*3.4. Radiosynthesis of [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115*

To a 1.5 mL low-protein-binding plastic tube containing either NOTA-PODS-ZEGFR:03115 or NODAGA-PODS-ZEGFR:03115 (lyophilized, 10–20 nmol), 2 mM AlCl3 (4.0–6.5 μL, 7–13 nmol) in 0.5 M sodium acetate buffer pH 4, 50 mM ascorbic acid in 25 mM sodium acetate pH 4.5 (to a final concentration of 1 mM), and aqueous non-purified [18F]fluoride (180–200 MBq) were added. A volume of ethanol was then added to achieve a final 1:1 *v*/*v* aqueous to organic solvent ratio. The mixture was heated at 100 ◦C for 15 min. After cooling to ambient temperature, the solution was purified by RP–HPLC using Gradient 2. The collected fraction containing the product was diluted with 0.1% aq TFA (3 mL) and loaded on an HLB-SPE cartridge (1 mL, 30 mg sorbent). The trapped radioactivity was washed with 0.1% aq TFA (4 mL) and then eluted with 60% ethanol/water (*v*/*v*, 120 μL). The product was quantified by measuring the UV absorbance at 280 nm. Synthesis time (from the beginning of the reaction) = ca. 45 min.

[ 18F]AlF-NOTA-PODS-ZEGFR:03115 = Analytical RP–HPLC (Gradient 2): Rt: 11:16 min:sec; RCY (decay corrected at the beginning of reaction): 11%–12.7%; apparent specific activity: 0.40–0.59 MBq/μg (apparent molar activity: 3.0–4.4 MBq/nmol).

[ 18F]AlF-NODAGA-PODS-ZEGFR:03115 = Analytical RP–HPLC (Gradient 2): Rt: 11:11 min:sec; RCY (decay corrected at the beginning of reaction): 4.3%–8.1%; apparent specific activity: 0.11–0.23 MBq/μg (apparent molar activity: 0.8–1.7 MBq/nmol).

#### *3.5. Determination of Distribution Coe*ffi*cient (LogD) at pH 7.4*

To 0.5 mL of PBS (pH 7.4), either [18F]AlF-NOTA-PODS-ZEGFR:03115 or [18F]AlF-NODAGA-PODS -ZEGFR:03115 (0.08 MBq) was added followed by 0.5 mL of *n*-octanol. The mixture was vortexed for 10 min followed by centrifugation at 100× *g* for 10 min. The experiments were performed in triplicate. Three 100 μL samples were taken from each layer and the amount of activity was measured in a 2480 WIZARD2 Automatic Gamma Counter (Perkin Elmer, Beaconsfield, UK) as counts per minutes (cpm). The distribution coefficient at pH 7.4 (logD7.4) was expressed as the mean ± standard deviation (SD) and calculated using the following formula (Equation 1):

$$\text{LogD} = \log[(\text{counts}\_{\text{octanol}}) / (\text{counts}\_{\text{PBS}})] \tag{1}$$

#### *3.6. In Vitro Serum Stability Assay*

The stability of [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 was assessed as previously described by incubating the purified [18F]AlF-radioconjugates (3.5–4 MBq) in mouse serum (500 μL) in a thermo shaker at 37 ◦C for 1 h (850 rpm) [17,24]. Each experiment was performed in triplicate. The data are expressed as the average of n = 3 measurements ± SD.

### *3.7. In Vivo Evaluation*

All experiments were performed in compliance with license issued under the UK Animals (Scientific Procedures) Act 1986 and following local ethical review (project license PCC916B22, Animals in Science Regulation Unit, Home Office Science, London, UK). The studies followed the United Kingdom National Cancer Research Institute Guidelines for Animal Welfare in Cancer Research [25]. Female NCr athymic mice (6–8 weeks old) were subcutaneously injected on the right shoulder with U87MGvIII cells (0.5 <sup>×</sup> 106/mouse) suspended in 30% Matrigel. Tumors were allowed to grow to 100 mm3. PET/CT studies were conducted on an Albira PET/SPECT/CT imaging system (Bruker, Coventry, UK). Mice were administered the radioconjugate (12 μg in 100 μL of 0.9% sterile saline, 1.1–1.8 MBq/mouse) by intravenous tail vein injection and were anesthetized using an isoflurane/O2 mixture (1.5%–2.0% *v*/*v*) approximately 5 min prior to imaging. Whole-body static PET images were acquired 1 h post-radioconjugate injection for the duration of 10 min, with a 358 to 664 keV energy window, followed by CT acquisition as previously described [26]. The image data were processed and reconstructed as previously reported [26].

Immediately after image data acquisition, the mice were euthanized by cervical dislocation for the biodistribution studies. The major organs/tissues were dissected and weighed, and the radioactivity was measured in 2480 WIZARD<sup>2</sup> Automatic Gamma Counter (Perkin Elmer, Beaconsfield, UK). The percentage of the injected dose per gram of tissue (%ID/g) was determined for each organ/tissue. The data are expressed as the average of *n* = 3 mice ± SD.

#### **4. Conclusions**

This study describes the preparation and attachment of two novel thiol-reactive PODS-bearing bifunctional chelators (NOTA-PODS and NODAGA-PODS) to the EGFR-targeting affibody molecule ZEGFR:03115. When radiolabeled with [18F]AlF, a RP–HPLC purification procedure followed by HLB-SPE was required to produce radioconjugates with a RCP > 98%. Overall, the radiolabeling efficiency for [ 18F]AlF-NODAGA-PODS-ZEGFR:03115 was found to be lower than for [18F]AlF- NOTA-PODS-ZEGFR:03115, a factor attributable to the chelator structure. Once purified, both radioconjugates showed a good serum stability. When injected in high EGFR-expressing tumor-bearing mice, [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 showed similar pharmacokinetics and allowed for clear visualization of the tumors already 1 h post-injection. Additionally, the radiolabeling procedure, purification requirements, in vitro stability and in vivo behavior (at 1 h) of both PODS- and maleimidebearing radioconjugates were found to be comparable. However, based on reports in the literature, it is possible that the benefits from the superior stability of PODS in vivo would be more noticeable at later time points. In conclusion, this investigation showed that PODS-based reagents are a viable alternative to maleimide for thiol-selective conjugation to cysteine-bearing proteins.

**Supplementary Materials:** The following are available online, Figure S1: RP–HPLC analysis (Gradient 1) of PODS (**A**), and the NODA-PODS (**B**) and NODAGA-PODS (**C**) reaction mixtures. NOTA-NHS and NODAGA-NHS elute with the mobile phase front, together with DMF (ca 3 min). The absorbance was recorded at the wavelength of 254 nm. The retention time (Rt) is indicated as min:sec. Figure S2: When the bifunctional chelators were purified by semi-preparative RPHPLC and subsequently dried using a speed-vacuum concentrator, the products showed clear signs of degradation RP–HPLC analysis (Gradient 1) of isolated NOTAPODS (**A**) and NODAGA-PODS (**B**). Each chromatogram shows the presence of one major degradation product (ca 10:40 min:sec). The absorbance was recorded at the wavelength of 254 nm. ESI–MS analysis of NOTA-PODS and the degradation product shows the expected mass of *m*/*z* 827 and a peak having a smaller mass (*m*/*z* 765) which could be associated to the hydrolysis derivative at the sulfone group (**C**). The prolonged presence of TFA in solution together with the type of drying process were possibly the cause. Figure S3: RP–HPLC analysis (Gradient 1) of pure NOTA-PODS (**A**) and NODAGAPODS (**B**) isolated by semi-preparative RP–HPLC using formic acid in the mobile phase instead of TFA. The absorbance was recorded at the wavelength of 254 nm. Figure S4: ESI–HRMS of NOTA-PODS (top) and NODAGA-PODS (bottom). Figure S5: RP–HPLC analysis of solutions of NOTA-PODS and NODAGA-PODS in DMF after being stored at −20 ◦C. Signs of degradation (peak at 10:36 min:sec) were detected already after 2 months for NOTA-PODS (**A**). Conversely, NODAGA-PODS showed good stability for at least 10 months (**B**). Figure S6: RP–HPLC analysis (Gradient 2) of NOTA-PODS-ZEGFR:03115 (A), and NODAGA-PODS-ZEGFR:03115 (**B**) reaction mixtures. NOTA-PODS and NODAGA-PODS elute with the mobile phase front, together with DMF (ca 3 min). The absorbance was recorded at the wavelength of 280 nm. The retention time (Rt) is indicated as min:sec. Figure S7: ESI–MS of purified NOTA-PODS-ZEGFR:03115 (top) and NODAGA-PODS-ZEGFR:03115 (bottom). Figure S8: Radiochromatograms (Gradient 2) of [18F]AlF-NOTA-PODS-ZEGFR:03115 (**A**), and [ 18F]AlF-NODAGA-PODS-ZEGFR:03115 (**B**) reaction mixtures. Free fluorine-18 elutes at ca 3 min. Labels on each peak on the chromatograms indicate the retention time (top) and the %ROI (bottom). Figure S9: Radiochromatograms (Gradient 2) of [18F]AlF-NOTA-PODS-ZEGFR:03115 (**A**), and [18F]AlF-NODAGA-PODS-ZEGFR:03115 (**B**) after purification by just HLB-SPE. As for [18F]AlF-NOTA-ZEGFR:03115 (**C**), the HLB-SPE-only purification step successfully removed the free fluorine-18 leaving the radioconjugate and the thermolysis products which elute at ca 3 min. The retention times (Rt) are expressed as min:sec. Figure S10: Representative radiochromatograms (Gradient 2) of [18F]AlF-NOTA-PODS-ZEGFR:03115 (**A**) and [18F]AlF-NODAGA-PODS-ZEGFR:03115 (**B**) after incubation in mouse serum for 1 h. The intact radioconjugates elute at ca 11 min. Activity non-associated with the conjugate elutes at ca 3 min. Labels on each peak on the chromatograms indicate the retention time (top) and the %ROI (bottom). Table S1: Summary of LogD7.4 values measured for the three radioconjugates. Table S2: Summary of serum stability determined by RP–HPLC. The three radioconjugates were incubated in mouse serum at 37 ◦C for 1 h. The data are shown as the mean values of *n* = 3 experiments ± SD. Statistical analysis was performed using one-way ANOVA with Tukey correction using GraphPad Prism v8. Table S3: Summary of serum stability determined by RP–HPLC. The three radioconjugates were incubated in mouse serum at 37 ◦C for 1 h. The data are shown as the mean values of *n* = 3 experiments ± SD. Statistical analysis was performed using one-way ANOVA with Tukey correction using GraphPad Prism v8.

**Author Contributions:** Conceptualization, C.D.P., G.K.-M. and G.S.; methodology, C.D.P, A.M. J.M. and S.T.; formal analysis, C.D.P., S.T. and G.K.-M.; investigation, C.D.P., A.M., J.M., S.T., G.K.-M.; resources, G.S. and G.K.-M.; data curation, C.D.P., S.T. and G.K.-M.; writing—original draft preparation, C.D.P.; writing—review and

editing, A.M., S.T., G.S. and G.K.-M.; visualization, C.D.P., S.T. and G.K.-M.; supervision, C.D.P. and G.K.-M.; project administration, G.S. and G.K.-M.; funding acquisition, A.M., G.S. and G.K.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Cancer Research UK-Cancer Imaging Centre (C1060/A16464) and German Cancer Aid (Deutsche Krebshilfe, project No: 70112043).

**Acknowledgments:** The authors gratefully thank Affibody AB for supplying the affibody molecule and the Structural Chemistry Facility (Cancer Therapeutics) for the provision of technical MS services.

**Conflicts of Interest:** The authors declare no conflict of interest.
