epidermal growth - factor receptor ( egfr ) 
 is overexpressed in a wide 
 variety of solid tumors and has served as a well - characterized target 
 for cancer imaging and therapy , and it 
 is associated with many human malignancies including head and neck , 
 colorectal , non - small cell lung , and breast cancers , among others . overexpression of egfr in tumors is correlated 
 with aggressive disease , poor prognosis , poor response , and resistance 
 to cytotoxic therapy in some tumor types . cetuximab ( c225 , erbitux ) is a recombinant human / mouse chimeric 
 immunoglobulin g1 ( igg1 ) monoclonal antibody ( mab ) that binds specifically 
 to the extracellular domain of egfr with high affinity . it was approved 
 by the us food and drug administration in 2004 for the treatment of 
 patients with egfr - expressing , metastatic colorectal cancer . cetuximab 
 blocks the activation of egfr by preventing ligand - mediated tyrosine 
 kinase phosphorylation and downstream signal transduction , which induces 
 the internalization and possible degradation of egfr . several clinical trials have shown that cetuximab , either 
 administered alone or combined with the standard first or second line 
 chemotherapy , improved treatment efficacy for metastatic colorectal 
 cancer without a significant increase in toxicity . there 
 has been significant interest in radiolabeled cetuximab since 
 the unlabeled drug was fda - approved for the treatment of colorectal 
 cancer . cetuximab has been radiolabeled with tc and in for spect imaging and cu , y , and zr for pet imaging . copper-64 ( t1/2 = 12.7 h ) is particularly promising because 
 of its suitable half - life and unique decay characteristics :  ( 656 kev , 17.4% ) and  ( 573 kev , 
 38.5% ) , and the beta - decay makes this radionuclide attractive for 
 therapy . radiolabeling at 37 c or below is critical to maintain the 
 biological integrity of antibodies , therefore dota has been the most 
 commonly used chelator for cu radiolabeling of cetuximab 
 and other antibodies . however , the in vivo behavior 
 of the cu - dota complex is less than optimal due to dissociation 
 of cu from the dota complex , and this might lead to relatively 
 poor tumor to nontumor ratios . cross - bridged macrocyclic chelators ( such as cb - te2a , cb - te1a1p , 
 and cb - te1k1p , figure 1 ) have shown impressive 
 kinetic inertness for cu complexes . the monocarboxylate monophosphonate 
 chelator cb - te1a1p can be labeled with cu at room temperature 
 with comparable in vivo stability to cb - te2a , and its peptide conjugate cu - cb - te1a1p - y3-tate 
 demonstrated improved tumor targeting and biodistribution compared 
 to cu - cb - te2a - y3-tate conjugate in a pancreatic tumor 
 bearing rat model . the newly developed 
 cb - te1k1p chelator , containing one methanephosphonate and one carboxylate 
 pendant group , has advantages due to the maintenance of the carboxylate 
 group postconjugation to biomolecules , and this chelator may be an 
 improved bifunctional chelator for the labeling of copper radionuclides 
 to monoclonal antibodies , such as cetuximab . structures 
 of dota , cb - te2a , cb - te1a1p , and cb - te1k1p . herein , the two cross - bridged chelators cb - te1a1p and cb - te1k1p 
 were conjugated to cetuximab via conventional nhs ( n - hydroxysuccinimide ) ester conjugation and/or strain - promoted azide  alkyne 
 cycloaddition ( spaac ) strategies , and the resulting conjugates were 
 evaluated in nude mice bearing hct116 human colorectal tumors . besides 
 comparison of the direct conjugation of the nhs ester of cb - te1a1p 
 to cetuximab using standard coupling techniques to the spaac conjugations 
 of cb - te1a1p and cb - te1k1p to cetuximab , we address whether the spaac 
 approach will achieve greater ease of radiolabeling in higher specific 
 activity ( sa ) compared to the conventional nhs - ester conjugation strategies . we also investigated whether conjugating the cb - te1k1p chelator to 
 cetuximab will result in improved targeting and biodistribution compared 
 to cb - te1a1p that utilizes one of the carboxylates for conjugation . unless specified , all chemicals were purchased 
 from sigma - aldrich 
 chemical co. ( st . louis , mo ) . louis , mo ) and the university of wisconsin ( madison , 
 wi ) . dbco - peg4-nhs ester and dbco - amine were purchased 
 from click chemistry tools ( scottsdale , az ) . cb - te1k1p - peg4-dbco , cb - te1a1p - dbco , and 3-azidopropionic acid succinimidyl ester 
 were synthesized as previously reported . size - exclusion hplc was performed on a superose 12 hr 10/300 
 column ( amersham biosciences , uppsala , sweden ) , attached to a waters 
 600e ( milford , ma ) chromatography system with a waters 991 photodiode 
 array detector and an ortec model 661 ( eg&g instruments , oak ridge , 
 tn ) radioactivity detector . the mobile phase was 20 mm hepes , 150 
 mm nacl , ph 7.3 eluted at a flow rate of 0.5 ml / min . multiscreen 96-well microtiter plates for receptor binding assays 
 were counted on a 1450 microbeta trilux liquid scintillation and luminescence 
 counter ( perkinelmer life sciences ) . the nhs - ester 
 aided conjugation of cb - te1a1p 
 to cetuximab is shown in scheme 1 . cb - te1a1p 
 ( 5.8 mg , 15 mol ) was mixed with sulfo - n - hydroxysuccinimide 
 ( snhs,1.7 mg , 15 mol ) and hobt ( 2.0 mg , 15 mol ) in 1 
 ml of dmf . the coupling reagent 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide 
 ( edc , 2.9 mg , 15 mol ) was added to the mixture to start the 
 reaction . after 30 min of stirring on ice , the reaction mixture was 
 evaporated to dryness . the concentrated cetuximab ( 20 mg / ml ) was mixed 
 with activated cb - te1a1p in a 100:1 molar ratio ( cb - te1a1p : cetuximab ) , 
 followed by incubation at 4 c for 7 days with end - over - end rotation . the conjugate was then transferred to a centricon 100 , washed twice 
 with 0.1 m ammonium acetate ( ph 8.0 ) , and concentrated before being 
 loaded onto a desalting column . unreacted small molecules were removed 
 by passing the reaction mixture 23 times through desalting 
 columns . purity and concentration of the conjugated cetuximab were 
 determined by size - exclusion hplc . the number of chelators per antibody 
 was determined by titration of cb - te1a1p  cetuximab with [ cu]copper acetate / cu - acetate using a published 
 method . for radiolabeling , cucl2 was added to cb - te1a1p  cetuximab in 0.1 m 
 ammonium acetate ( ph 8.0 ) , followed by incubation at 40 c for 
 2 h. the radiochemical purity of cu - cb - te1a1p  cetuximab 
 was determined by size - exclusion hplc , and desalting columns were 
 used for purification if necessary . the 
 synthesis of cb - te1a1p and 
 cb - te1k1p attached cetuximab conjugates via spaac is shown in scheme 2 . cetuximab ( 2.0 mg / ml , 20.0 ml ) was functionalized 
 with azide groups by mixing with 3-azidopropionic acid succinimidyl 
 ester ( 1.27 mg in 1.0 ml of dmf ) in na2hpo4 buffer 
 ( 0.1 m , ph = 8.2 , 5.0 ml ) overnight at 4 c , and then the mixture 
 was transferred to a centricon 100 and washed with 0.1 m ammonium 
 acetate ( ph 8.0 ) 56 times . cetuximab 
 ( 2.0 mg / ml ) was then mixed with 100-fold excess cb - te1k1p - peg4-dbco or cb - te1a1p - dbco and incubated for 30 min at 37 c , 
 followed by incubation overnight at 4 c with end - over - end rotation . the reaction was transferred to a centricon 100 , washed with 0.1 m 
 ammonium acetate ( ph 8.0 ) 34 times , and loaded onto a desalting 
 column to remove the unreacted dbco - chelator . the purity and concentration 
 of the chelator cetuximab with [ cu]copper / cu - acetate was conducted to measure the number 
 of chelators per antibody . copper-64-labeled click - chelator cetuximab 
 conjugates were prepared by adding cucl2 to 
 the conjugate in 0.1 m ammonium acetate ( ph 8.0 ) , followed by incubation 
 at 37 c for 30 min for cb - te1k1p - peg4-dbco - conjugated 
 antibody and 40 c for 60 min for cb - te1a1p - dbco  cetuximab . purity of the cu - click - chelator cetuximab conjugates 
 was confirmed by size - exclusion hplc . bert vogelstein ( johns hopkins university ) and were cultured 
 in dmem medium supplemented with 10% fbs and 0.1% gentamicin in a 
 37 c humidified 95% air , 5% co2 incubator . all animal 
 experiments were conducted in compliance the institutional care and 
 use committees ( iacuc ) at washington university and the university 
 of pittsburgh . female athymic nude mice ( 56 weeks ) were purchased 
 from the nci ( frederick , md ) . for biodistribution studies and small 
 animal pet / ct imaging , 4  10 hct116 cells in 100 
 l of 0.9% saline were implanted subcutaneously into the dorsal 
 flank of each animal , and the mice were subjected to biodistribution 
 and/or pet / ct imaging when the tumor volume reached 100 to 200 mm ( 23 weeks ) . the affinity 
 of cu - labeled cetuximab conjugates for egfr was determined 
 by a saturation binding assay based on previously published methods . hct116 cell membrane preparations were diluted 
 in binding buffer [ 0.1% bovine serum albumin , 50 mm tris - hcl ( ph 7.4 ) , 
 5.0 mmol / l mgcl2 , 0.5 g / ml aprotinin , 200 g / ml 
 bacitracin , 10 g / ml leupeptin , and 10 g / ml pepstatin 
 a ] , and 15 g of membrane was added to each well of a 96-well 
 filtration plate ( multiscreen durapore ; millipore ; billerica , ma ) . membranes were incubated with increasing concentrations of cu - labeled cetuximab conjugates for 2 h at room temperature . when equilibrium was reached , unbound radioactivity was filtered off 
 and the membranes were washed twice with 200 l of binding buffer . 
 bound radioactivity was measured with a liquid scintillation and luminescence 
 plate reader ( 1450 microbeta ; perkinelmer ; waltham , ma ) . total binding 
 sites ( bmax ) and binding affinity ( kd ) were determined by a nonlinear regression 
 fit of bound peptides per mg of protein versus concentration of radioligand 
 using graphpad prism v 5.0 ( san diego , ca ) . copper-64-labeled cetuximab ( 0.74 mbq , 1.06.0 
 g in 150 l ) was injected via tail vein into hct116 tumor - bearing 
 mice ( 79 weeks ) . at 24 and 48 h after injection , mice were 
 sacrificed and selected organs were removed , weighed , and counted 
 on a  counter ( beckman 8000 ; irvine , ca ) . a blocking study 
 was also conducted to examine the specificity of in vivo uptake . cetuximab ( 1 mg ) was preinjected 24 h prior to cu - labeled cetuximab conjugates into hct116 tumor - bearing mice via 
 tail vein . hct116 tumor - bearing female 
 nude mice ( 79 weeks ) were injected with cu - labeled 
 cetuximab ( 3.7 mbq , 630 g in 150 l ) . 
 at 24 and 48 a blocking study was conducted in two tumor - bearing 
 mice as described above . the blocking mice were each paired with nonblocked 
 mice and imaged at the same time points . tumor standard uptake values 
 ( suv ) were generated by measuring regions of interest from pet / ct 
 images and calculated with the formula suv = [ nci / ml ]  [ animal 
 weight]/injected dose [ nci ] . the data are expressed as mean 
  sd . when more than two data sets were compared , two - way anova 
 analysis with bonferroni post - tests was used . we initially performed conjugation of cb - te1a1p 
 and cetuximab via the conventional nhs - aided reaction to form an amide 
 bond between the carboxylic acid of cb - te1a1p and the primary amines 
 of cetuximab . the sa of cu - cb - te1a1p  cetuximab 
 made by the nhs - aided reaction ranged from 60 to 129 mbq / mg ( 870019,000 
 mbq/mol ) corrected to end - of - bombardment ( eob ) for copper-64 . the cb - te1a1p : cetuximab ratio was 1.5 for cb - te1a1p  cetuximab 
 synthesized via nhs - aided reactions , compared to 5 chelators / mab for 
 previously reported dota  cetuximab . the low ratio of the chelator to antibody is likely responsible 
 for the lower sa of cu - cb - te1a1p  cetuximab compared 
 to the dota and cb - te1k1p - peg4-dbco conjugates . in the conjugation of 
 cb - te1k1p - peg4-dbco to azide cetuximab , a 100-fold 
 of excess cb - te1k1p - peg4-dbco was used to ensure that all 
 the azides in cetuximab were conjugated with chelator . the resulting 
 cb - te1k1p - peg4-click - cetuximab conjugate was purified by 
 centricon 100 and a desalting column , and then radiolabeled with cu at a yield > 95% after incubation for 30 min at 37 c . the sa of the cu - cb - te1k1p - peg4-click - cetuximab 
 ranged from 548 to 684 mbq / mg ( 80,000 to 100,000 mbq/mol ) . the conjugation ratio averaged 9 chelators per cetuximab , approximately 
 6 times greater than for cb - te1a1p  cetuximab . even at this 
 high chelator : mab ratio , the binding affinity of the click chemistry 
 conjugate was not compromised . cb - te1a1p - dbco ( 100 equiv ) was used 
 to react with the azido cetuximab . the resulting cb - te1a1p - click - cetuximab 
 conjugate ( 9 chelators per mab ) was purified by centricon 100 and 
 a desalting column , and the radiolabeling yield was > 90% after 
 incubation 
 for 60 min at 40 c . the sa of the cu - cb - te1a1p - click - cetuximab 
 ranged from 206 to 321 mbq / mg ( 30,000 to 47,000 mbq/mol ) . aggregation 
 was observed after radiolabeling of cb - te1a1p - click - cetuximab , as 
 evidenced by size - exclusion hplc . the dissociation 
 constant ( kd ) values of cu - cb - te1a1p  cetuximab , cu - cb - te1a1p - click - cetuximab , cu - cb - te1k1p - peg4-click - cetuximab , and cu - dota cetuximab 
 for egfr were 2.00  0.60 nm , 1.32  0.42 nm , 1.43  
 0.41 nm , and 1.89  0.66 nm respectively ( table 1 ) , and these values are similar to the previous reported kd of dota - cetuximab . bmax : 
 total number of binding sites . due to the 
 aggregation of cu - cb - te1a1p - click - cetuximab , only cu - cb - te1a1p cetuximab and cu - cb - te1k1p - peg4-click - cetuximab were evaluated in biodistribution studies . the biodistributions were conducted in female nude mice bearing hct116 
 tumors . as shown in figure 2 , both agents demonstrated 
 little to no blood clearance from 24 to 48 h ( 8.2  1.1% id / g 
 at 24 h to 8.3  1.1% id / g at 48 h for cu - cb - te1a1p  cetuximab , 
 and 11.5  2.5% id / g at 24 h to 11.9  0.8% id / g at 48 h 
 for cu - cb - te1k1p - peg4-click - cetuximab ) . the 
 radioactivity in the liver also showed either no clearance or slightly 
 increased uptake , from 10.4  0.8% id / g at 24 h to 9.5  
 0.2% id / g at 48 h for cu - cb - te1a1p  cetuximab , 
 and 8.7  2.5% id / g at 24 h to 13.1  2.6% id / g at 48 h 
 for cu - cb - te1k1p - peg4-click - cetuximab . for 
 both agents , the amount of activity localized in the kidney , heart , 
 and muscle were lower than the liver , and did not show significant 
 clearance from 24 to 48 h. biodistribution of cu - cb - te1k1p - peg4-click - cetuximab 
 ( a ) and cu - cb - te1a1p  cetuximab ( b ) was conducted 
 in hct116 tumor - bearing female nude mice . separate blocking studies 
 were performed to confirm the specificity of probes for egfr , respectively . data are presented as percent injected dose per gram ( n = 35 for each data point ; bars  sd ) . cu - cb - te1a1p  cetuximab and cu - cb - te1k1p - peg4-click - cetuximab showed increasing 
 tumor uptake from 24 to 
 48 h. the tumor uptake of cu - cb - te1k1p - peg4-click - cetuximab at 48 h postinjection ( 36.5  1.9% id / g ) was 
 three times higher than the tumor uptake at 24 h postinjection ( 9.6 
  1.2% id / g ) . the tumor uptake of cu - cb - te1a1p  cetuximab 
 also increased 1.5 times from 24 h ( 14.1  1.8% id / g ) to 48 h 
 ( 20.2  6.1% id / g ) after the administration of agents . the % 
 injected dose in the tumor was greatest at 48 h postinjection for 
 both cu - cb - te1k1p - peg4-click - cetuximab and cu - cb - te1a1p  cetuximab ( p s < 0.05 compared to values at other time points ) . the specificity of cu - cb - te1a1p  cetuximab and cu - cb - te1k1p - peg4-click - cetuximab for egfr was 
 confirmed by separate blocking studies performed at 24 h postinjection . preinjecting 1.0 mg of cold cetuximab reduced tumor uptake of cu - cb - te1a1p  cetuximab from 14.1  1.78% id / g 
 to 8.68  1.03% id / g ( p < 0.05 ) , and that 
 of cu - cb - te1k1p - peg4-click - cetuximab from 
 9.5  1.2% id / g to 4.9  0.5% id / g ( p < 
 0.05 ) . the comparison of tumor : nontumor ratios at 48 h postinjection 
 among cu - cb - te1k1p - peg4-click - cetuximab , cu - cb - te1a1p  cetuximab , and cu - dota  cetuximab is shown in figure 3 . 
 the tumor : muscle and tumor : liver ratios of cu - cb - te1k1p - peg4-click - cetuximab were significantly higher than those of cu - dota  cetuximab ( p < 0.001 , p < 0.01 respectively ) , whereas the tumor : blood showed 
 a higher trend but the differences were not significant ( p = 0.09 ) . on the other hand , compared to cu - dota cetuximab , cu - cb - te1a1p  cetuximab showed comparable tumor : blood 
 and tumor : liver ratios , but significantly lower tumor : muscle and tumor : kidney 
 ratios ( p < 0.05 for both ) , which could be attributed 
 to the lower specific activity ( 60 to 129 mbq / mg ) of cu - cb - te1a1p  cetuximab that may have resulted in lower tumor 
 uptake at 48 h postinjection compared to cu - cb - te1k1p - peg4-click - cetuximab and cu - dota  cetuximab . comparisons 
 of tumor : blood , tumor : muscle , tumor : liver , and tumor : kidney 
 ratios of cu - cb - te1k1p - peg4-click - cetuximab , cu - cb - te1a1p  cetuximab , and cu - dota  cetuximab 
 at 48 h postinjection in hct116 tumor - bearing female nude mice ( * p < 0.05 , * * p < 0.01 , * * * p < 0.001 vs cu - dota  cetuximab ) . small animal pet / ct imaging 
 was performed at 24 and 48 h after injection of cu - cb - te1a1p  cetuximab 
 and cu - cb - te1k1p - peg4-click - cetuximab in hct116 
 tumor bearing mice . for both radiotracers , tumor xenografts were clearly 
 visible at 24 and 48 h postinjection . mice receiving a blocking dose 
 of unlabeled cetuximab showed a significant reduction in uptake of 
 the tracers ( figure 4 ) . the average standardized 
 uptake values ( suvavg ) and the tumor - to - muscle ratios were 
 determined for both the blocked and unblocked tumors through region - of - interest 
 ( roi ) analysis . the suvs of the tumor and muscle for cu - cb - te1a1p  cetuximab were 1.75 and 0.24 respectively , while 
 the tumor and muscle suvs for cu - cb - te1k1p - peg4-click - cetuximab were 1.5 and 0.18 . in cohorts injected with a blocking 
 dose , the tumor - to - muscle ratio of cu - cb - te1a1p  cetuximab 
 was reduced from 7.72  1.59 to 4.44  0.11 , and that of cu - cb - te1k1p - peg4-click - cetuximab was reduced 
 from 8.07  0.52 to 3.27  0.17 ( p < 
 0.05 ) ( figure 4c ) , which further confirmed 
 the specificity of the two cross - bridge chelator attached cetuximab 
 conjugates for the egfr in the hct116 tumor . small animal pet / ct maximum 
 intensity projection images of hct116 
 tumor bearing female nude mice at 48 h postinjection of cu - cb - te1k1p - peg4-click - cetuximab ( a ) and cu - cb - te1a1p  cetuximab ( b ) . one group of mice was pretreated 
 with 16633 equiv of unlabeled cetuximab 24 h prior 
 to probe injection ( a and b right panels ) while the other group was 
 not pretreated ( a and b left panels ) . calibration bars are indicative 
 of bq / ml . ( c ) comparison of tumor - to - muscle uptake ratios between 
 blocked and unblocked mice treated with cetuximab probes . the 
 first attempt to conjugate cb - te1a1p with cetuximab was performed 
 in an aqueous reaction with the assistance of sulfo - nhs . the carboxylate 
 group in cb - te1a1p was activated by edc to form an unstable reactive 
 intermediate . because the hydrolysis of the o - acylisourea 
 ester was more prevalent than the coupling reaction with sulfo - nhs , 
 we were not able to produce any conjugated cetuximab by this method . we next tested reactions using dmf as the solvent , with the assistance 
 of nhs and initiated by edc as the coupling reagent . activated cb - te1a1p 
 reacted with nhs to form a semistable amine - reactive ester , which 
 could then form a stable amide bond with the antibody . dmf was evaporated 
 under vacuum before cetuximab aqueous solution was added to the activated 
 cb - te1a1p . although conjugation occurred , only an average of 1.5 chelators 
 per cetuximab resulted via this synthetic route , due to the low efficiency 
 of the nhs - aided cross - linking reaction between the cb - te1a1p and 
 the primary amines of the antibody . previous studies have shown that 
 the reaction between phosphonic acid groups and amines can occur , 
 but the products quickly hydrolyze and are stable only under strong 
 basic conditions . we postulate that the hydrolysis 
 of phosphoramides leads to lower conjugation efficiency between cb - te1a1p 
 and cetuximab , resulting in low sa of cu - cb - te1a1p  cetuximab . synthesis of radiopharmaceuticals often requires rapid kinetics 
 because of the short half - life . click chemistry has attractive characteristics , 
 including modularity , reliability , selectivity , rapidity , and efficiency . alkyne cycloaddition ( cuaac ) has been 
 widely used for f labeled peptides and small molecules 
 and has demonstrated advantages over conventional conjugation methodologies . however , cuaac is not ideal for conjugation 
 of chelators because cu(i ) ions may prevent formation of the radiometal the development of metal - free spaac has expanded the use 
 of click chemistry for applications with radiometals . to improve the conjugation efficiency , we applied spaac to our newly 
 developed chelators . here we demonstrate the synthesis of cross - bridged 
 chelator  cetuximab conjugates through spaac that allows radiolabeling 
 at physiological temperature in high sa . cb - te1a1p was first modified 
 with commercially available dbco - amine to form dbco the resulting cu - cb - te1a1p - click - cetuximab 
 was produced at 40 c within 60 min in moderate sa ( 206 to 321 
 mbq / mg ) . the binding assay showed comparable binding affinity and bmax values of the cb - te1a1p - click - cetuximab 
 with cb - te1a1p  cetuximab . however , this conjugate showed different 
 degrees of aggregation from batch to batch . to solve this problem , 
 another cross - bridged macrocyclic chelator , cb - te1k1p - peg4-dbco , with an additional carboxylate pendant arm and a peg4 linker the cb - te1k1p - peg4-click - cetuximab 
 conjugate was radiolabeled at 37 c in 30 min in higher sa ( 548 
 to 684 mbq / mg ) . after conjugation of cb - te1a1p to cetuximab , only 
 one methanephosphonic acid moiety is available for chelating cu(ii ) . 
 due to the loss of the carboxylate group and the short linker between 
 the chelator and the antibody , cb - te1a1p cetuximab requires 
 40 c and relatively longer radiolabeling times ( 12 h ) , which may also account for some of the aggregation problems . dbco - peg4 was attached to the primary amine of cb - te1k1p 
 and was then conjugated to azide , we 
 were able to avoid hydrolysis of phosphoramides , which resulted in 
 much higher chelator : mab ratios . the ratio of azide - nhs ester : cetuximab 
 was 20:1 , and 45% of the nhs esters reacted with primary amines 
 in cetuximab , while the other 55% nhs esters hydrolyzed . the ratio 
 of cb - te1k1p - peg4-dbco : azide  cetuximab was 100:1 , 
 driving all azide groups to react with the cb - te1k1p - peg4-dbco , resulting in a chelator : antibody ratio of 9 , which increased 
 the sa by 6-fold compared to the sa obtained from the cb - te1a1p  cetuximab 
 conjugate formed via a nhs - aided reaction . the in vivo performance of cu radiopharmaceuticals 
 is driven in part by the stability of the cu - bfc complex 
 and the sa of the radiotracer . cu - cb - te1k1p and cu - cb - te1a1p demonstrated comparable stability to cu - cb - te2a , both as the metal chelate and after conjugation to peptides.cu-cb-te1a1pcetuximab 
 showed improved biodistribution compared to cu - dota cetuximab , 
 as evident by more rapid blood clearance and the lower uptake in the 
 lung , liver , spleen , and kidney . in vivo transchelation 
 of cu(ii ) is one factor for nonspecific uptake in normal tissues , 
 and forming stable metal chelates improves biodistribution of peptide 
 conjugates ; however , there are no definitive data that this is true 
 for chelator  mab conjugates . in fact , the tumor : muscle and 
 tumor : kidney ratios of cu - cb - te1a1p  cetuximab 
 were not significantly better than those of cu - dota  cetuximab 
 ( p values > 0.05 ) . the low specific activity of cu - cb - te1a1p  cetuximab may have led to receptor saturation , 
 reducing tumor uptake of the radiotracer . the application of click 
 chemistry enabled increasing sa by enhancing radiolabeling efficiency 
 with a longer linkage between the chelator and mab and a higher chelator : antibody 
 ratio . cu - cb - te1k1p  cetuximab demonstrated significantly 
 higher tumor : muscle ( p < 0.001 ) and tumor : liver 
 ( p < 0.01 ) ratios compared to cu - dota  cetuximab . cu - cb - te1a1p  cetuximab was prepared using conventional 
 nhs - aided conjugation , and cu - cb - te1a1p - click - cetuximab 
 and cu - cb - te1k1p - peg4-click - cetuximab were 
 prepared using spaac , and the resulting cetuximab conjugates were 
 evaluated as receptor - targeted agents for pet imaging with future 
 applications for radioimmunotherapy ( rit ) of tumors overexpressing 
 egfr . the click chemistry strategy successfully increased the number 
 of chelators that were attached to each antibody and the sa of the 
 corresponding radiopharmaceutical . cu - cb - te1k1p - peg4cetuximab also demonstrated higher tumor : nontumor 
 ratios than cu - cb - te1a1p  cetuximab . these results 
 provide opportunities for using click chemistry to stably conjugate cu with a wide variety of heat - sensitive biological molecules . cu - cb - te1k1p - peg4-click - cetuximab has potential 
 for simultaneously imaging and treating egfr - positive tumors .
epidermal growth - factor receptor 
 ( egfr ) is overexpressed in a wide 
 variety of solid tumors and has served as a well - characterized target 
 for cancer imaging and therapy . 
 cetuximab was the first mab targeting 
 egfr approved by the fda for the treatment of metastatic colorectal 
 and head and neck cancers . 
 previous studies showed that 64cu ( t1/2 = 12.7 h ; + ( 17.4% ) ) labeled dota 
 cetuximab showed promise for pet imaging 
 of egfr - positive tumors ; however the in vivo stability 
 of this compound has been questioned . in this study , 
 two recently 
 developed cross - bridged macrocyclic chelators ( cb - te1a1p and cb - te1k1p ) 
 were conjugated to cetuximab using standard nhs coupling procedures 
 and/or strain - promoted azide 
 alkyne cycloaddition ( spaac ) methodologies . 
 the radiolabeling and in vitro / vivo evaluation of the resulting cetuximab conjugates were compared . 
 improved cu-64 labeling efficiency and high specific activity ( 684 
 kbq/g , decay corrected to the end of bombardment ) were obtained 
 with the cb - te1k1p - peg4-click - cetuximab conjugate . 
 saturation 
 binding assays indicated that the prepared cetuximab conjugates had 
 comparable affinity ( 1.322.00 nm ) in the hct116 human colorectal 
 tumor cell membranes . 
 in the subsequent in vivo evaluation , 64cu - cb - te1k1p - peg4-click - cetuximab demonstrated 
 more rapid renal clearance with a higher tumor / nontumor ratio than 
 other 64cu - labeled cetuximab conjugates , and it shows the 
 greatest promise for imaging and therapy of egfr - positive tumors .