Versatile Bispidine‐Based Bifunctional Chelators for 64CuII‐Labelling of Biomolecules

Abstract Bifunctional chelators as parts of modular metal‐based radiopharmaceuticals are responsible for stable complexation of the radiometal ion and for covalent linkage between the complex and the targeting vector. To avoid loss of complex stability, the bioconjugation strategy should not interfere with the radiometal chelation by occupying coordinating groups. The C9 position of the very stable CuII chelator 3,7‐diazabicyclo[3.3.1]nonane (bispidine) is virtually predestined to introduce functional groups for facile bioconjugation as this functionalisation does not disturb the metal binding centre. We describe the preparation and characterisation of a set of novel bispidine derivatives equipped with suitable functional groups for diverse bioconjugation reactions, including common amine coupling strategies (bispidine‐isothiocyanate) and the Cu‐free strain‐promoted alkyne–azide cycloaddition. We demonstrate their functionality and versatility in an exemplary way by conjugation to an antibody‐based biomolecule and validate the obtained conjugate in vitro and in vivo.


Introduction
The design of tailor-madeb ifunctional chelating agents (BFCs) for radioactive metal ions for nuclear medical applications as well as acquisition of reliable informationa bout the biodistribution of biological objects/biomaterials represents an intensive and rapidlyd eveloping field of research. [1] For theranostic applications( simultaneous therapeutic and diagnostic competencies), BFCs that form very stable complexes with radiometal ions and also allow the simple introduction of vector mole-cules for pharmaceutical targeting under physiological conditions are of special interest. Bispidine derivatives are such multifunctional ligands with interesting pharmacological [1d] and metalb inding properties. [1c, 2] The preorganised and for Cu II complementary geometry of bispidines,a dopting ad oublechair conformation and endo-endo configuration of the C2/C4 substituents, leads to metal complexes with high thermodynamics tabilitya nd kinetic inertness. In recent years, 64 Cu II -labelledb ispidines have gainedi mportance as imaging agents for positron emission tomography( PET). [3] For this purposea variety of bispidine ligands bearing in particularp endant pyridine [3a,f] but also imidazole, [4] pyridazine, [3e] picolinic acid, [5] oxine, [6] and phosphonate [7] groups as well as bispidine dioxotetraaza macrocycycles [3d] are available.
The bispidine scaffold also offers the possibility of incorporating fluorescent molecules [8] for optical imaging as well as providing as ite for the attachment of biological vector molecules,s uch as peptidesa nd biotin. [3f,g, 7a, 8b] With respect to a minors teric influence on the metal binding centre, the C9-position of bispidines is particularly well suited for the introduction of biomolecules. However,t his position is relatively chemically inert. Recently,w eh ave reportedt he synthesis of ab ispidine carbonate that easily allows the formation of carbamates using amine-functionalised molecules. [8b] Ar elevant bispidine-BODIPY (boron-dipyrromethene)urethane derivativew as sufficiently stable in vitro. An alternative synthesis strategy for the preparation of chemically more stable ether-linked bispidine derivatives is the reductive alkylation of bispidoles. So far, there is only one example in the literature, namely the preparation of 9-methoxy and 9-fluorodecyloxy bispidine derivatives. [9] In this article, we report the synthesis of novel BFCs based on the hexadendateb is(amine)tetrakis(pyridine) bispidine-9-ol (1)e quipped with suitable functionalities for diverse bioconjugation reactions (Scheme 1). Biomolecules possessing amine or carboxylate groups can be coupled to acetic acid-functionalised 2 and amine-terminated 3 bispidines, respectively,t op roduce bioconjugatesw ith standard peptidec oupling. The alkyne-containing bispidine 4 can be used for conjugationt o azide-functionalised biomolecules forming stable triazole rings, exploitingb iorthogonal click chemistry.U sing 3 as ak ey intermediate, novel isothiocyanate-terminated 5 and dibenzocyclooctyne( DBCO)-functionalised bispidine 6 can be generated. The amine-reactive derivative 5 can be readily applied for classical protein modification exploiting the reactivity of lysine functionalities present on the protein surface. However,a st his bioconjugation strategy is nonspecific,i tt ypically resultsi na mixtureo fc onjugates labelledt ov arious extents and at different positions. Conjugation to crucial residues next to the antigen-binding site of antibodies or active sites of enzymes may heavily affect the affinity and immunoreactivity of the former or diminish the activity of the latter. Thus,t he conjugates may differ in their enzymatica ctivities, solubility,c harge, pharmacokinetic profile and antigen-binding characteristics.
In contrast, site-specific protein modification results in a more homogenous product population with defined and tunable properties. For that reason, we designed the DBCO-functionalised bispidine 6 applicable in ag eneric two-stepc hemo-enzymatic protein modification approachf or the generation of highly defined radioimmunotracers as demonstrated herein in an exemplary way for an epidermal growth factor receptor (EGFR)-specific camelid single-domain antibody( sdAb) fragment.T his protein is in the first place equipped with au nique biorthogonal azide handle in as ite-specific Sortase A-mediated conjugation.T he single-conjugated immunoconjugate is completed thereafter by as train-promoted azide-alkyne cycloaddition (SPAAC) reaction using the azide-functionalised intermediate in combination with the DBCO-containing chelator 6.T he functionality of the obtainedc onjugate was validated in vitro and its tumour-targeting capability was evaluated by small animal PETimaging.

Synthesis of bispidine derivatives
The bispidine-9-olp recursor 1 was synthesised through classical double-Mannich reactions with reportedp rotocols. [3f, 10] The reduction of bispidine was carriedo ut with NaBH 4 in dioxanewater leading exclusively to the anti-product 1 (OH group in the anti-p ositiont oC 2/C4 substituents, see the Supporting Information,F igure S1). Ar ange of exploitable organic functionalities were introduced to intermediate 1 to develop novel C9 modified bispidines 2-6.U nder basic conditions using sodium hydride, 1 was reactedt oi odoacetic acid, tert-butyl (3-bromopropyl)carbamate and propargyl bromide, respectively,t oy ield bispidine-acetic acid 2,b ispidine-amine 3 and bispidinealkyne 4 derivatives. The reactions proceed by the generation of bispidine-alkoxides, undergoing nucleophilic substitution pathways and subsequente limination of the halide to yield the desired bispidines. The amine-functionalised bispidine 3 was coupled to p-phenylene diisothiocyanate to produce the isothiocyanate-functionalised bispidine 5,f orming at hiourea bond.D BCO-terminated bispidine 6,o nt he other hand, was synthesised by aD BCO-NHS ester coupled to 3 by as table amide linkage. The synthesised ligands were purified by semipreparative reversed phase HPLC. Due to similar polarity and solubility properties of products andb yproducts,t his method has proventob ethe most suitable for the mg-scale. Bispidines were analysed by ESI-MS and furtherc haracterisedb y 1 H, 13 CNMR and IR spectroscopy (see the Supporting Information, Figure S2-S6). The molecular ion peaks at m/z:6 53.1 (2), 652.1 (3), 633.4 (4), 843.3 (5)a nd 939.1(6)c onfirmed the identities of synthesised ligands. In the 1 HNMR spectra,t he characteristic protons inglet observed between d = 4.6-5 ppm confirms the modifications of the C9 carbon atom of 2, 3,a nd 4.T he protons from the propylc hain of 3 appear in the range of d = 3.7-1.9 ppm in the 1 HNMR whereas the 13 CNMR displays ac haracteristic signal for C9 at d = 72.6 ppm. The 1 HNMR of 4 shows the methylene protons at d = 3.7 ppm positioned next to the alkyneg roup. The 13 CNMR of 5 shows aromatic carbon atoms in the range of d = 150-125 ppm and the characteristic resonance for the isothiocyanate carbon was observed at d = 182 ppm. The FTIR spectrum of 2 shows the carboxylic acid OÀHs tretcha sabroad band between3 300-2700 cm À1 and the C=Os tretch between 1720-1690cm À1 .T he FTIR of 3 displays strong NÀHs tretching from 3500-2900 cm À1 and aw eak bending peak at 1126 cm À1 .T he characteristicc arbonyls tretching vibrations for esters (O=CÀO) at C1 and C5 is positioned at 1727 cm À1 whereas the CÀOs tretchi sa t1 200 cm À1 .T he characteristic terminal alkyne with as trong CÀHs tretch at % 3000 cm À1 and aw eak -CC-stretcha t% 2200 cm À1 confirms the alkyne derivative 4.T he FTIR spectrum for 5 shows ac haracteristic N=C=Ss tretcha t2 140 cm À1 .T he 1 H NMR of 6 shows ac luster of aromatic peaks between d = 7.6-7.2 ppm, characteristic for protons of the dibenzylic group.

Cu II -labelling experiments
Bispidinel igandsform stable complexes very quicklyw ith 64 Cu II under mild conditions. [3a,f] This was also confirmed for the new bispidines 2-6 with additional functional groups in the C9-position. Quantitative yields (> 99 %) of 64 Cu II complexes with 2-6 were obtained within 5min at room temperature. Radio-HPLC and radio-TLC experiments show high radiochemical yield and purity,r espectively,w ith ligand concentrations ! 5 mm at high activity concentrations( 100 MBq/200 mL). The molar activity obtained( see the Experimental Section) was highert han 100 MBq nmol À1 for bispidines 2-6,w hereby the acetic acidfunctionalised bispidine 2 gives the highest value of > 500 MBqnmol À1 (Table 1). The distribution ratio (log D o/w )o f 64 Cu II -bispidine complexesw as determinedi natwo-phase 1octanol/buffer system at physiologically relevantp Hv alues (see the Experimental Section). This allows for information about the hydrophilic or lipophilic character of the complexes and thusp ermits the reliable evaluation of properties such as adsorption,d istribution,m etabolism, and excretion( ADME) in living systems. [11] As summarised in Ta ble 1, 64 Cu II complexes of bispidines 2-6 are hydrophilic in characterw ith log D values ranging from À4( 64 Cu II -2)t oÀ1.2 ( 64 Cu II -5).
The rapid complexation kinetics under mild conditions, high molar activities anda ppropriate hydrophilic properties of 64 Cu II -labelled bispidines make these BFCs attractive for further in vitro and in vivo experiments.

Stability assessment by ligand-exchange studies
To estimate the stability of the 64 Cu II -labelled bispidine BFCs 2-6,l igand exchange reactions in the presenceo fa1 000-folds excess of the competitive ligandsE DTAa nd DOTAf or up to 24 ha t3 7 8Cw ere studied. The evaluation was conducted using radio-TLC by which the studied complexes were easily distinguished from [ 64 Cu]Cu-EDTAa nd [ 64 Cu]Cu-DOTA, as the radiolabelled bispidine derivatives 2-6 have ar etentionf actor (R f )o f0 .6-0.7 and 64 Cu II coordinated to EDTAa nd DOTAm oves with the solventf ront( Supporting Information Figure S7). The percent of 64 Cu II exchanged between ligands is tabulated for 1, 2, 4, and 24 hi nt he SupportingI nformation, Ta ble S1. At a 1000-fold molar excess of EDTAo rD OTA, the different bispidine derivatives shown ot ranschelation throughout the period of investigation, confirming their excellent Cu II complex stabilities. The modification of the bispidine backbone at the C9-position has obviously no influence on the stabilitya nd inertness of the 64 Cu II complexes formed.
Synthesis and radiolabellingofb ispidine-cetuximab conjugate (5') For the classical, non-specific conjugation using the isothiocyanate-functionalised bispidine derivative 5,t he well-studied, FDA-approved therapeuticm onoclonala ntibody (mAb)c etuximab (C225;E rbitux, ImCloneL LC) served as am odel protein. [12] This chimeric human murine IgG1 molecule binds specifically to the extracellulard omain of EGFR, ar eceptor tyrosine kinase that is often overexpressed in human malignancies. [13] Cetuximab containsi nt otal 86 lysine residues, eleven in each light and 32 in each heavy chain. [14] Similart oaprevious study, [15] the mAb was modified with 5 via random isothiocyanate-mediated coupling. After purification,t he degree of conjugation was determined by MALDI-TOF MS analysis (Supporting Information, FigureS8). Twot ot hree molecules of bispidine were on average conjugated to cetuximab as each chelator subunit adds approximately 843 gmol À1 to the molecular weightoft he mAb. The bispidine-cetuximab conjugate 5' was successfully labelled with 64 Cu (Supporting Information, Figure S9) and afterwards analysed by gel electrophoresis and autoradiography ( Figure 1). The detection of the light and the heavy chains in the autoradiograph prove the attachment of bispidine chelators to both structuralelements of the antibody.
These findings illustrate that random bioconjugation to the side-chain e-amine of lysine residues inevitably leads to at wofold heterogeneity.F irst, the product population contains conjugates with av ariable bispidine-to-antibody ratio. Second, conjugates with the same bispidine-to-antibody ratio are likely regioisomers as many surface-accessible lysine residues are potential sites for conjugation with 5.I no ther words,i nt he obtained product population the conjugates do not only differ in the amount of bispidines they are bearing, buta lso in the sites where the chelators are attached. Consequently,e ach conjugate of that vastly heterogeneous mixture can have its own set of properties regarding key parameters such as pharmacokinetics, solubility,s tability,b iological potency,a ntigen affinity and immunoreactivity. [16] Regarding the latter,exhaustive modification of lysine residues at or next to the antigen-binding site may lead to steric hindrance of antigen recognition and interaction. [17] In particular,f or small-sized antibodyf ormats such as single-chain variable fragments (scFvs) or single-domain antibodies (sdAbs), the risk of affecting key amino acids is huge due to their compact structure and limited number of available functional reactive residues outside the antigen-binding site. These severe drawbacks of randomc onjugation strategiesr epresent major challenges in the clinical translation and market approvalo fa ntibody-derived conjugatesa st hey impede consistent and reproducible manufacturing as well as straightforward characterisation. Hence, significant efforts are made to obtain ah omogeneous product population by pursuings ite-specific conjugation strategies. [16a, 17b, 18] With this in mind, we herein adapted the two-steps ite-specific modificationi ntroduced recently,c ombining chemoenzymatic bioconjugation and click chemistry. [19] The first step of our approachu ses Sortase-mediated bioconjugation for regioselectivei ncorporation of an azide-containing linker into an EGFR-specific sdAb. The second step of this modular approachi nvolves the modification of the azide-tagged antibodyf ragment with the DBCO-modified bispidined erivative 6 via Cu-free strain-promoted alkyne-azide cycloaddition (Scheme 2).

Strain-promoted azide-alkyne cycloaddition
The site-specifically azide-modified sdAb 7C12-Strep-N 3 was reacted with 6 for up to 4h at 25 8C. To optimise the reaction, the molar rations of sdAb and 6 were varied ( Figure 2).
Ar eaction with am olar ratio sdAb:6 of 1:5w as identified as being ideal.A fter purification of the reaction mixture by size exclusion chromatography,t he obtained conjugate was analysed by MALDI-TOF MS ( Figure 3). The mass spectra of the final purified product showed ah omogeneous population of a single-conjugated 6' with am olecular mass of % 17.4 kDa ( Table S2,Supporting Information).

Radiolabellingo fbispidine-modified sdAb6 '
Bispidine-sdAb 6' (1 nmol) was radiolabelledw ith [ 64 Cu]CuCl 2 with activities ranging from 0.5 to 10 MBq, and the extento f radiometal complexation was assessedb yr adio-TLC (Supporting Information, Figure S11). As expected, the amount of radiolabelled protein correlated with the activity of 64 Cu II added. In addition to radio-TLC, 64 Cu II -labelling of 6' was confirmed by SDS-PAGE and subsequent autoradiography.As ingle predominant protein species corresponding to the mononuclear 64 Cu IIlabelleds dAb is clearly visible in the Coomassie stained gels as well as in the autoradiographic images ( Figure 4). Free 64 CuCl 2 is detectable at the upper part of the gel, whereas the reference [ 64 Cu]Cu II -6 migrates with the gel front. The reactionm ixture was subsequently purified by spin filtration to completely remove free 64 CuCl 2 until 64 Cu II -6' was finally obtained with a radiochemical yield of % 99 %.

In vitro characterisation of single-conjugatedsdAbs
The functionality of the functionalised 6' after the two-step site-specific modification was evaluated by cell binding studies using the human epithelial cell line A431 originating from an epidermoid carcinoma of the skin. These squamous carcinoma cells express approximately 2 10 6 EGFR molecules per cell, which represents ah igh expression level. [21] In as aturation binding assay,i nw hicht he amount of radioligand required to saturatet hese receptors is measured, the equilibrium dissociation constant K d and the total number of receptors expressed on the cell surface B max were determined (Figure5).
Scheme2.Overview of the two-step site-specific antibody modification and conjugation using ac ombination of enzyme-mediated bioconjugation and click chemistry. The first step uses enzymaticbioconjugation with the transpeptidase Sortase Afor site-specific incorporation of af unctional azide group and the second step involves the azide-alkyne cycloaddition click reaction.  Nonlinear regression analysiso ft he saturation curve data revealed a K d value of 53.8 AE 8.6 nm andaB max of 45.8 AE 2.7 pmol mg À1 protein. The apparent affinity determinedi si n accordance with previously obtained data for 99m Tc -labelled 7C12. [22] The bispidine conjugationv ia the two-step site-specific modification procedure does obviously not affect the binding properties of the sdAb.
To investigate the binding and localisation of these immunoconjugates at the cellular level, fluorescence microscopic imaging was performed ( Figure 6). Confocali maging of A431 cells showedb inding of the bispidine-functionalised sdAbs to the plasma membrane of these human epidermoid carcinoma cells and co-localisation with EGFR indicating the preserved targeting ability of 7C12 after site-specific modification. As observed earlier, 6' showedapredominant membrane staining even after 24 ho fi ncubation, and only very little intracellular fluorescence was observed (Supporting Information, Figure S12). Theseo bservations are in agreement with former studies, [22,23] indicating that bispidine conjugation has no detrimental effect on the EGFR binding behaviour or on the specificity of the sdAb.

PET imaging of single-conjugatedsdAbs in A431 tumour xenografts
The promising in vitro results strongly encouraged us to investigate the in vivo distribution of this 64 Cu II -labelled bispidine-   Chem. Eur.J.2020, 26,1989 -2001 www.chemeurj.org 2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim sdAb conjugate 6' in at umour mouse.T umourx enograft (epidermoid carcinoma) was obtained after subcutaneous injection of A431 cells into the right thigh of af emaleN MRI nu/nu mouse.Adynamic PET imaging study was performed when the tumour was about 10 mm. Conjugate 6' was labelled with [ 64 Cu]CuCl 2 for 60 min at room temperature, resulting in ar adiochemical yield of 91.9 %and molar activity of 27.7 GBq mmol À1 withoutf urtherp urification. 39 MBq (2 nmol 64 Cu II -6')w ere injected into at ail vein of A431 tumour bearing mice (n = 2). As shown in Figure 7, the 64 Cu II -labelled bispidine-sdAb conjugate 6' was rapidlycleared via the renal pathwaywith abloodactivity half-life of 28.2 min, which is expected due to the hydrophilic character and the relatively low molecular weight for a protein. Liver uptake was likely elicited by free 64 Cu II in the injection solution. The tumouri sc learly visible after 5min and the activity peak wasr eached after 22.5 min. After one hour, most of the activity was removed from the blood and the tumourw as sharplyd elimited, leading to at umour-to-muscle ratio above 13. The tumour is visible with high contrastb etween 20 min and2h. Overall, the pharmacokinetic properties of 64 Cu II -6' are as expected and similar to the data obtained for a 99m Tc-labelled sdAb 7C12. [24] Conclusion Bispidines with their excellent metal chelation properties and promisingb ioconjugation strategies are outstanding bifunctional imaging agents. Due to their high 64 Cu II selectivity,e ase of biofunctionalisationa nd high in vivo stability,t he hexaden-tate bispidine derivatives reveals an enormousp otential as PET imaginga gent forc ancerd iagnosis. The C9-modified bispidines (2-6)t hat can be readily coupled to cancert argeting moietiesh aveb een synthesised and characterised. The DBCOfunctionalised ligand 6 was designed to undergo strain-promoted azide-alkyne coupling to single-domaina ntibodies, and the corresponding site-specifically single-conjugated sdAb derivativew ithu ncompromised functionalityw as obtained by a combination of enzyme-mediated bioconjugation and click chemistry.T he introduction of the azide handlea tt he C-terminal end of the sdAb makes this strategy ag eneric approach for aw ider ange of dibenzocyclooctyne-containing substrates. As the herein targeted antigen EGFR is present in practically all squamousc ell and some other cancers occurring in the clinic, the bispidine-based selective and highly specific antibodyf ragments labelled with 64 Cu II hold ap romise to deliver important therapy planning and monitoring optionsf or head and neck, oesophageal, gastric, bladder and other mucosal cancerp atients.

Molecular cloning
AD NA fragment coding for a( GGGGS) 3 spacer followed by a Strep-tag, the LPETGG sortase motif and another (GGGGS) 3 spacer was synthesised including a5' restriction site for HindIII and a3' restriction site for XhoI, respectively.T he % 150 nt fragment was digested with appropriate restriction endonucleases and ligated inframe into HindIII/XhoI-linearised pET-28b:7C12 plasmid. [22] The ligation reactions were transformed into chemically competent E. coli NEB 5-alpha cells. The DNA sequences of the resulting recombinant construct pET-28b:7C12-Strep-sortag-6HIS were checked by Sanger sequencing.

Cultivation and expressiono frecombinant proteins
Freshly transformed E. coli SHuffle T7 Express or E. coli BL21(DE3) harbouring the plasmids pET-28b:7C12-Strep-sortag-6HIS or pGBMCS-SortA were inoculated in 10 mL of LB broth containing 50 mgmL À1 of kanamycin or 100 mgmL À1 of ampicillin, respectively, and cultivated at 30 8Co vernight in an orbital shaker with 50 mm offset and shaking speed of 200 rpm. After that, 5mLo ft his preculture were transferred into 100 mL MagicMedia E. coli Expression Medium (Life Te chnologies) in 500 mL baffled-bottom glass flasks and grown at 30 8Cf or 24 h. For final harvest, cultures were chilled on ice for 5min and centrifuged for at least 15 min at 6000 ga nd 4 8C. After removal of the supernatant, cell pellets were either stored at À20 8Co rs ubjected to the purification procedure immediately.

Purification of recombinant proteins
Ah igh-capacity Ni-iminodiacetic acid (IDA) resin in combination with an ¾KTAp ure chromatography system (GE Healthcare) was used for purification of hexahistidine tagged proteins by immobilised metal affinity chromatography (IMAC) under native conditions.
Efficientc ell lysis was achieved by addition of 1mLR IPAc ell lysis buffer (G-Biosciences) supplemented with EDTA-free protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific), 500 mgl ysozyme (Sigma-Aldrich) and 25 Ue ndonuclease (Thermo Scientific Pierce) per 200 mg bacterial cell pellet. Prior to incubation on ice for at least 15 min, the pelleted cells were resuspended completely by vortexing or pipetting up and down until no cell clumps remained. After centrifugation at 10 000 ga nd 4 8Cf or 20 min to remove cellular debris, the clarified supernatant was loaded using an automated sample pump with af low rate of 0.5 mL min À1 .I MAC was performed on ap refilled 5mLH is60 Ni Superflow cartridge (Clontech Laboratories) at af low rate of 5mLmin À1 in equilibration buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.5).
Removal of imidazole and buffer exchange after IMAC was achieved by dialysis against sortase buffer (50 mm Tris-HCl, 150 mm NaCl and 10 mm CaCl 2 ,p H7.5) using ac ellulose ester membrane with am olecular weight cut-off of 3.5-5 kDa (Spectrum Laboratories).

Gel electrophoresis
Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to as tandard protocol. [27] For each gel, PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as molecular weight ladder standard. After electrophoresis, gels were stained with PageBlue protein staining solution (Thermo Fisher Scientific) according to the manufacturer's instructions.

Protein determination
Protein concentration was determined with the DC Protein Assay (Bio-Rad Laboratories) according to the manufacture's microplate assay protocol using bovine serum albumin as protein standard.

Purificationofconjugation reactions
In the first purification step, all remaining hexahistidine tagged proteins were eliminated from the reaction mixture by IMAC using prepacked His60 Ni Gravity Columns (Clontech Laboratories). After collection of the flow-through, the gravity-flow column was washed twice with equilibration buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.5). These wash fractions as well as the flow-through were analysed for the presence of the azide-functionalised sdAb conjugate by SDS-PAGE.
The remaining unconjugated (Gly) 3 -Lys-N 3 was removed in a second purification step by affinity chromatography using aS trep-Ta ctin XT system (IBA Lifesciences) in combination with an ¾KTA pure chromatography system (GE Healthcare). The flow through as well as the wash fractions of the first purification step were loaded using an automated sample pump with af low rate of 0.5 mL min À1 .A ffinity chromatography was performed on ap refilled 5mLS trep-Tactin XT Superflow cartridge (IBA lifesciences) at a flow rate of 5mLmin À1 in buffer W( 100 mm Tris-HCl, 150 mm NaCl, pH 8.0). Before elution of the Strep tagged proteins by addition of 8CVe lution buffer BXT (100 mm Tris-HCl, 150 mm NaCl, 1mm EDTA, 50 mm biotin, pH 8.0), the column was washed with 8 CV buffer W.

Strain-promoted azide-alkyne cycloaddition
Small-scale reactions were set up in 50 mLw ith variable molar ratios of azide-functionalised sdAb and 6.T he optimal conditions and molar ratios (1:5) were upscaled and the reaction mixture was composed of 275 nmol azide-functionalised sdAb and 1375 nmol of 6.R eactions were incubated at 25 8Cf or up to 4h with gentle shaking.
Non-conjugated 6 was removed by size-exclusion chromatography using Zeba Spin Desalting Columns (7 KM WCO, Thermo Scientific) with elution in MES/NaOH buffer (100 mm,1 50 mm NaCl, pH 6.5). The purified conjugate 6' was sterile filtered using Whatman Puradisc FP 30 cellulose acetate syringe filter units with ap ore size of 0.2 mm( GE Healthcare Life Sciences) and stored at 4 8C.
Protein samples were desalted by chloroform/methanol protein precipitation as described elsewhere. [29] The dried protein pellets were dissolved in 50-100 mL0 .1 %T FA solution. A2mLa liquot of this desalted protein sample was mixed with 2 mLo f2%T FA solution. After addition of 2 mLo fm atrix solution, the mixture was pipetted up and down until the crystallisation started and the solution became cloudy.F inally,0 .5 mLo ft he crystal suspension was spotted onto the ground steel target plate and the droplet was air-dried completely at room temperature.
Spectra were acquired with an autoflex II TOF/TOF (Bruker Daltonik) in positive linear mode in combination with the flexControl software (Version 3.3, Bruker Daltonik) and analysed with the flexAnalysis software (Version 3.3, Bruker Daltonik). Theoretical molecular weights were calculated using the Compute pI/Mw tool on the ExPASy Server.

Cu-labelling experiments
The production of 64 Cu was performed via proton irradiation of enriched 64 Ni at aT R-Flex cyclotron from Advanced Cyclotron Systems Inc (ACSI, Canada) and module-assisted separation as described in detail recently. [30] For 64 Cu II labelling, the pH of the [ 64 Cu]CuCl 2 solution was adjusted by adding one volume of 1 m MES/NaOH, pH 6.0. The radiochemical yield and purity were deter-mined by radio-HPLC and radio-TLC. As olution of bispidine ligands 2-6 (40 nmol in 40 mL1 00 mm MES/NaOH-buffer of pH 6.5) spiked with 40 MBq [ 64 Cu]CuCl 2 was stirred for 30 min at room temperature. To determine the maximum molar activity, 64 Cu-labelling of bispidine ligands 2-6 was investigated in the concentration range of 0.1 to 10 nmol/200 mLM ES-NaOH buffer (pH 6.5), containing 100 MBq [ 64 Cu]CuCl 2 at different time points (5, 30, 60 min). The radiochemical yields were analysed using radio-TLC. The results presented are the average of two independent experiments.

Stability assessment by ligand challenge
Ligands 2-6 (40 nmol) were radiolabelled with 40 MBq [ 64 Cu]CuCl 2 in at otal of 40 mL1 00 mm MES-NaOH, pH 6.5 at 25 8Cf or 30 min. The formation of the complexes were analysed by radio-TLC using glass microfiber chromatography paper impregnated with silica gel (iTLC-SG) as stationary phase and a2 m NH 4 OAc/MeOH (1:2 v/v) mixture as mobile phase. The radiochemical yield was higher than 99.9 %, otherwise the labelling reaction was discarded. For ligand challenge, 10 nmol of the radiolabelled ligands (10 mL) were incubated with a1 000-fold molar excess of the challenging ligands EDTAo rD OTA, respectively,i n1 00 mm HEPES-NaOH, pH 7.4 at 37 8Cf or up to 24 h. At each time point, the ratio between transchelated 64 Cu as [ 64 Cu]Cu-EDTAo r[ 64 Cu]Cu-DOTA( R f = 1), respectively,a nd [ 64 Cu]Cu-bispidine (R f = % 0.7) was determined by radio-TLC. The chromatography stripes were air-dried and exposed to a high-resolution phosphor-imaging plate (GE Healthcare). The exposed plates were scanned with an Amersham Typhoon 5S canner (GE Healthcare), and the images were analysed using the Image-Quant TL software (Version 8.1, GE Healthcare).
For subsequent in vitro studies using [ 64 Cu]Cu-6',t his labelling reaction was upscaled. Accordingly,1 00 nmol of 6' were radiolabelled with 100 MBq of [ 64 Cu]CuCl 2 in MES/NaOH buffer (100 mm, 150 mm NaCl, pH 6.5). The extent of 64 Cu II complexation was assessed by radio-TLC, using glass microfiber chromatography paper impregnated with silica gel (iTLC-SG) as stationary phase and 2 m NH 4 OAc/MeOH (1:2 v/v) mixture as mobile phase. As ac ontrol, radio-TLC analysis of [ 64 Cu]Cu-6 was also performed under the same conditions. Individual solutions (5 mL) were first mixed with 5 mLo fE DTA( 500 mm,p H7.4) and % 1 mLw as spotted at the origin. After TLC, the chromatography stripes were air-dried and exposed to ah igh-resolution phosphor-imaging plate (GE Healthcare). The exposed plates were scanned with an Amersham Typhoon 5S canner (GE Healthcare). Free 64 Cu II (as EDTAc omplex) migrated to the solvent front (R f = 1), whereas the radiolabelled proteins remained at the origin (R f = 0). [ 64 Cu]Cu II -6 has an R f of % 0.7. In addition to radio-TLC, 64 Cu II -labelling of 6' was confirmed by SDS-PAGE and subsequent radioluminography.
If necessary,t he radiolabelled proteins were purified prior to further use via spin filtration with Amicon Ultra-0.5 centrifugal filter devices with am olecular weight cutoff of 3K (Amicon Ultra 3K device, Merck-Millipore).

Cell culture
Cell culture flasks, dishes and plates (CELLSTARS) were supplied by Greiner Bio-One GmbH. The adherent human tumour cell lines A431 (ATCC number:C RL-1555 was maintained as previously reported. [22,31] The cell line was confirmed to be mycoplasma-negative using the Venor GeM Advance Mycoplasma Detection Kit (Minerva Biolabs) and was tested monthly.

Confocal microscopy
At otal of 100 000 A431 cells were seeded in 35 mm imaging dishes (IBIDI) in 2mLD MEM supplemented with 10 %f oetal calf serum (FCS), and incubated in ah umidified atmosphere of 95 % air/5 %C O 2 at 37 8C. After 24 ho fi ncubation, the medium was refreshed and cells were incubated with 100 nm of 6' at 37 8Cf or up to 24 h. Afterwards, cells were washed thrice with ice-cold PBS, fixed with 4% paraformaldehyde and 2.5 %s ucrose in PBS, and permeabilised with 0.25 %T ritonX-100 in PBS for 10 min. To prevent unspecific antibody binding, cells were incubated with 10 % FCS in PBS overnight at 4 8C. Cells were then incubated with rabbit anti-EGFR (D38B1) Alexa Fluor 647 monoclonal antibody (Cell Signaling Te chnology) and with StrepMAB-Classic Chromeo 488 conjugate (IBA Lifesciences) for 2h at room temperature in the dark. Cells were again washed three times with PBS, and the nuclei were stained using Hoechst 33258. Fluorescence microscopy was performed with aF luoview 1000 confocal laser scanning microscope (Olympus) using a60 (NA 1.35) oil objective.

Cell binding studies
At otal of 40 000 A431 cells were seeded in 48-well plates in 250 mL DMEM supplemented with 10 %f oetal calf serum (FCS) and incubated in ah umidified atmosphere of 95 %a ir/5 %C O 2 at 37 8C. After 48 ho fi ncubation, the medium was replaced by serum-free DMEM and cultivation was continued for % 4h.T he plates were pre-incubated for 30 min at 4 8Cb efore the addition of different concentrations of [ 64 Cu]Cu II -6' ranging from 12 pm to 400 nm.T o control wells only,anexcess of unlabelled sdAb (4 mm final concentration) was added to determine the non-specific binding. The cell culture microplates were further incubated for 1h at 4 8C. Following treatment with [ 64 Cu]Cu II -6',c ells were washed three times with ice-cold PBS to ensure removal of loosely attached proteins from the cell membrane. Finally,c ell lysis was achieved by the addition of 1% SDS/0.1 m NaOH and incubation for 30 min at room temperature with vigorous shaking. The cell-associated radioactivity was quantified using an automated gamma counter (Perkin-Elmer Life and Analytical Sciences). To tal protein concentration in cell extracts was determined as described above. The equilibrium dissociation constant K d as well as the maximum specific binding B max were determined from the measured data using the Prism software (Version 7, GraphPad).

PET studies
In vivo experiments were performed at the Semmelweis University Department of Biophysics and Radiation Biology according to the National Institute of Food Chain Safety (Licence No.:X IV-I-001/29-7/2012 and PE/EA/50-2/2019). Naval Medical Research Institute (NMRI)-Foxn1 nu/nu mice were used for the in vivo experiments and were purchased from JANVIER LABS (JANVIER LABS, Saint-Berthevin Cedex, France). A431 cells were grown in culture as described earlier.F or the pre-clinical experiments of tumour binding in PET diagnostics, one million A431 cells in 0.2 mL of medium were injected to the right flank of the mice. Four weeks after tumour cell inoculation, tumours of circa 3mmi nd iameter were formed. Mice were then used for the PET imaging dynamic study to establish the tumour binding kinetics of [ 64 Cu]Cu II -6'.T herefore, approximately 2nmol with 39 MBq of the respective protein were injected into a lateral tail vein of NMRI-Foxn1 nu/nu mice. This was followed by dynamic PET scans for 2h and repetitive scans after 1.7, 16.7, 41.7 and 63.3 hu sing am icroPET P4 scanner (Siemens). Data evaluation was carried out using the ROVER software (ABX GmbH). Quantitative time activity curves were expressed as mean of the standardised uptake value (SUV) AE SEM. SUV was estimated by the following equation:[ tissue concentration (MBq mL À1 ) the body weight (g)]/injected dose (MBq).