-specific affinity of the radioconjugates towards the serum proteins (Table S2). However, amongst the tested compounds, the maleimide-bearing [18F]AlF-NOTA-ZEGFR:03115 demonstrated significantly higher protein-associated radioactivity than [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 (30.5 <sup>±</sup> 2.1% vs. 24.2 ± 3.4% and 20.4 ± 0.7%, respectively). Studies to determine whether this effect is connected to the maleimide linker are under investigation.

#### *2.4. In Vivo Studies*

To investigate the chelator effect on the pharmacokinetics and targeting properties of the conjugates, the two PODS-bearing radioactive agents were assessed in vivo in high EGFR-expressing U87MGvIII tumor-bearing mice. To determine the influence of PODS, a comparison to the maleimide derivative [ 18F]AlF-NOTA-ZEGFR:03115 was performed. Previous studies found that 12 μg of radiolabeled EGFR-targeting affibody conjugate with an apparent specific activity of 0.09–0.15 MBq/μg was able to partially saturate the endogenous EGFR expression (e.g., liver) and allowed for clear visualization of the tumors 1 h post-injection [22]; therefore, a dose of 12 μg (1.1–1.8 MBq) of each radioconjugate was injected. The biodistribution data indicate that the nature of the linker (i.e., PODS or maleimide) minimally influenced the radioconjugate pharmacokinetics, since similar distribution profiles were observed by both the NOTA-based products [18F]AlF-NOTA-PODS-ZEGFR:03115 and [ 18F]AlF-NOTA-ZEGFR:03115 (Figure 3, Table S3). However, compared to the NOTA-bearing products, [ 18F]AlF-NODAGA-PODS-ZEGFR:03115 showed an inconsistent radioactivity accumulation in the liver. This result suggests that the chelator can influence the radioconjugate pharmacokinetics and that chelator-customized injected protein doses and specific activities should be investigated further by dose-escalation studies. Among the non-targeted organs, the highest uptake of all three conjugates was found in the kidneys, which is due to renal excretion of the affibody molecule, and the reabsorption of the radioactive metabolites followed by their retention in the proximal tubular cells [23]. Based on this effect, the kidney uptake value for [18F]AlF-NODAGA-PODS-ZEGFR:03115 suggests a possible higher renal clearance of the NODAGA conjugate compared to [18F]AlF-NOTA-PODS-ZEGFR:03115. Moreover, both PODS-bearing radioconjugates show significantly lower kidney uptakes than the maleimide analogue, indicating a possible effect of the PODS linker on the renal retention (Figure 3 and Table S3). More experiments will be needed to confirm this observation. High accumulation of the conjugates was found in the tumors, with uptakes of 14.1 ± 5.3 and 16.7 ± 4.5 %ID/g for [ 18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115, respectively (Figure 4, Table S3). The favorable tumor-to-muscle ratios for the two PODS-bearing radioconjugates resulted in high-contrast PET images already 1 h post-injection (Table 2). In contrast to Adumeau et al., there was no difference in both the biodistribution profile and the tumor-to-tissue ratios between the [18F]AlF PODS-bearing conjugates and the malemide-based product (Table 2, Table S3) [15].

**Figure 3.** Biodistribution results for [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS -ZEGFR:03115 at 1 h p.i. [18F]AlF-NOTA-ZEGFR:03115 was used as a control. The data are reported as the mean percentage of the injected dose per gram of tissue (%ID/g) ± SD (for each group, *n* = 3).

**Figure 4.** PET/CT images of U87MGvIII tumor-bearing mice using [18F]AlF-NOTA-PODS-ZEGFR:03115 and [ 18F]AlF-NODAGA-PODS-ZEGFR:03115. [18F]AlF-NOTA-ZEGFR:03115 was used as control. High-contrast images were acquired as early as 1 h p.i. The tumors are indicated by a white circle.

**Table 2.** Tumor-to-organ ratios at 1 h p.i. of the three examined [18F]AlF conjugates. No differences were identified.


#### **3. Materials and Methods**

#### *3.1. General Materials and Methods*

Chemicals and solvents were purchased and used without further purification unless otherwise stated. The 2,2 -(7-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1,4,7-triazonane-1,4-diyl)diacetic acid (NOTA-NHS) and 2,2 -(7-{1-carboxy-4-[(2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl]-1,4,7-triazonane-1,4 diyl}diacetic acid (NODAGA-NHS) were purchased from Chematech (Dijon, France). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) was purchased from Thermo Fisher Scientific (Loughborough, UK). Ethanol (EtOH) and HPLC grade acetonitrile, trifluoroacetic acid (TFA) and formic acid were purchased from Thermo Fisher Scientific (Loughborough, UK). Phosphate-buffered saline (PBS) was purchased from Gibco (Life Technologies, Paisley, UK). Ethylenediaminetetraacetic acid (EDTA), *N*,*N*-diisopropylethylamine (DIPEA), mouse serum, dimethylformamide (DMF), *n*-octanol and Iso-Disc PVDF syringe filters (13 mm, 0.2 μm) were purchased from Sigma-Aldrich (Gillingham, UK). Aluminum chloride hexahydrate (AlCl3, 99.9995%) was purchased from Alfa Aesar (Heysham, UK). Sodium acetate (AnalR Normapur) was purchased from VWR International (Lutterworth, UK). The ZEGFR:03115-Cys affibody molecule was provided by Affibody AB (Solna, Sweden http://www.affibody.com) as a solution in 0.2 M sodium acetate. Phenyloxadiazolyl methylsulfone (PODS) was synthesized as described in the literature [15]. The maleimide-bearing affibody analogue NOTA-ZEGFR:03115 was prepared and radiolabeled as previously reported [22]. Low-protein-binding microcentrifuge tubes (1.5 mL) were purchased from Eppendorf (Stevenage, UK). Dry bifunctional chelators and affibody conjugates were achieved using a Concentrator Plus (Eppendorf, Stevenage, UK). Incubation of the reaction mixtures was performed using a Grant Bio thermo shaker (Camlab, Stevenage, UK). Protein conjugate concentration was determined by measuring the UV absorbance at 280 nm on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Loughborough, UK) using a molar extinction coefficient (ε280) of 36345 cm−<sup>1</sup> M−1. Oasis HLB (1 mL, 30 mg sorbent) SPE cartridges were purchased from Waters

(Elstree, UK). [18F]Fluoride was produced a GE PETrace cyclotron by 16 MeV irradiation of an enriched [ 18O]H2O target, supplied by Alliance Medical Radiopharmarcy Ltd. (Warwick, UK) and used without further purification.

Analytical and semi-preparative HPLC were carried out on an Agilent Infinity 1260 quaternary pump system equipped with a 1260 Diode array (Agilent Technologies, Didcot, UK). Elution profiles were recorded using Laura 4 software (Lablogic, Sheffield, UK, 2013). NOTA-PODS and NODAGA-PODS were analyzed on an Eclipse XDB C18 column, 4.6 × 150 mm, 5 μm (Agilent Technologies, Didcot, UK) using Gradient 1: 0–20 min 3%–60% B, at flow rate of 1 mL/min with 0.1% TFA in water as eluent A and 0.1% TFA in acetonitrile as eluent B. The bifunctional chelators were purified by semi-preparative RP–HPLC on a Ultracarb ODS C18 column, 10 × 250 mm, 7 μm (Phenomenex, Macclesfield, UK), using Gradient 1a: 0–20 min 3%–60% B, at a flow rate of 3mL/min with 0.1% formic acid in water as eluent A and 0.1% formic acid in acetonitrile as eluent B and. Affibody conjugates and radioconjugates were analyzed on a Zorbax 300SB C18 column, 4.6 × 250 mm, 5 μm (Agilent Technologies, Didcot, UK) using Gradient 2: 0–20 min. 30%–65% B, with 0.1% TFA in water as eluent A and 0.1% TFA in acetonitrile as eluent B at a flow rate of 1 mL/min. Affibody conjugates were purified by semi-preparative RP–HPLC on a Jupiter C18 column, 10 × 250 mm, 10 μm, 300 Å (Phenomenex, Macclesfield, UK), using Gradient 2 at a 3 mL/min flow rate. The radioactivity of the eluate was monitored using an IN/US Systems Gamma-ram Model 4 NaI radiodetector (Lablogic, Sheffield, UK). Retention times (Rt) are expressed as minutes:seconds (min:sec). Electro spray ionization high-resolution mass spectrometry (ESI–HRMS) was performed using an Agilent 1200 series LC pump with a 6210 time-of-flight (TOF) mass analyzer. Protein MS was performed on 6520 a Series qToF mass spectrometer fitted with a dual ESI ionization source (Agilent, Santa Clara, CA, USA).
