research article cf750-a33scfv-fc-based optical...

Research ArticleCF750-A33scFv-Fc-Based Optical Imaging ofSubcutaneous and Orthotopic Xenografts of GPA33-PositiveColorectal Cancer in Mice

Danfeng Wei1 Qing Fan1 Huawei Cai2 Hao Yang1 Lin Wan1 Lin Li2 and Xiaofeng Lu1

1Key Laboratory of Transplant Engineering and Immunology Regenerative Medical Center West China Hospital Sichuan UniversityChengdu 610041 China2Department of Nuclear Medicine West China Hospital Sichuan University Chengdu 610041 China

Correspondence should be addressed to Xiaofeng Lu xiaofengluyahoocom

Received 5 January 2015 Accepted 24 April 2015

Academic Editor Dominic Fan

Copyright copy 2015 Danfeng Wei et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Antibody-based imaging agents are attractive as adjuvant diagnostic tools for solid tumors GPA33 is highly expressed in mosthuman colorectal cancers and has been verified as a diagnostic and therapeutic target Here we built an A33scFv-Fc antibodyagainst GPA33 by fusing A33scFv to the Fc fragment of human IgG1 antibodies The A33scFv-Fc specifically binds GPA33-positivecolorectal cancer cells and tumor tissues After the intravenous injection of mice bearing subcutaneous GPA33-positive LS174Ttumor grafts with near-infrared fluorescence probe CF750-labeled A33scFv-Fc (CF750-A33scFv-Fc) high contrast images of thetumor grafts could be kinetically documented within 24 h using an optical imaging system However GPA33-negative SMMC7721tumor grafts could not be visualized by injecting the same amount of CF750-A33scFv-FcMoreover in subcutaneous LS174T tumor-bearingmice tissue scanning revealed that the CF750-A33scFv-Fc accumulated in the tumor grafts other than the kidney and liverIn mice with orthotopic tumor transplantations excrescent LS174T tumor tissues in the colon were successfully removed underguidance by CF750-A33scFv-Fc-based optical imagingThese results indicate that CF750-A33scFv-Fc can target GPA33 suggestingthe potential of CF750-A33scFv-Fc as an imaging agent for the diagnosis of colorectal cancer

1 Introduction

Colorectal cancer is one of themost commonmalignancies inthe world as the third most common cancer in men and thesecond in women [1] Although colorectal cancer incidencerates are stabilizing or even declining in historically high-riskareas (United States New Zealand and Canada) they arerapidly increasing in several historically low-risk countries(China Japan Korea and Eastern European countries) [2 3]Colorectal cancer mainly results from a series of geneticchanges leading to the progressive and irreversible loss ofthe normal control of cell growth and differentiation [4] Inaddition several environmental factors mostly related to dietand lifestyle have been identified and seem to play a certainrole in the development of colorectal cancer [5]

The development of colorectal cancer has been revealedas an ordered process spanning three main phases initiation

promotion and progression [6] Clinical data from colorec-tal cancer in high-resource countries have demonstratedthat the mortality of colorectal cancer can be reduced byearly treatment [7ndash9] Consequently early diagnosis playsimportant role in reducing the burden of colorectal cancerHowever the current diagnosis of colorectal cancer is limitedby fecal occult blood tests (FOBTs) flexible sigmoidoscopyand colonoscopy [8 10] As a result it is urgent to developnovel tools for the early diagnosis of colorectal cancer

Noninvasive molecular imaging has become popularin recent years for the diagnosis of solid tumors [11 12]Antibodies against tumor cell-specific surface markers areideal for tumor imaging because of their high specificity andaffinity for antigens [13] GPA33 a 43 kDa membrane glyco-protein is highly expressed in over 95 of human colorectalcancers [14] In addition no circulating GPA33 antigen hasbeen detected [15]These results suggest that GPA33might be

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 505183 11 pageshttpdxdoiorg1011552015505183

2 BioMed Research International

a candidatemarker for the diagnosis and therapy of colorectalcancer Consequently many murine antibodies and theirhumanized antibodies against GPA33 have been developedin the past decades Of these antibodies A33 (a murinemonoclonal antibody against GPA33) [16] and huA33 (thehumanized A33) [17] have been widely used as imaging toolsfor mice with human colon cancer xenografts and for coloncancer patients [16 18 19] However these antibodies arelimited by their low affinity for GPA33 [20]

It was well known that the affinity of rabbit antibodies ishigher than that of murine antibodies Consequently a rabbitantibody against GPA33 has been developed and humanized[20] As expected the affinity of this humanized rabbitantibody against GPA33 (hurA33) was much higher thanthat of the murine antibody A33 A HurA33-derived singlechain fragment of variable antibody (A33scFv) was recentlydeveloped for use in drug delivery [21 22] Considering theslow tumor targeting of large hurA33 and the low affinityof small A33scFv it is better to develop divalent diabodiesminibodies or scFv-Fc antibodies against GPA33 as imagingtools In addition taking into account the risk of radiationinjury by radioactive antibodies it is urgent to developnonradioactive probes for antibody labeling In fact opticaltumor imaging with near-infrared (NIR) fluorescence probe-labeled antibodies has become more and more popular inrecent years [23 24]

In this paper we first produced a divalent antibodyA33scFv-Fc againstGPA33 by fusing the humanizedA33scFvto the Fc fragment of hIgG1 antibodies Subsequently wedetermined the immunoreactivity of CF750- 131I- andFITC-labeled A33scFv-Fc Moreover we evaluated CF570-A33scFv-Fc by optically imaging mice with subcutaneousxenografts of human colon cancer We also performed ortho-topic tumor tissue dissections under the guidance of opticalimaging with CF750-A33scFv-Fc Finally we analyzed thebiodistribution of 131I-labeled A33scFv-Fc in a subcutaneousxenograft mouse model

2 Materials and Methods

21 Construction of a pPIC9K-A33scFv-Fc Expression PlasmidThe genes encoding the variable heavy (VH) and light (VL)chains of intact antibody against A33 antigen were designedbased on their amino acid sequences [20] The single chainfragment of variable antibody against A33 antigen (A33scFv)was constructed in the format VH-(Gly4Ser)3-VL with anadditional 6His tag at the N-terminus EcoRI and AvrIIrestriction sites were added at the 51015840 and 31015840 ends of thegene encoding A33scFv which was synthesized by GenscriptCorporation (Nanjing China) To construct the expressionplasmid for A33scFv-Fc an EcoRIAvrII double-digestedA33scFv gene was ligated to a pPIC9K-hIgG1Fc plasmidcontaining a gene encoding an hIgG1 Fc fragment [25]The sequence-verified plasmid was designated as pPIC9K-A33scFv-Fc

22 Expression and Purification of the A33scFv-Fc To expressthe A33scFv-Fc we linearized the plasmid of pPIC9K-A33scFv-Fc using SalI restriction enzymes transforming

them into Pichia pastoris GS115 competent cells by elec-troporation (15 kV 200Ω and 25 120583F) according to theinstructions for the P pastoris expression kit (InvitrogenCA USA) His+ transformants were selected on histidine-deficient minimal dextrose (MD) plates Subsequently pos-itive colonies were screened through PCR using the 120572-factor primer (51015840-TACTATTGCCAGCATTGCTGC-31015840) andthe 31015840 AOX1 primer (51015840-GCAAATGGCATTCTGACATCC-31015840) Moreover the A33scFv-Fc-producing colonies wereidentified by western blot with an anti-His tag antibodyThe expression and purification of the A33scFv-Fc wereperformed similarly according to our previous work [26]Briefly cells derived from a single colony were inoculatedinto 25mL buffered glycerol-complex medium (BMGY) andincubated at 28∘C with shaking (260 rpm) overnight Thenthe culture was transferred into 1 L fresh BMGY and all ofthe cells were collected by centrifugation at room temperature(3500 g for 5min) until the 119860

600 nm of the culture reachedapproximately 6 at which point it was resuspended in 100mLof buffered methanol-complex medium (BMMY) Methanol(3) was added to the media daily to induce the expressionof the A33scFv-Fc antibody The culture supernatant wascollected by centrifugation at 4∘C (15000 g for 15min) afteran induction period and the supernatant was dialyzedagainst binding buffer (50mM Tris-HCl 05M NaCl and10mM imidazole pH 80) at 4∘C overnight To improve theyield ofantibody the expression conditions including theinitial pH values of the medium and the induction timewere optimized The A33scFv-Fc was purified using Ni-NTAsuperflow (Qiagen CA USA) Subsequently the purifiedantibody was dialyzed against phosphate-buffered saline(PBS 8 g Lminus1 NaCl 02 gLminus1 KCl 349 gLminus1Na

2HPO4sdot12H2O

and 02 gLminus1 KH2PO4) at 4∘C overnight The protein concen-

tration was measured using the Bradford method

23 SDS-PAGE and Western Blot Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and westernblots were performed according to the descriptions in ourprevious work [27] with some modifications Briefly theproteins were separated on 10 gels and visualized byCoomassie Brilliant Blue For western blots the separatedproteins were transferred onto polyvinylidene difluoride(PVDF)membranes (Bio-Rad CA USA) and incubated witha horseradish peroxidase- (HRP-) labeled antibody againstthe 6-His tag (Qiagen CA USA) Finally the target proteinswere visualized using a chemiluminescent substrate for HRPdetection (Thermo IL USA)

24 Labeling of A33scFv-Fc

241 Labeling of A33scFv-Fc with FITC The labeling of theantibody with fluorescein isothiocyanate (FITC) was per-formed according to the EZ-Label FITC protein labeling kit(Pierce CAUSA) Briefly a 24-foldmolar excess of FITCwasadded to the antibody (1mgmL pH 85) After incubationat room temperature for 1 h in the darkness the mixture wasdialyzed against PBS with several buffer changes to removethe unconjugated FITC

BioMed Research International 3

242 Labeling of A33scFv-Fc with CF750 The labeling ofthe antibody with CF750 was performed according to ourprevious work [26] Briefly CF750 dye dissolved in dimethylsulfoxide (DMSO) was added to the antibody (1mgmLpH 83) at a 12 1 molar ratio of dye to antibody Afterincubation at room temperature for 1 h in darkness themixture was dialyzed against PBS with several buffer changesto remove the excessive CF750 dye The degree of labeling(DOL) was calculated according to the following formulaDOL = (119860

755 nm times molecular weight of antibody times dilutionfactor)(120576 times concentration of antibody)Themolar extinctioncoefficient (120576) of CF750 is 250000

243 Labeling of A33scFv-Fc with Radionuclide 131I Thelabeling of the antibody with radionuclide 131I was describedin our previous work [26] Briefly the mixture containing50120583g antibody (1mgmL) 74MBq of 131I-labeled sodiumiodide (specific radioactivity ge 28MBq120583L) and 75 120583g of N-bromosuccinimide (1mgmL) was incubated at room tem-perature with shaking for 5min A PB-10 desalting columnwas used to remove the free sodium iodide and thin layerchromatography (TLC) was used to determine the degree oflabeling (DOL)

25 Cell Culture The GPA33 antigen-positive LS174TCOLO205 cells and GPA33 antigen-negative SMMC7721AGS cells were all purchased from the American TypeCulture Collection (ATCC VA USA) Cells were culturedin medium (Dulbeccorsquos Modified Eagle Medium for LS174TRPMI-1640 for COLO205 and SMMC7721 and F-12K forAGS) containing 10 fetal bovine serum (FBS) at 37∘C in ahumidified atmosphere containing 5 CO

2

26 Immunoreactivity Assays

261 Immunoreactivity Assay for FITC-A33scFv-Fc To eval-uate the immunoreactivity of FITC-labeled A33scFv-Fcapproximately 3 times 105 cells were incubated with 20 nM FITC-labeled antibody in 100 120583L PBS containing 05 calf serumat 37∘C for 1 h in darkness followed by analysis using a flowcytometer (Cytomics FC 500 Beckman Coulter CA USA)A FITC-labeled isotype antibody was utilized as a control inthese assays

262 Immunoreactivity Assay for CF750-A33scFv-Fc Theimmunoreactivity of CF750-A33scFv-Fc was analyzed bydose-dependent cell binding assays Briefly approximately3 times 105 LS174T cells were incubated with CF750-A33scFv-Fcantibody at different concentrations (1 2 4 10 and 20 nM)in 100 120583L PBS containing 05 calf serum at 37∘C for 1 h indarkness After two washes with PBS containing 05 calfserum the cells were transferred onto a 96-well plate andscanned by the IVIS optical imaging system (Caliper LifeSciences CA USA) The fluorescence signal intensities ofcells were analyzed

263 Immunoreactivity Assay for 131I-A33scFv-Fc Dose-dependent cell binding assays were used to evaluate

the immunoreactivity of the 131I-labeled antibody Brieflyapproximately 6 times 105 LS174T cells were incubated with the131I-A33scFv-Fc antibody at different concentrations (1 255 10 20 30 and 60 nM) in 200 120583L PBS containing 05calf serum at 37∘C for 1 h After incubation the cells werewashed with PBS containing 05 calf serum twice and thetotal radioactivity of the bound antibody was counted usinga FJ-2008PS Gamma counter (Xirsquoan Nuclear InstrumentFactory Shanxi China) The free 131I+A33-scFv-Fc was usedas a control

27 Immunofluorescence Histochemistry The tumor xeno-grafts and liver tissues obtained frommice were immediatelycut into frozen sections and stained with the FITC-labeledantibody in the dark at 4∘C overnight The cell nucleiwere visualized using DAPI before being observed underfluorescence microscopy A FITC-labeled isotype antibodywas utilized as a negative control

28 Optical Imaging of Mice Bearing Subcutaneous TumorXenografts All of the protocols used in this experimentwere approved by the University Animal Care and UseCommittee LS174T COLO205 or SMMC7721 (1 times 106cellsmouse) were subcutaneously implanted at the rightflank of female BALBC nunu mice (4sim6 weeks) Tumorgrowth was monitored every day and tumor volumes werecalculated according to the following formula width2 timeslength times 05 To monitor the tumor uptake of antibody themice (119899 = 3) bearing subcutaneous tumor xenografts (100sim200mm3) were intravenously injected with CF750-A33scFv-Fc (100 120583g in 100 120583L) andwere followed by kinetic scans usingthe IVIS optical imaging system at different time points afterinjection To compare the uptake of CF750-A33scFv-Fc by thetumor and the other organstissues the injected mice (119899 = 3)bearing LS174T xenografts were sacrificed and the tumorheart liver spleen lung kidney stomach colon pancreassmall intestine brain and muscle were scanned at differenttime points after injectionThe fluorescence signal intensitiesin the tumor and the other organstissues were analyzedIn the control group the mice were injected with the sameamount of unconjugated CF750 dye

29 Optical Imaging of Mice with Orthotopic Tumor Trans-plantations LS174T cells (1 times 106 cellsmouse) were sub-cutaneously injected into female BALBC nunu mice (4sim6 weeks) When the tumor volume reached approximately200sim300mm3 the tumor grafts were dissected and imme-diately cut into small granules (siml mm3) followed by thetransplantation of these tissues into the colons of other nudemice Once the tumor grafts were palpable the mice (119899 =3) were injected with CF750-A33scFv-Fc (100 120583g in 100 120583L)followed by imaging using the IVIS optical imaging systemat 5 h after injection Subsequently the mice were sacrificedand laparotomywas performedThe orthotopic tumor tissueswere identified and dissected under guidance by CF750-A33scFv-Fc antibody-based optical imaging


AOX1 mmA33scFv hIgG1 Fc

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Figure 1 Expression and purification of A33scFv-Fc (a) Schematic diagram of the pPIC9K-A33scFv-Fc expression vector (b) Effects of theinitial pH (4sim8) of the inductive medium and inductive time (24sim96 h) on the production of A33scFv-Fc (c) SDS-PAGE analysis of purifiedA33scFv-Fc (6120583g) M protein markers

210 Biodistribution of 131I-A33scFv-Fc To evaluate thebiodistribution of 131I-A33scFv-Fc mice (119899 = 3) bearingsubcutaneous LS174T tumor xenografts were intravenouslyinjected with 131I-A33scFv-Fc (185 kBqg body weight) andsacrificed at different time points The tumor grafts andnormal organstissues were taken out weighed and countedfor radioactivity using the FJ-2008PS Gamma counter Theresults are expressed as the percent injected dose per gram oftissue (IDg)

3 Results

31 Production of A33scFv-Fc To construct the pPIC9K-A33scFv-Fc expression plasmid an EcoRIAvrII doubledigested A33scFv gene was ligated to the pPIC9K-hIgG1Fcvector The schematic diagram of the pPIC9K-A33scFv-Fcexpression plasmid is shown in Figure 1(a) To produce theA33scFv-Fc antibody the pPIC9K-A33scFv-Fc plasmid wastransformed into P pastoris GS115 competent cells followedby their identification with PCR and western blot (data notshown) To improve the production of A33scFv-Fc the initial

pH of inductive media and inductive time were optimizedAs shown in Figure 1(b) A33scFv-Fc was expressed in mediawith an initial pH of 40sim80 and a peak at pH 70 More-over time-dependent analysis demonstrated that A33scFv-Fcaccumulated in the medium throughout the induction timeof 24sim96 h Finally the expression ofA33scFv-Fcwas inducedfor 96 h by daily additions of 3 methanol to a mediumwith an initial pH of 70 Figure 1(c) showed that A33scFv-Fc was recovered with approximately 90 purity from thesupernatant using Ni-NTA affinity chromatography

32 Immunoreactivity of A33scFv-Fc The immunoreactivityof the FITC- CF750- and 131I-labelled A33scFv-Fc wasexamined using cell binding and immunofluorescence histo-chemistry As shown in Figure 2(a) flow cytometry analysisdemonstrated that the binding rates of FITC-A33scFv-Fc toGPA33-positive LS174T and COLO205 were 94 and 938respectively In contrast the binding rates of FITC-A33scFv-Fc to GPA33-negative SMMC7721 and AGS cells were as lowas 10 and 17 A33scFv-Fc has been indicated to be able tospecifically bind GPA33-positive cells Immunofluorescence


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Figure 2 Immunoreactivity and specificity of A33scFv-Fc (a) Cell binding assays of FITC-labeled A33scFv-Fc GPA33-positive (LS174TCOLO205) and GPA33-negative (SMMC7721 AGS) cells were incubated with FITC-A33scFv-Fc followed by flow cytometric analysis (b)Immunofluorescence chemical assay for the tissue binding of FITC-A33scFv-Fc LS174T COLO205 tumor tissues and liver tissues werestained with FITC-A33scFv-Fc and observed under a fluorescence microscope The cell nuclei were stained with DAPI and an isotypeantibody was used as a negative control (c) The cell binding of 131I-labeled A33scFv-Fc LS174T cells were incubated with 131I-A33scFv-Fc at different concentrations (1sim60 nM) followed by counting the radioactivity of cells using a gamma counter The mixture containing thesame amount of unconjugated 131I and A33scFv-Fc was used as a control (d) Cellular binding of CF750-labeled A33scFv-Fc SMMC-7721and COLO205 cells were incubated with the CF750-A33scFv-Fc at the indicated molar concentrations (0sim20 nM) and followed by scanningwith the IVIS optical imaging system The fluorescence intensities of the cells were calculated and compared


(h)1 3 5 9 24 48

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Figure 3 CF750-A33scFv-Fc-mediated optical imaging of mice bearing subcutaneous LS174T xenografts Mice (119899 = 3) were intravenouslyinjected with CF750-A33scFv-Fc (100120583g) and scanned at 1 3 5 9 24 and 48 h after injection The LS174T tumor xenografts are indicated byblue circle

histochemistry further verified the specificity of A33scFv-Fc for the GPA33 antigen As shown in Figure 2(b) FITC-A33scFv-Fc bound GPA33-positive LS174T and COLO205tumor tissues but not GPA33-negative liver tissue

The immunoreactivity of 131I-labeled A33scFv-Fc wasalso determined by cell binding assays LS174T cells wereincubated with either 131I-A33scFv-Fc or free 131I+A33-scFv-Fc followed by counting the cell-bound radioactive signalAs shown in Figure 2(c) after incubation with 131I-A33scFv-Fc the radioactive signals of the cells increased along withthe dose of the antibody In contrast dose-dependent bindingwas not observed in cells incubated with free 131I+A33-scFv-Fc These results demonstrate that 131I-labeled A33scFv-Fc isactive in cell binding

To examine the immunoreactivity of CF750-labeled anti-body LS174T cells and SMMC7721 cells were incubatedwith antibodies followed by being transferred into a 96-wellplate and scanned using an IVIS optical imaging systemFigure 2(d) showed that the fluorescence intensity of theLS174T cells increased alongside the antibody On the otherhand only low level of fluorescence intensity was detected inSMMC7721 cells incubated with the same amount of CF750-A33scFv-FcThese data indicate that CF750-labeledA33scFv-Fc is able to specifically bind GPA33-positive LS174T cells

33 Subcutaneous Tumor Targeting of CF750-A33scFv-Fc Tomonitor the tumor uptake of the antibody mice bearingsubcutaneous LS174T xenografts were intravenously injectedwith CF750-A33scFv-Fc followed by kinetic scanning usingan optical imaging system at 1 3 5 9 24 and 48 h afterinjection As shown in Figure 3 LS174T tumor grafts were

visible at 1 h after injection indicating the rapid tumor uptakeof A33scFv-Fc Moreover A33scFv-Fc accumulated in thetumor grafts with time peaking at 5 h High-contrast imagesof the subcutaneous LS174T tumor grafts were obtained at3sim9 h after injection Although the tumor uptake of A33scFv-Fc was reduced by 24 h it was still detectable at 48 h afterinjection To further compare the antibody uptake miceinjected with CF750-A33scFv-Fc were sacrificed and thetumor grafts and other organstissues were scanned at 5 924 and 48 h after injection As shown in Figures 4(a) and4(b) fluorescence signals were mainly detected in the kidneyliver and tumor grafts at all time points The uptake rates ofA33scFv-Fc at each time point were as follows (from highto low) kidney gt liver gt tumor gt spleen gt lung gt heartgt stomach gt colon gt pancreas gt small intestine gt brain gtmuscleThe uptake ratios at 5 9 24 and 48 h were as followstumor-to-muscle 198 plusmn 53 155 plusmn 25 174 plusmn 56 and 170 plusmn51 tumor-to-colon 91 plusmn 19 110 plusmn 02 134 plusmn 15 and78 plusmn 35 and tumor-to-small intestine 191 plusmn 73 218 plusmn 48246plusmn55 and 208plusmn21These results demonstrated the highpersistence of CF750-A33scFv-Fc in GPA33-positive LS174Ttumor xenografts

However injecting the same amount of CF750-A33scFv-Fc did not allow the visualization of the subcutaneousGPA33-negative SMMC 7721 tumor grafts (Figure 5(a)) indicatingthe high specificity of A33scFv-Fc Surprisingly we failedto obtain high-contrast images of mice with GPA33-positiveCOLO205 tumor grafts after they had been injected withthe same amount of CF750-A33scFv-Fc (Figure 5(b)) Tofurther compare the antibody uptakes by the COLO205 andLS174T xenografts we inoculated COLO205 and LS174T


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Figure 4 Tissue distribution of CF750-A33scFv-Fc in mice bearing subcutaneous LS174T xenografts (a) Optical imaging of the tumor graftsand organ tissues at different time points Mice (119899 = 3) were intravenously injected with CF750-A33scFv-Fc (100120583g) and sacrificed at 5 9 24and 48 h after injection Tumor grafts and other normal organstissues were taken out and scanned using the optical imaging system (b)Thefluorescence intensities of CF750-A33scFv-Fc in the tumor grafts and the organstissues at the indicated time points

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Figure 5 Optical imaging of mice bearing single SMMC7721 (a) COLO205 (b) or dual LS174T and COLO205 (c) xenografts Mice (119899 = 3)bearing tumor grafts were intravenously injected with CF750-A33scFv-Fc (100120583g) and scanned at 0 1 3 and 5 h after injection SMMC7721COLO205 and LS174T xenografts are indicated with red circle blue circle and red square respectively After the last scans (5 h) the micewere sacrificed and the organstissues were removed and scanned 1 heart 2 liver 3 spleen 4 lung 5 kidney 6 muscle 7 SMMC7721tumor graft 8 COLO205 tumor graft and 9 LS174T tumor graft


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(a) (b) (c) (d) (e)

Figure 6 CF750-A33scFv-Fc-based optical imaging-directed orthotopic tumor tissue dissection LS174T tumor tissues derived fromxenografts were inoculated into colons of mice Once the tumor tissues were palpable the mice (119899 = 3) were injected with CF750-A33scFv-Fc(100120583g) followed by scanning and tumor tissue dissection at 5 h after injection Before laparotomy themice were first imaged using an opticalimaging systemwith white light (a) andNIR filter settings (b) Next laparotomywas performed and the excrescent tumor tissues in the colonwere identified by an optical imaging system (c and d) Finally the orthotopic tumor tissues were removed under the guide of optical imaging(e) LS174T xenografts are indicated with blue circle

cells in the same mice As shown in Figure 5(c) after theinjection of CF750-A33scFv-Fc into mice with dual tumorgrafts the fluorescence signal in the COLO205 xenograftwas significantly lower than that in the LS174T xenograftBecause both COLO205 and LS174T cells were GPA33-positive these results suggest that CF750-A33scFv-Fc mightnot be effectively delivered into COLO205 tumor grafts

34 Orthotopic Tumor Targeting of CF750-A33scFv-Fc Toevaluate the targeting of CF750-A33scFv-Fc in an orthotopicLS174T tumor transplantation mice model the injected micewere first imaged using the optical imaging system fromthe abdomen without laparotomy As shown in Figures 6(a)and 6(b) the bladders of three mice were visible underthe optical imaging system at 5 h after injection but tumorgrafts were palpable in only two mice Once the laparotomywas performed excrescent tumor tissues in the colon weredistinguished fromnormal tissues in threemice usingCF750-A33scFv-Fc (Figures 6(c) and 6(d)) Moreover tumor tissueswere accurately dissected under the guidance of an IVIS opti-cal imaging system (Figure 6(e)) These results demonstratethat the CF750-A33scFv-Fc can target orthotopic LS174Ttumor grafts

35 Biodistribution Analysis of 131I-A33scFv-Fc In a biodis-tribution assay mice bearing subcutaneous LS174T tumorxenografts (300sim400mm3) were intravenously injected with

131I-A33scFv-Fc Subsequently the mice were sacrificed at008 05 2 5 8 and 24 h after injection followed by countingthe radioactivity of tumor and organstissues As shown inTable 1 the radioactivity of 131I-A33scFv-Fc in the tumor at5min was 412 plusmn 076 indicating a rapid tumor uptake thatincreased with time and peaked at 5 h after injection with anactivity of 545 plusmn 125 Thereafter the tumorrsquos radioactivitybegan to decrease but persisted at a high level at 24 h afterinjection The radioactivity in the tumor was 203 plusmn 051 at24 h after injection compared to 068 plusmn 019 016 plusmn 005047 plusmn 016 and 038 plusmn 011 in the blood muscle smallintestine and colon respectively As shown in Figure 7 theratios of tumor-to-muscle were 43plusmn03 47plusmn04 and 126plusmn1at 5 8 and 24 h after injection respectively The ratio oftumor-to-blood increased from 15 plusmn 01 at 5 h to 30 plusmn 03at 24 h In addition the ratios of tumor-to-small intestineand tumor-to-colon were as high as 44 plusmn 07 and 53 plusmn 05respectively at 24 h after injectionThese results demonstratethat the 131I-A33scFv-Fc was able to specifically localize atthe LS174T tumor

4 Discussion

In this study divalent A33scFv-Fc against GPA33 was pre-pared by fusing A33scFv to the Fc fragment of an hIgG1antibody In vitro cell binding assays demonstrated that


Table 1 Biodistribution of 131I-A33scFv-Fc in nude mice bearing LS174T xenografts (119899 = 3 at each time point)

Tissue 008 h 05 h 2 h 5 h 8 h 24 hBlood 2098 plusmn 187 1092 plusmn 243 636 plusmn 191 286 plusmn 014 161 plusmn 028 068 plusmn 019Heart 1036 plusmn 274 547 plusmn 117 316 plusmn 090 141 plusmn 010 081 plusmn 014 039 plusmn 015Liver 1091 plusmn 180 952 plusmn 110 653 plusmn 078 328 plusmn 021 268 plusmn 070 212 plusmn 088Spleen 2162 plusmn 333 1663 plusmn 296 990 plusmn 131 503 plusmn 048 323 plusmn 022 272 plusmn 128Lung 3516 plusmn 1071 1748 plusmn 257 856 plusmn 249 339 plusmn 034 166 plusmn 018 073 plusmn 023Kidney 3035 plusmn 240 2714 plusmn 154 2051 plusmn 146 1350 plusmn 074 1148 plusmn 096 761 plusmn 219Stomach 1159 plusmn 328 1607 plusmn 232 2963 plusmn 902 1193 plusmn 064 461 plusmn 118 128 plusmn 043Small intestine 882 plusmn 172 671 plusmn 103 436 plusmn 252 286 plusmn 096 200 plusmn 144 047 plusmn 016Pancreas 996 plusmn 117 725 plusmn 060 937 plusmn 318 438 plusmn 044 150 plusmn 032 029 plusmn 005Colon 639 plusmn 167 379 plusmn 087 547 plusmn 137 207 plusmn 028 095 plusmn 019 038 plusmn 011Brain 145 plusmn 002 085 plusmn 009 081 plusmn 020 027 plusmn 002 015 plusmn 001 003 plusmn 001Muscle 400 plusmn 026 247 plusmn 012 257 plusmn 041 098 plusmn 008 062 plusmn 012 016 plusmn 005Tumor 428 plusmn 037 499 plusmn 006 545 plusmn 125 421 plusmn 029 290 plusmn 037 203 plusmn 051IDg the percent of injected dose per gram tissue

0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

Time after injection (h)

TumorbloodTumorsmall intestine

TumorcolonTumormuscle

Figure 7 The 131I-A33scFv-Fc uptake ratio of tumor to normal tis-sues at different time points Mice (119899 = 3) bearing subcutaneousLS174T tumor xenografts were intravenously injected with 131I-A33scFv-Fc (185 kBqg body weight) and then sacrificed at 00805 2 5 8 and 24 h after injection respectively The radioactivityof the organtissue was counted and the ratios of tumor-to-bloodtumor-to-small intestine tumor-to-colon and tumor-to-musclewere calculated

the A33scFv-Fc produced by P pastoris can bind GPA33-positive but not GPA-negative tumor cells In mice bearingsubcutaneous xenografts CF750-labeled A33scFv-Fc wasaccumulated rapidly in GPA33-positive LS174T xenograftsbut not in GPA33-negative SMMC7721 xenograftsMoreoverorthotopic LS174T tumor tissues were dissected successfullyunder the guidance of an optical imaging system usingCF750-labeled A33scFv-Fc as an indicator Biodistributionanalysis revealed a rapid tumor uptake and a high level persis-tence of A33scFv-Fc in the xenografts These results suggest

that A33scFv-Fc could be developed as a useful imaging agentfor colorectal cancer

Due to their high specificity and affinity for antigensantibody-based imaging agents are ideal for tumor diag-nostics Taking into account the slow tumor targeting andserum persistence of large intact antibodies approachesfor building small antibodies including scFv-Fc minibodydiabody and scFv have been constructed in the past decades[28 29] Building scFv was the first step toward engi-neering a small antibody In fact we began by preparingA33scFv and evaluating its potential as an imaging agentfor GPA33-positive colorectal cancer As shown in supple-mentary Figure 1(a) (in Supplementary Material availableonline at httpdxdoiorg1011552015505183) A33scFv canalso bind GAP33-positive LS174T cells However the affinityof A33scFv-Fc for LS174T cells was much higher than thatof A33scFv For example the mean fluorescence intensities(MFI) of cells incubated with 10 and 20 nM FITC-A33scFv-Fc were 45- and 42-fold of that of A33scFv respectively(supplementary Figure 1(b)) Moreover the injection ofCF750-A33scFv-Fc produced high-contrast images of micebearing LS174T xenografts (Figure 3) but the injection ofthe same amount of CF750-A33scFv gave images withlow contrast (supplementary Figure 2(a)) Further tissuescanning demonstrated that the tumor uptake of CF750-A33scFv-Fc (Figure 4(a)) was obviously higher than that ofCF750-A33scFv (supplementary Figure 2(b)) The fluores-cence intensity of CF750-A33scFv-Fc in tumor grafts at 9 h(Figure 4(b)) was approximately 3sim5 times higher than thatof CF750-A33scFv at 7 h (supplementary Figure 2(c)) Inaddition the CF750-A33scFv-Fc uptake ratios of tumor-to-muscle tumor-to-colon and tumor-to-small intestine were155 plusmn 25 110 plusmn 02 and 218 plusmn 48 respectively comparedto 75 plusmn 19 32 plusmn 15 and 54 plusmn 21 among CF750-A33scFv These results suggest that divalent CF750-A33scFv-Fc is more properly used than monovalent CF750-A33scFvas an imaging agent for colorectal cancer Neverthelessbecause an Fc fragment might introduce such side effects as


antibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) [30] it might bebetter to produce a GPA33-recognizing diabody or minibodywithout an Fc fragment as an imaging agent in the future

It is known that NIR fluorescence probes have lowautofluorescence and low absorbance in normal tissue withintheNIR region (700sim900 nm) which can potentially increasethe sensitivity and specificity of a cancer diagnosis [31]Consequently NIR fluorescence probe-labeled antibodieswithout radiation injury have become attractive as noveltools for tumor imaging Usually mice bearing subcutaneoustumor xenografts are used for the preclinical biological eval-uation of tumor imaging agents In our experiment we firstevaluated CF750-A33scFv-Fc in mice bearing subcutaneousLS174T tumor grafts Because an injection of CF750-A33scFv-Fc produced high-contrast images in subcutaneous tumor-bearing mice (Figure 3) we further investigated whetherthe orthotopic tumor grafts could be dissected under theguidance of CF750-A33scFv-Fc-based optical imaging Asshown in Figure 6 after the laparotomy of the mice injectedwith CF750-A33scFv-Fc optical imaging demonstrated thata fluorescence signal was predominantly detectable in tumortissues and the liver In particular the excrescent tumortissue was distinguished from that of the normal colonusing the optical imaging system Finally the tumor tissuewas accurately dissected under the guidance of the opticalimaging system These results suggest that CF750-A33scFv-Fc-based imaging agent might facilitate the surgical removalof colorectal cancer while keeping both patients and surgeonsfree from radiation

However unlike the LS174T tumor grafts the COLO205tumor grafts in mice injected with the same amountof CF750-A33scFv-Fc were visualized with low contrast(Figure 5(b)) suggesting a difference between COLO205 andLS174T tumor grafts in uptake of CF750-A33scFv-Fc Toreduce the bias caused by individual differences betweenmiceand batch variations of CF750-A33scFv-Fc we further usedmice bearing both COLO205 and LS174T tumor grafts toevaluate the uptake of CF750-A33scFv-Fc Scanning the miceand the tissues revealed that the uptake of CF750-A33scFv-Fc by the COLO205 tumor graft was significantly less thanthat by the LS174T tumor graft (Figure 5(c)) Flow cytometricanalysis with FITC-A33scFv-Fc demonstrated that GPA33was expressed on the surfaces of COLO205 and LS174T cells(Figure 2(a)) Further there was no obvious difference ineither cell line in MFI after incubation with the same amountof FITC-A33scFv-Fc (data not shown) Immunofluorescencehistochemistry revealed that even though membrane GPA33was expressed in both COLO205 and LS174T tumor graftsat a similar level (Figure 2(b)) the LS174T xenografts werehypervascular [32] The expression of vascular endothelialgrowth factor (VEGF) in LS174T cells was much higherthan that in COLO205 suggesting a difference between thexenografts of LS174T and COLO205 in vascularization [3334] In fact we found blood vessel-rich areas in LS174T butnot in COLO205 tumor xenografts These different vascular-izations may have led to the differences between COLO205and LS174T tumor xenografts in their delivery of A33scFv-Fc Consequently CF750-A33scFv-Fc might be limited in

utility for imaging blood vessel-rich colorectal cancer Stillthe injection of a small tumor-homing peptide producedclear images of COLO205 tumor xenografts (data not shown)suggesting that the tissue-penetrating ability of A33scFv-Fcis poor A33scFv-based diabody or minibody with reducedmolecular weight but retained affinity might possess a highertissue-penetrating ability Moreover the tissue penetration ofA33scFv-Fc might also be improved by fusion with tissue-penetrating peptides [35]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Danfeng Wei and Qing Fan contributed equally to this work

Acknowledgments

This work was supported by the National Natural ScienceFund of China (81273419) and the National Key ClinicalProgram

References

[1] A Jemal M M Center C DeSantis and E M Ward ldquoGlobalpatterns of cancer incidence and mortality rates and trendsrdquoCancer Epidemiology Biomarkers amp Prevention vol 19 no 8 pp1893ndash1907 2010

[2] M M Center A Jemal R A Smith and E Ward ldquoWorldwidevariations in colorectal cancerrdquo CA Cancer Journal for Clini-cians vol 59 no 6 pp 366ndash378 2009

[3] M M Center A Jemal and E Ward ldquoInternational trendsin colorectal cancer incidence ratesrdquo Cancer EpidemiologyBiomarkers amp Prevention vol 18 no 6 pp 1688ndash1694 2009

[4] B Vogelstein E R Fearon S R Hamilton et al ldquoGeneticalterations during colorectal-tumor developmentrdquo The NewEngland Journal of Medicine vol 319 no 9 pp 525ndash532 1988

[5] J D Potter ldquoColorectal cancer molecules and populationsrdquoJournal of the National Cancer Institute vol 91 no 11 pp 916ndash932 1999

[6] R AWeinberg ldquoOncogenes antioncogenes and themolecularbases of multistep carcinogenesisrdquo Cancer Research vol 49 no14 pp 3713ndash3721 1989

[7] A G Zauber I Lansdorp-Vogelaar A B Knudsen J WilschutM Van Ballegooijen and K M Kuntz ldquoEvaluating test strate-gies for colorectal cancer screening a decision analysis for theUS Preventive Services Task ForcerdquoAnnals of InternalMedicinevol 149 no 9 pp 659ndash669 2008

[8] J S Mandel J H Bond T R Church et al ldquoReducingmortalityfrom colorectal cancer by screening for fecal occult bloodrdquoTheNew England Journal of Medicine vol 328 no 19 pp 1365ndash13711993

[9] A D Muller and A Sonnenberg ldquoPrevention of colorectalcancer by flexible endoscopy and polypectomy a case-controlstudy of 32 702 veteransrdquo Annals of Internal Medicine vol 123no 12 pp 904ndash910 1995


[10] S Winawer R Fletcher D Rex et al ldquoColorectal cancerscreening and surveillance clinical guidelines and rationalemdashupdate based on new evidencerdquo Gastroenterology vol 124 no2 pp 544ndash560 2003

[11] U Iqbal H Albaghdadi Y Luo et al ldquoMolecular imaging ofglioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodiesrdquo British Journal ofCancer vol 103 no 10 pp 1606ndash1616 2010

[12] V Sarangthem E A Cho S M Bae et al ldquoConstruction andapplication of elastin like polypeptide containing IL-4 receptortargeting peptiderdquo PLoS ONE vol 8 no 12 Article ID e818912013

[13] S Kaur G Venktaraman M Jain S Senapati P K Garg and SK Batra ldquoRecent trends in antibody-based oncologic imagingrdquoCancer Letters vol 315 no 2 pp 97ndash111 2012

[14] P Garin-Chesa J Sakamoto S Welt F X Real W J Rettigand L J Old ldquoOrgan-specific expression of the colon cancerantigen A33 a cell surface target for antibody-based therapyrdquoInternational Journal of Oncology vol 9 no 3 pp 465ndash4711996

[15] J K Heath S J White C N Johnstone et al ldquoThe human A33antigen is a transmembrane glycoprotein and anovelmember ofthe immunoglobulin superfamilyrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no2 pp 469ndash474 1997

[16] S Welt C R Divgi F X Real et al ldquoQuantitative analysisof antibody localization in human metastatic colon cancer aphase I study of monoclonal antibody A33rdquo Journal of ClinicalOncology vol 8 no 11 pp 1894ndash1906 1990

[17] D J King P Antoniw R J Owens et al ldquoPreparation andpreclinical evaluation of humanisedA33 immunoconjugates forradioimmunotherapyrdquo British Journal of Cancer vol 72 no 6pp 1364ndash1372 1995

[18] F T Lee C Hall A Rigopoulos et al ldquoImmuno-PET ofhuman colon xenograft-bearing BALBc nude mice using 124I-CDR-grafted humanized A33 monoclonal antibodyrdquo Journal ofNuclear Medicine vol 42 no 5 pp 764ndash769 2001

[19] J A Carrasquillo N Pandit-Taskar J A OrsquoDonoghue et alldquo124I-huA33 antibody PET of colorectal cancerrdquo Journal ofNuclear Medicine vol 52 no 8 pp 1173ndash1180 2011

[20] C Rader G Ritter S Nathan et al ldquoThe rabbit antibodyrepertoire as a novel source for the generation of therapeutichuman antibodiesrdquoThe Journal of Biological Chemistry vol 275no 18 pp 13668ndash13676 2000

[21] L M Damasceno I Pla H-J Chang et al ldquoAn optimizedfermentation process for high-level production of a single-chainFv antibody fragment in Pichia pastorisrdquo Protein Expression andPurification vol 37 no 1 pp 18ndash26 2004

[22] L M Damasceno K A Anderson G Ritter J M CreggL J Old and C A Batt ldquoCooverexpression of chaperonesfor enhanced secretion of a single-chain antibody fragment inPichia pastorisrdquoAppliedMicrobiology and Biotechnology vol 74no 2 pp 381ndash389 2007

[23] P Zou S Xu S P Povoski et al ldquoNear-infrared fluorescencelabeled anti-TAG-72 monoclonal antibodies for tumor imagingin colorectal cancer xenograft micerdquo Molecular Pharmaceuticsvol 6 no 2 pp 428ndash440 2009

[24] D Li S Liu R Liu et al ldquoEphB4-targeted imaging with anti-body h131 h131-F(ab1015840)

2and h131-FabrdquoMolecular Pharmaceutics

vol 10 no 12 pp 4527ndash4533 2013[25] L Wan S Zhu J Zhu et al ldquoProduction and characterization

of a CD25-specific scFv-Fc antibody secreted from Pichia

pastorisrdquo Applied Microbiology and Biotechnology vol 97 no 9pp 3855ndash3863 2013

[26] Q Fan H Cai H Yang et al ldquoBiological evaluation of 131I- andCF750-labeled Dmab(scFv)-Fc antibodies for xenograft imag-ing of CD25-positive tumorsrdquo BioMed Research Internationalvol 2014 Article ID 459676 11 pages 2014

[27] L Wan H Cai H Yang et al ldquoHigh-level expression of afunctional humanized single-chain variable fragment antibodyagainst CD25 in Pichia pastorisrdquo Applied Microbiology andBiotechnology vol 81 no 1 pp 33ndash41 2008

[28] A M Wu ldquoEngineered antibodies for molecular imaging ofcancerrdquoMethods vol 65 no 1 pp 139ndash147 2014

[29] P Holliger and P J Hudson ldquoEngineered antibody fragmentsand the rise of single domainsrdquo Nature Biotechnology vol 23no 9 pp 1126ndash1136 2005

[30] E Kaneko and R Niwa ldquoOptimizing therapeutic antibodyfunction progress with fc domain engineeringrdquo BioDrugs vol25 no 1 pp 1ndash11 2011

[31] E M Sevick-Muraca ldquoTranslation of near-infrared fluores-cence imaging technologies emerging clinical applicationsrdquoAnnual Review of Medicine vol 63 pp 217ndash231 2012

[32] T Fujii M Tachibana D K Dhar et al ldquoCombination therapywith paclitaxel and thalidomide inhibits angiogenesis andgrowth of human colon cancer xenograft in micerdquo AnticancerResearch vol 23 no 3 pp 2405ndash2411 2003

[33] D T Dang S Y Chun K Burkitt et al ldquoHypoxia-induciblefactor-1 target genes as indicators of tumor vessel response tovascular endothelial growth factor inhibitionrdquoCancer Researchvol 68 no 6 pp 1872ndash1880 2008

[34] R SWarren H YuanM RMatli N A Gillett andN FerraraldquoRegulation by vascular endothelial growth factor of humancolon cancer tumorigenesis in a mouse model of experimentallivermetastasisrdquoThe Journal of Clinical Investigation vol 95 no4 pp 1789ndash1797 1995

[35] T-H Shin E-S Sung Y-J Kim et al ldquoEnhancement of thetumor penetration of monoclonal antibody by fusion of aneuropilin-targeting peptide improves the antitumor efficacyrdquoMolecular Cancer Therapeutics vol 13 no 3 pp 651ndash661 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


a candidatemarker for the diagnosis and therapy of colorectalcancer Consequently many murine antibodies and theirhumanized antibodies against GPA33 have been developedin the past decades Of these antibodies A33 (a murinemonoclonal antibody against GPA33) [16] and huA33 (thehumanized A33) [17] have been widely used as imaging toolsfor mice with human colon cancer xenografts and for coloncancer patients [16 18 19] However these antibodies arelimited by their low affinity for GPA33 [20]

It was well known that the affinity of rabbit antibodies ishigher than that of murine antibodies Consequently a rabbitantibody against GPA33 has been developed and humanized[20] As expected the affinity of this humanized rabbitantibody against GPA33 (hurA33) was much higher thanthat of the murine antibody A33 A HurA33-derived singlechain fragment of variable antibody (A33scFv) was recentlydeveloped for use in drug delivery [21 22] Considering theslow tumor targeting of large hurA33 and the low affinityof small A33scFv it is better to develop divalent diabodiesminibodies or scFv-Fc antibodies against GPA33 as imagingtools In addition taking into account the risk of radiationinjury by radioactive antibodies it is urgent to developnonradioactive probes for antibody labeling In fact opticaltumor imaging with near-infrared (NIR) fluorescence probe-labeled antibodies has become more and more popular inrecent years [23 24]

In this paper we first produced a divalent antibodyA33scFv-Fc againstGPA33 by fusing the humanizedA33scFvto the Fc fragment of hIgG1 antibodies Subsequently wedetermined the immunoreactivity of CF750- 131I- andFITC-labeled A33scFv-Fc Moreover we evaluated CF570-A33scFv-Fc by optically imaging mice with subcutaneousxenografts of human colon cancer We also performed ortho-topic tumor tissue dissections under the guidance of opticalimaging with CF750-A33scFv-Fc Finally we analyzed thebiodistribution of 131I-labeled A33scFv-Fc in a subcutaneousxenograft mouse model

2 Materials and Methods

21 Construction of a pPIC9K-A33scFv-Fc Expression PlasmidThe genes encoding the variable heavy (VH) and light (VL)chains of intact antibody against A33 antigen were designedbased on their amino acid sequences [20] The single chainfragment of variable antibody against A33 antigen (A33scFv)was constructed in the format VH-(Gly4Ser)3-VL with anadditional 6His tag at the N-terminus EcoRI and AvrIIrestriction sites were added at the 51015840 and 31015840 ends of thegene encoding A33scFv which was synthesized by GenscriptCorporation (Nanjing China) To construct the expressionplasmid for A33scFv-Fc an EcoRIAvrII double-digestedA33scFv gene was ligated to a pPIC9K-hIgG1Fc plasmidcontaining a gene encoding an hIgG1 Fc fragment [25]The sequence-verified plasmid was designated as pPIC9K-A33scFv-Fc

22 Expression and Purification of the A33scFv-Fc To expressthe A33scFv-Fc we linearized the plasmid of pPIC9K-A33scFv-Fc using SalI restriction enzymes transforming

them into Pichia pastoris GS115 competent cells by elec-troporation (15 kV 200Ω and 25 120583F) according to theinstructions for the P pastoris expression kit (InvitrogenCA USA) His+ transformants were selected on histidine-deficient minimal dextrose (MD) plates Subsequently pos-itive colonies were screened through PCR using the 120572-factor primer (51015840-TACTATTGCCAGCATTGCTGC-31015840) andthe 31015840 AOX1 primer (51015840-GCAAATGGCATTCTGACATCC-31015840) Moreover the A33scFv-Fc-producing colonies wereidentified by western blot with an anti-His tag antibodyThe expression and purification of the A33scFv-Fc wereperformed similarly according to our previous work [26]Briefly cells derived from a single colony were inoculatedinto 25mL buffered glycerol-complex medium (BMGY) andincubated at 28∘C with shaking (260 rpm) overnight Thenthe culture was transferred into 1 L fresh BMGY and all ofthe cells were collected by centrifugation at room temperature(3500 g for 5min) until the 119860

600 nm of the culture reachedapproximately 6 at which point it was resuspended in 100mLof buffered methanol-complex medium (BMMY) Methanol(3) was added to the media daily to induce the expressionof the A33scFv-Fc antibody The culture supernatant wascollected by centrifugation at 4∘C (15000 g for 15min) afteran induction period and the supernatant was dialyzedagainst binding buffer (50mM Tris-HCl 05M NaCl and10mM imidazole pH 80) at 4∘C overnight To improve theyield ofantibody the expression conditions including theinitial pH values of the medium and the induction timewere optimized The A33scFv-Fc was purified using Ni-NTAsuperflow (Qiagen CA USA) Subsequently the purifiedantibody was dialyzed against phosphate-buffered saline(PBS 8 g Lminus1 NaCl 02 gLminus1 KCl 349 gLminus1Na

2HPO4sdot12H2O

and 02 gLminus1 KH2PO4) at 4∘C overnight The protein concen-

tration was measured using the Bradford method

23 SDS-PAGE and Western Blot Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and westernblots were performed according to the descriptions in ourprevious work [27] with some modifications Briefly theproteins were separated on 10 gels and visualized byCoomassie Brilliant Blue For western blots the separatedproteins were transferred onto polyvinylidene difluoride(PVDF)membranes (Bio-Rad CA USA) and incubated witha horseradish peroxidase- (HRP-) labeled antibody againstthe 6-His tag (Qiagen CA USA) Finally the target proteinswere visualized using a chemiluminescent substrate for HRPdetection (Thermo IL USA)

24 Labeling of A33scFv-Fc

241 Labeling of A33scFv-Fc with FITC The labeling of theantibody with fluorescein isothiocyanate (FITC) was per-formed according to the EZ-Label FITC protein labeling kit(Pierce CAUSA) Briefly a 24-foldmolar excess of FITCwasadded to the antibody (1mgmL pH 85) After incubationat room temperature for 1 h in the darkness the mixture wasdialyzed against PBS with several buffer changes to removethe unconjugated FITC






2











6His tag

EcoRI AvrII NotI

120572-factor

(a)

24

(pH)

(h)967248

4 86 75

(b)

662

450

1160

350

250

184

144

(kD

a)

M A33scFv-Fc

(c)



3 Results






Even

ts

10010

110

210

010

110

210

010

110

210

010

110

2

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

(a)


Con

trol

A33

scFv

-Fc

(b)


25

20

15

10

5

0

1 25 5 10 20 30 60

Upt

ake o

f131I(times103 C

PM)


(c)

SMMC7721

LS174T

SMMC7721LS174T

Inte

nsity

(nM)

50

40

30

20

10

0

0 1 2 4 10 20

01 2 4 10 20

Inte

nsity

(times106)


(d)



(h)1 3 5 9 24 48

Mou

se 1

Mou

se 2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity









(h)

5

9

24

48In

tens

ityIn

tens

ityIn

tens

ityIn

tens

ity

Hea

rtLi

ver

Sple

enLu

ng

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

Smal

lin

testi

ne

(a)

100

2545

Hea

rt

Live

r

Sple

en

Lung

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

5101520

6080

0

Smal

lin

testi

ne

5h9h

24h48h

Inte

nsity

(times108)

(b)


1 3 50(h)

Inte

nsity

SMM

C772

1

2 3

4 5 6 7

1Organtissue (5h)

(a)

COLO

205

Inte

nsity

1 2 3

4 5 6 8

(b)

LS17

4T-C

OLO

205

Inte

nsity

2 3 4

6 8 9

22222 33333333333333333333333333333333333333333333333333333 44

666 88888 99

1 2 3

4 5 6 8 9

(c)



Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2











6His tag

EcoRI AvrII NotI

120572-factor

(a)

24

(pH)

(h)967248

4 86 75

(b)

662

450

1160

350

250

184

144

(kD

a)

M A33scFv-Fc

(c)



3 Results






Even

ts

10010

110

210

010

110

210

010

110

210

010

110

2

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

(a)


Con

trol

A33

scFv

-Fc

(b)


25

20

15

10

5

0

1 25 5 10 20 30 60

Upt

ake o

f131I(times103 C

PM)


(c)

SMMC7721

LS174T

SMMC7721LS174T

Inte

nsity

(nM)

50

40

30

20

10

0

0 1 2 4 10 20

01 2 4 10 20

Inte

nsity

(times106)


(d)



(h)1 3 5 9 24 48

Mou

se 1

Mou

se 2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity









(h)

5

9

24

48In

tens

ityIn

tens

ityIn

tens

ityIn

tens

ity

Hea

rtLi

ver

Sple

enLu

ng

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

Smal

lin

testi

ne

(a)

100

2545

Hea

rt

Live

r

Sple

en

Lung

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

5101520

6080

0

Smal

lin

testi

ne

5h9h

24h48h

Inte

nsity

(times108)

(b)


1 3 50(h)

Inte

nsity

SMM

C772

1

2 3

4 5 6 7

1Organtissue (5h)

(a)

COLO

205

Inte

nsity

1 2 3

4 5 6 8

(b)

LS17

4T-C

OLO

205

Inte

nsity

2 3 4

6 8 9

22222 33333333333333333333333333333333333333333333333333333 44

666 88888 99

1 2 3

4 5 6 8 9

(c)



Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















6His tag

EcoRI AvrII NotI

120572-factor

(a)

24

(pH)

(h)967248

4 86 75

(b)

662

450

1160

350

250

184

144

(kD

a)

M A33scFv-Fc

(c)



3 Results






Even

ts

10010

110

210

010

110

210

010

110

210

010

110

2

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

(a)


Con

trol

A33

scFv

-Fc

(b)


25

20

15

10

5

0

1 25 5 10 20 30 60

Upt

ake o

f131I(times103 C

PM)


(c)

SMMC7721

LS174T

SMMC7721LS174T

Inte

nsity

(nM)

50

40

30

20

10

0

0 1 2 4 10 20

01 2 4 10 20

Inte

nsity

(times106)


(d)



(h)1 3 5 9 24 48

Mou

se 1

Mou

se 2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity









(h)

5

9

24

48In

tens

ityIn

tens

ityIn

tens

ityIn

tens

ity

Hea

rtLi

ver

Sple

enLu

ng

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

Smal

lin

testi

ne

(a)

100

2545

Hea

rt

Live

r

Sple

en

Lung

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

5101520

6080

0

Smal

lin

testi

ne

5h9h

24h48h

Inte

nsity

(times108)

(b)


1 3 50(h)

Inte

nsity

SMM

C772

1

2 3

4 5 6 7

1Organtissue (5h)

(a)

COLO

205

Inte

nsity

1 2 3

4 5 6 8

(b)

LS17

4T-C

OLO

205

Inte

nsity

2 3 4

6 8 9

22222 33333333333333333333333333333333333333333333333333333 44

666 88888 99

1 2 3

4 5 6 8 9

(c)



Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Even

ts

10010

110

210

010

110

210

010

110

210

010

110

2

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

IsotypeA33scFv-Fc

(a)


Con

trol

A33

scFv

-Fc

(b)


25

20

15

10

5

0

1 25 5 10 20 30 60

Upt

ake o

f131I(times103 C

PM)


(c)

SMMC7721

LS174T

SMMC7721LS174T

Inte

nsity

(nM)

50

40

30

20

10

0

0 1 2 4 10 20

01 2 4 10 20

Inte

nsity

(times106)


(d)



(h)1 3 5 9 24 48

Mou

se 1

Mou

se 2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity









(h)

5

9

24

48In

tens

ityIn

tens

ityIn

tens

ityIn

tens

ity

Hea

rtLi

ver

Sple

enLu

ng

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

Smal

lin

testi

ne

(a)

100

2545

Hea

rt

Live

r

Sple

en

Lung

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

5101520

6080

0

Smal

lin

testi

ne

5h9h

24h48h

Inte

nsity

(times108)

(b)


1 3 50(h)

Inte

nsity

SMM

C772

1

2 3

4 5 6 7

1Organtissue (5h)

(a)

COLO

205

Inte

nsity

1 2 3

4 5 6 8

(b)

LS17

4T-C

OLO

205

Inte

nsity

2 3 4

6 8 9

22222 33333333333333333333333333333333333333333333333333333 44

666 88888 99

1 2 3

4 5 6 8 9

(c)



Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(h)1 3 5 9 24 48

Mou

se 1

Mou

se 2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity









(h)

5

9

24

48In

tens

ityIn

tens

ityIn

tens

ityIn

tens

ity

Hea

rtLi

ver

Sple

enLu

ng

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

Smal

lin

testi

ne

(a)

100

2545

Hea

rt

Live

r

Sple

en

Lung

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

5101520

6080

0

Smal

lin

testi

ne

5h9h

24h48h

Inte

nsity

(times108)

(b)


1 3 50(h)

Inte

nsity

SMM

C772

1

2 3

4 5 6 7

1Organtissue (5h)

(a)

COLO

205

Inte

nsity

1 2 3

4 5 6 8

(b)

LS17

4T-C

OLO

205

Inte

nsity

2 3 4

6 8 9

22222 33333333333333333333333333333333333333333333333333333 44

666 88888 99

1 2 3

4 5 6 8 9

(c)



Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(h)

5

9

24

48In

tens

ityIn

tens

ityIn

tens

ityIn

tens

ity

Hea

rtLi

ver

Sple

enLu

ng

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

Smal

lin

testi

ne

(a)

100

2545

Hea

rt

Live

r

Sple

en

Lung

Kidn

ey

Panc

reas

Col

on

Brai

n

Mus

cle

Tum

or

Stom

ach

5101520

6080

0

Smal

lin

testi

ne

5h9h

24h48h

Inte

nsity

(times108)

(b)


1 3 50(h)

Inte

nsity

SMM

C772

1

2 3

4 5 6 7

1Organtissue (5h)

(a)

COLO

205

Inte

nsity

1 2 3

4 5 6 8

(b)

LS17

4T-C

OLO

205

Inte

nsity

2 3 4

6 8 9

22222 33333333333333333333333333333333333333333333333333333 44

666 88888 99

1 2 3

4 5 6 8 9

(c)



Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mou

se 1

M

ouse

2

Mou

se 3

Inte

nsity

Inte

nsity

Inte

nsity

(a) (b) (c) (d) (e)






4 Discussion





0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















0

2

4

6

8

10

12

14

008 05 2 5 8 24

Upt

ake r

atio

















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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