receptor tyrosine kinases 
 ( rtks ) are a class of protein kinases 
 that play a critical role in the development and progression of various 
 types of cancer .
recently , approximately 20 different rtk classes 
 have been identified , with axl belonging to one of these classes .
the axl receptor tyrosine kinase was isolated as a transforming 
 gene from primary human myeloid leukemia cells .
axl is implicated in vascular remodeling , regulation of 
 smooth muscle cells , and migration of endothelial cells . in recent years
, the significant role of axl in tumor initiation 
 and metastases has been reinforced by the fact that axl is overexpressed 
 in multiple cancer types including prostate , breast , lung , gastric , glioblastoma , and kaposi sarcoma .
several studies 
 also indicate that the expression level of axl is highly correlated 
 with lung tumor progression and invasiveness 
 of breast cancer cells .
in addition , 
 axl is identified as a potential therapeutic target for overcoming 
 egfr inhibitor resistance and for lapatinib 
 and trastuzumab resistance in breast cancer cells . in order to hasten the pace of developing anti - axl 
 based cancer 
 therapy for clinical trials , it is desirable to establish effective 
 methods to quantify axl expression in vivo .
molecular 
 imaging techniques have been widely used for diagnosis and therapy 
 management through evaluation of molecular marker and receptor expression in vivo . in particular ,
positron emission tomography ( pet ) 
 has gained a remarkable amount of attention due to its quantitative 
 imaging properties and high sensitivity .
in fact , there is a considerable 
 amount of interest in development of imaging probes for pet that can 
 serve as companion diagnostics for novel therapeutics .
however , to 
 the best of our knowledge , no pet probe targeting axl has been reported 
 in previous literature . recently
, monoclonal antibodies of murine 
 origin that are specific 
 for axl have been developed . one of these antibodies , m173 , appears 
 to bind to the first fibronection domain . to enhance clinical application , humanized 173 ( h173 )
because of the strong and 
 specific binding of h173 to axl , we hypothesized that radiolabeled 
 h173 could depict the axl receptor distribution in vivo by pet .
the data obtaining by pet imaging could be used to confirm 
 the presence of axl , which would be important clinical information 
 in determining the utility of axl - targeted chemo- and radiotherapy 
 in receptor positive patients . in this study
, we radiolabeled h173 
 with cu to create an antibody based pet probe to noninvasively 
 quantify axl expression in vivo .
human small cell lung cancer a549 cell line and 
 nonsmall cell lung cancer cell line nci - h249 , obtained from american 
 type culture collection ( manassas , va ) , were grown in rpmi-1640 supplemented 
 with 10% fetal bovine serum . for facs analysis ,
aliquots of cells were blocked with 10% normal goat serum , incubated 
 with anti - axl primary antibody h173 ( kindly provided by vasgene therapeutics 
 inc .
, los angeles , ca ) produced using composite human antibody technology and goat antihuman alexa fluor 488 ( invitrogen , 
 paisley , scotland ) in sequence .
subsequently , cells were measured 
 by flow cytometry ( cyan analyzer , beckman coulter ) .
proteins extracted from a549 
 and nci - h249 tumor cells were fractionated using 420% sds - page 
 ( bio - rad ) . after protein transfer ,
polyvinylidene difluoride membrane 
 was blocked with 5% nonfat dry milk , probed with anti - axl primary 
 antibody ( cat # 8661 , cell signaling technology , beverly , ma ) and secondary 
 antibody , and developed .
h173 was conjugated with 
 dota - nhs synthesized in situ ( molar ratio , 1:20 ) 
 through amino groups to form dota - h173 .
negative control 
 antibody , human normal immunoglobulin g ( higg ) , was purchased from 
 rockland ( gilbertsville , pa ) .
control probe dota - higg was also synthesized 
 using the same procedure . after cu ( purchased from washington 
 university , st .
louis ) labeling , probes 
 were used for further in vitro and in vivo experiments .
axl binding 
 activity of dota - h173 
 and dota - higg was performed through a bead - based binding assay with 
 axl - alkaline phosphatase ( ap ) ( kindly provided by vasgene therapeutics 
 inc .
cell uptake of probes in a549 and 
 nci - h249 tumor cells was performed as described previously .
5  10 cells were suspended in 200 l of complete cell culture media , 
 and 37 kbq of cu - dota - h173 and cu - dota - higg 
 was added .
after 1.5 h of incubation , unbound probes were removed 
 by washing twice with cold pbs .
finally , cells were sedimented by 
 centrifugation , and the radioactivity in each cell pellet was counted . 

all animal experiments 
 were performed under a protocol approved by the university of southern 
 california institutional animal care and use committee ( iacuc ) . to 
 establish a lung tumor xenograft model ,
2  10 of 
 a549 or nci - h249 cells were subcutaneously injected in the right shoulder 
 of nude mice as previous reported .
the 
 tumor - bearing mice were injected with 3.77.4 mbq of cu probes via tail veins . for each probe , 3 randomly selected mice 
 were used .
multiple static scans were obtained at 3 , 16 , 28 , and 45 
 h postinjection ( p.i . ) . pet imaging and analysis
were conducted by 
 using a siemens micropet r4 rodent model scanner as described previously .
antibody distribution was localized by 
 using secondary antibody goat antihuman alexa fluor 568 ( invitrogen , 
 paisley , scotland ) .
all of the 
 quantitative data are 
 given as means  sd of three independent measurements .
h173 and higg 
 were conjugated with cu chelator dota 
 through amino groups , which lead to dota - h173 and dota - higg . after cu labeling , the radiochemical yields for cu - dota - h173 
 and cu - dota - higg were 44.5% and 57.6% , respectively . 

the specific activity of cu - dota - h173 and cu - dota - higg was estimated to be 1.482.96 gbq / mg antibody . 
 to investigate the influence of dota conjugation on axl binding ability , 
 a binding activity assay was conducted .
axl binding activity was preserved 
 with dota - h173 ( 98.27%  1.29% ) . on the contrary ,
we used a549 and 
 nci - h249 human lung cancer cell lines for this study . 

a western blot was performed to detect axl expression in these two 
 cell lines .
as shown in figure 1a , a549 overexpressed 
 axl , while nci - h249 was negative .
fluorescence - activated cell sorting 
 ( facs ) data demonstrated that the percentage of axl positive in a549 
 and nci - h249 was 84.40  1.56% and 2.43  0.27% , respectively 
 ( figure 1b ) .
cell uptake study was also conducted 
 ( figure 1c ) . in a549 cells ,
the cell uptake 
 of cu - dota - h173 ( 1.96  0.10% ) was significantly 
 higher than cu - dota - higg ( 0.36  0.04% ) ( p < 0.05 ) . the cell uptake of both cu - dota - h173 
 ( 0.32  0.05% ) and cu - dota - higg ( 0.30  0.05% ) 
 in nci - h249 cells was low and showed no significant difference between 
 them ( p > 0.05 ) .
the above in vitro data demonstrated that cu - dota - h173 probe was axl - specific .
( a ) western blot of axl in a549 and ncl - h249 tumor 
 cells .
( b ) facs analysis of a549 and ncl - h249 tumor cells using h173 
 as the primary antibody .
( c ) cell uptake assay of cu - dota - higg 
 and cu - dota - h173 on a549 and ncl - h249 tumor cells ( n = 3 , mean  sd ) .
the in vivo micropet imaging study was performed on both a549 
 and nci - h249 tumor xenograft mice after the injection of cu - dota - h173 or cu - dota - higg .
representative decay - corrected 
 coronal images are shown in figure 2 . at an 
 early time point ,
both cu - dota - h173 and cu - dota - higg demonstrated high heart uptake because of the relative 
 long circulation half - life of antibodies in vivo . 
 similarly , the liver also had relatively high uptake , which could 
 be attributed to the nonspecific uptake of tracers through the reticular - endothelial 
 system .
as for other organs , such as the lung , their uptake was not 
 significantly different from the background such as muscle .
the uptake 
 in the tumor or other organs was calculated from the roi analysis 
 and shown in figure 3 .
the a549 tumor uptake 
 of cu - dota - h173 was 5.02  1.00 , 8.32  0.89 , 
 11.37  1.53 , and 13.37  1.53 % id / g ( percentage of injected 
 dose per gram of tissue ) at 3 , 16 , 28 , and 45 h p.i . , respectively . 

the nci - h249 tumor uptake of cu - dota - h173 was 3.66  
 0.64 , 4.52  1.08 , 2.65  0.36 , and 2.21  0.30 % id / g 
 at 3 , 16 , 28 , and 45 h p.i .
cu - dota - h173 
 uptake in a549 tumors was significantly higher than in nci - h249 tumors 
 ( p < 0.05 , figure 3d ) at 
 16 , 28 , and 45 h time points .
the a549-tumor to lung ratios were 1.21 
  0.25 , 2.42  0.36 , 4.35  1.80 , and 6.53  2.10 
 % id / g at 3 , 16 , 28 , and 45 h p.i .
cu - dota - higg was also imaged in a549 tumor models to validate 
 the target specificity of cu - dota - h173 .
the a549 tumor 
 uptake of cu - dota - higg at all time points examined was 
 comparatively low ( 3.26  0.94 , 3.59  0.54 , 2.09  
 0.55 , and 1.76  0.58 % id / g at 3 , 16 , 28 , and 45 h p.i .
, respectively ) , 
 which is similar to the muscle uptake ( 4.28  1.23 , 3.35  
 0.65 , 2.52  0.98 , and 1.69  0.66 % id / g at 3 , 16 , 28 , and 
 45 h p.i . ,
decay - corrected whole - body coronal micropet 
 images from a static 
 scan at 3 , 16 , 28 , and 45 h p.i . of ( a )
( b ) cu - dota - h173 into ncl - h249 
 tumor bearing mice , and ( c ) cu - dota - higg into a549 tumor 
 bearing mice .
major organ radioactivity accumulation quantification from a static 
 scan at 3 , 16 , 28 , and 45 h p.i . of ( a )
( b ) cu - dota - h173 into ncl - h249 
 tumor bearing mice , and ( c ) cu - dota - higg into a549 tumor 
 bearing mice .
( d ) time course of a549 and ncl - h249 tumor uptake of cu - dota - h173 from 3 to 45 h p.i .
probe 
 distribution analysis in tumor tissues was conducted through immunofluorescence 
 staining . as shown in figure 4 , more h173 accumulated 
 in axl - positive a549 tumors than higg control . in axl - negative nci - h249 
 tumors
therefore , both in vitro and in vivo data indicated that cu - dota - h173 could be applied for noninvasive imaging of 
 axl expression .
up until now , 20 distinct subfamilies of 
 rtks have been found and 
 categorized according to their identities in their amino acid sequence 
 and structural similarities in their extracellular regions .
one of 
 these , the subfamily tam , is composed of axl , sky , and mer .
axl and 
 its ligand , gas6 , have been found to play a pivotal role in the survival 
 and metastasis of multiple cancers and other diseases . in this study ,
the novel cu - labeled 
 h173 antibody 
 was synthesized and characterized to demonstrate that imaging axl 
 expression with pet is possible .
attachment of cu to 
 a targeting molecule required the use of a bifunctional chelator ( bfc ) . 
 in our experiment ,
binding activity assay showed that dota - h173 preserved 98.27%  
 1.29% axl binding activity .
facs ( figure 1b ) 
 and the cell uptake study ( figure 1c ) further 
 confirmed that h173 could bind to axl - positive a549 lung cancer cells , 
 but not to axl - negative nci - h249 tumors , indicating that h173 antibody 
 was axl - specific .
western detects both cell surface and cytoplasm 
 axl while flow cytometry detects only cell surface axl .
therefore , 
 the receptor expression difference in flow cytometry was not as drastic 
 as the western data .
the in vivo micropet imaging 
 study was performed 
 on both a549 and nci - h249 tumor xenograft mice ( figures 2 and 3 ) .
cu - dota - h173 
 uptake in a549 tumors increased with time , and a better tumor - to - background 
 contrast was displayed at late time points ( 28 h , 45 h p.i . ) . both 
 active uptake ( contributed by h173-axl interaction ) and passive uptake 
 ( contributed by the enhanced permeability and retention effect )
in contrast , 
 tumor accumulation of cu - dota - higg was only caused by 
 passive uptake .
the a549 tumor uptake of cu - dota - higg 
 at all time points examined was comparatively low .
thus , the target 
 specificity of cu - dota - h173 was confirmed . on the contrary ,
cu - dota - h173 uptake in nci - h249 tumors was significantly 
 lower than in a549 tumors ( p < 0.05 , figure 3d ) .
as h173 only recognizes human axl , it does not 
 target murine vasculature , which also expresses axl in tumor xenograft 
 mouse models , according to the published studies
. therefore , the difference in tumor uptake should reflect the axl 
 expression levels of different lung tumor cells .
we would like to 
 point out that tumors without axl expression could still show some 
 tracer uptake due to the presence of nonspecific accumulation . in 
 order to accurately reflect axl receptor expression level with immunopet , 

the contribution from  passive  targeting needs to be 
 considered during data analysis . however , in humans , use of cu - dota - h173 for axl - targeted imaging might provide higher contrast 
 from surrounding tissues due to the fact that it recognizes both tumor 
 cells and associated vasculature .
in addition , highly selective localization 
 of h173 to the tumor bed could lead to applications with therapeutic 
 radionuclides , such as y , lu , or bi for targeted radiation therapy .
we have developed a receptor - targeted pet probe for detection of 
 axl - positive tumors .
this study shows that cu - dota - h173 , 
 based on a humanized axl - specific monoclonal antibody , displays high 
 target specificity both in vitro and in vivo .
the positive correlation between axl expression levels and tumor 
 invasiveness in multiple cancer types makes cu - dota - h173 
 potentially clinically useful for staging axl - positive cancers and 
 managing patients with axl - targeted therapy .
 accumulating 
 experimental evidence indicates that overexpression 
 of the oncogenic receptor tyrosine kinase , axl , plays a key role in 
 the tumorigenesis and metastasis of various types of cancer . 
 the objective 
 of this study is to design a novel imaging probe based on the monoclonal 
 antibody , h173 , for micropet imaging of axl expression in human lung 
 cancer . 
 a bifunctional chelator , dota , was conjugated to h173 , followed 
 by radiolabeling with 64cu . 
 the binding of dota - h173 to 
 the axl receptor was first evaluated by a cell uptake assay and flow 
 cytometry analysis using human lung cancer cell lines . 
 the probe 64cu - dota - h173 was further evaluated by micropet imaging , and ex vivo histology studies in the axl - positive a549 tumors . in vitro cellular study 
 showed that axl probe , 64cu - dota - h173 , was highly immuno - reactive with a549 cells . 
 western 
 blot analysis confirmed that axl is highly expressed in the a549 cell 
 line . 
 for micropet imaging , the a549 xenografts demonstrated a significantly 
 higher 64cu - dota - h173 uptake compared to the nci - h249 xenograft 
 ( a negative control model ) . 
 furthermore , 64cu - dota - h173 
 uptake in a549 is significantly higher than that of 64cu - dota - higg . 
 
 immuno - fluorescence staining was consistent with the in vivo micro - pet imaging results . in conclusion , 64cu - dota - h173 
 
 could be potentially used as a probe for noninvasive imaging of axl 
 expression , which could collect important information regarding tumor 
 response to axl - targeted therapeutic interventions . 