Anti-CD24 Antibody Nitric Oxide Conjugate Selectively and ...However, a targeted delivery system for NO donors has remained elusive, creating a bottleneck that limits its druggability

Tumor Biology and Immunology

Anti-CD24 Antibody–Nitric Oxide ConjugateSelectively and Potently Suppresses HepaticCarcinomaFumou Sun1,YangWang1, Xiaojun Luo2, Zhaoxiong Ma1,Yao Xu1, Xinrong Zhang1,Tian Lv2,Yihua Zhang2, Min Wang1, Zhangjian Huang2, and Juan Zhang1

Abstract

Nitric oxide (NO) has a wide range of potential applica-tions in tumor therapy. However, a targeted delivery systemfor NO donors has remained elusive, creating a bottleneckthat limits its druggability. The antibody–drug conjugate(ADC) is a targeted drug delivery system composed of anantibody linked to an active cytotoxic drug. This designmay compensate for the weak targeting ability and variousbiological functions of the NO donor. In this study, wedesigned the NO donor HL-2, which had a targeted, cleaveddisulfide bond and an attachable maleimide terminal. Weconjugated HL-2 with an antibody that targeted CD24through a thioether bond to generate an ADC-like immu-noconjugate, antibody-nitric oxide conjugate (ANC), whichwe named HN-01. HN-01 showed efficient internalization

and significantly increased the release of NO in hepaticcarcinoma cells in vitro. HN-01 induced apoptosis of tumorcells and suppressed tumor growth in hepatic carcinoma-bearing nude mice through antibody-dependent co-toxicity;HN-01 also increased NO levels in tumor cells. Collectively,this study expands the concept of ADC and provides aninnovative NO donor and ANC to address current chal-lenges in targeted delivery of NO. This new inspiration foran ANC design can also be used in future studies for othermolecules with intracellular targets.

Significance: This study is the first to expand the concept ofADC with an antibody-nitric oxide conjugate that suppresseshepatic carcinoma in vitro and in vivo.

IntroductionThe endogenous free radical nitric oxide (NO) is one of the

smallest and extensively distributed signaling messengers thatarbitrates numerous physiologic processes, which include neuro-transmission, immune response, and vasodilation (1–3). Fortumor, NO is a double-edged sword. Commonly, a proper con-centration of NO is preferable for the proliferation of cancer, but avery low intracellular level of NO that is induced by NOS inhi-bition, and high levels of NO released by NO donors, can induceinhibition of cancer cell growth (4–7). However, due to thevarious biological functions of NO in the entire body and dichot-omous effects on cancer biology, the ideal NO-based tumoricidalagents could store efficiently and release therapeutic concentra-tions of NO controllably for the desired period at the target siteswith minimal side effects.

Although many NO-based small molecules were developed asselective and potent anticancer agents, there is increasing interestin developing NO-releasing nanomaterials that include polymer-ic nanoparticles (8–10), dendritic polymers (11), liposomes (12),and silica nanoparticles (13). Compared with classical NOdonors, the NO-releasing nanomaterials load a higher level ofNO onto the high surface area and deliver NO to the desired sites,but it also increases the stability of the NO donor, which avoidsthe side effects of the off-target release of NO. However, someartificial nanoparticle materials could be accumulated in thebody, which would be difficult to excrete after long-term admin-istration. Additionally, the complexity of preparing nanoparticleslimits their druggability.

Antibody–drug conjugates (ADC) display an encouraging ther-apeutic approach for tumor therapy by utilizing antigen-specificmonoclonal antibodies to deliver cytotoxic chemotherapeuticdrugs (14). The FDA-approved brentuximab vedotin and trastu-zumab have validated this design of an "armed" antibody, whichforms the next generation of antitumor antibodies (15–19). Thedevelopment of ADC technology has attracted the attention ofresearchers in the biological and chemical synthesis fields inrecent years, which has resulted in more than 50 ADCs in clinicaltrials (20, 21). Improved conjugation methods have shown aremarkable advantage over traditional techniques; various typesof toxin payloads have emerged, which are crucial componentsof ADCs.

Inspired by the superiority of accurate targeting, internal sta-bility, and appropriate half-life of ADCs compared with nano-particles, we hypothesized that a NO-donor molecule could beconjugated to an antibody and then cleaved inside targeted tumorcells. The combination of the NO donor and antibody might be

1Antibody Engineering Laboratory, School of Life Science & Technology, ChinaPharmaceutical University, Nanjing, China. 2State Key Laboratory of NaturalMedicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases,Center of Drug Discovery, China Pharmaceutical University, Nanjing, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

CorrespondingAuthors: Juan Zhang, China Pharmaceutical University, Nanjing,Jiangsu 210009, China. Phone: 86-25-83271483; E-mail: [email protected];and Zhangjian Huang, E-mail: [email protected]

Cancer Res 2019;79:3395–405

doi: 10.1158/0008-5472.CAN-18-2839

�2019 American Association for Cancer Research.

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beneficial for NOdelivery that is targeted at cancer cells and for itsdruggability. We designed and synthesized a new NO-donorcompound, HL-2, which has the following characteristics: (i) themaleimide terminal reacts readily with the reduced cysteine ofantibodies to form a thioether bond and (ii) the disulfide bond isstable in the circulatory system and can be triggered by highconcentrations of glutathione (GSH) in tumor cells to generatediazeniumdiolate anion by a 1,6-elimination reaction, whichcould release two molecules of NO spontaneously in situ. Inaddition, a cluster of differentiation 24 (CD24) is expressed rarelyin normal cells, but it is overexpressed inmany solid tumors, suchas hepatocellular carcinoma (HCC; refs. 22–24), in which thechemotherapy drugs and targeted drugs have only limited effects.Antibody G7mAb can target the human CD24 specifically, and itcan induce efficient internalization, which is an appropriateantibody for ADC design (25–27). Using this approach, HL-2was conjugated to G7mAb to provide a previously unknownantibody–nitric oxide conjugation (ANC), HN-01. We proposedthat after being injected, HN-01 would exhibit stability in circu-lation and would target CD24þ tumor cells selectively. Afterinternalization into tumor cells that was mediated by CD24,HN-01was trigged in the presence of high concentrations of GSH.Subsequent cleavage of the disulfide bond liberated a diazenium-diolate anion through a 1,6-elimination reaction, which releasedtwo molecules of NO spontaneously in situ; this exerted antipro-liferative and apoptosis-inducing activity to generate anticancereffects synergistically in vitro and in vivo (Fig. 1).

Materials and MethodsHN-01 preparation

Hybridoma technology was used to generate the anti-CD24monoclonal antibody G7mAb. Hybridoma cells were injectedintraperitoneally into mice. About 1 week later, ascites wascollected and purified by a protein G affinity chromatographycolumn (GE Healthcare), as previously performed (25). MouseIgG1 control antibody (Genscript Biotech Corporation, CatalogNo. 18K001612) was used as the isotype antibody. Please refer to

the Supplementary Materials for the method for synthesizingNO donor HL-2. G7mAb was reduced partially with TCEP*HCl(ThermoFisher Scientific) to 1mLG7mAb at 10mg/mL, dissolvedin PBS solution, and 33.8 mL TCEP at 1 mg/mLwas added. This wasfollowed by incubation on ice for 1 hour, and the TCEP wasremoved through Ultrafree-15 centrifugal filter devices (molecu-lar weight cutoff of Mr 30,000; Millipore). The reaction wasstopped and the partially reduced G7mAb was stored in 1 mLPBS that included antioxidants. Next, 47.6 mL HL-2 (10mg/mL inDMSO)was added slowly, then reacted on ice for 30minutes, andthe excess HL-2 was removed by centrifugal ultrafiltration. Theconjugated product HN-01 was filtered through a 0.2 mm filterunder sterile conditions, stored in PBS at�80�C, and identified bySDS-PAGE electrophoresis. Accordingly, an isotype antibody-nitric oxide conjugate (IgG-NO) was generated and used as anisotype control group in subsequent experiments.

Analysis of drug-to-antibody ratioThe drug-to-antibody ratio (DAR) of HN-01 was analyzed by

hydrophobic interaction chromatography (HIC) as previouslyperformed (28). 2.5 mm particle size in a dimension of 4.6 mm�35mmwaspacked in thenonporousTSK-gelButyl-NPRcolumn(Tosoh Bioscience). We equilibrated the column with mobilephase A at 0.5 mL/min flow rate until the baseline (monitoredat 280 nm) was achieved. Then, we injected 10 mL of the HN-01(10 mg/mL) sample and eluted with the gradient mix of mobilephase A (25 mmol/L sodium phosphate, 1.5 M ammoniumsulfate,pH6.95)andmobilephaseB(75%(v/v)aqueous solutionof 25 mmol/L sodium phosphate (pH 6.95) and 25 % (v/v)isopropyl alcohol. The eluted results were detected at 280 nm,and DAR was calculated according to the percentage peak area.

Cell culture and animalsHepatocellular carcinoma BEL-7402, Huh7, and normal

human liver HL-7702 cell lines were bought from CobioerBiosciences Co., Ltd., and preserved in our lab between passages2 and 20. The cells were cultured in DMEM medium that con-tained 10% (v/v) FBS, 5%CO2, 37�C. All cells were authenticatedby short tandem repeat (STR) profiling and examinedMycoplasmaroutinely by Mycoplasma Detection Kit (ThermoFisher Scientific,Catalog No. M7006). The human peripheral bloodmononuclearcells (PBMC) were collected from 15 healthy donors. Writteninformed consents were obtained from donors. The BALB/cnude mice (female) of 5 weeks old were purchased fromthe Comparative Medicine Centre of Yangzhou University(Yangzhou, China). All animals were raised and treatedaccording to the standards of the Comparative Medicine Centreof Yangzhou University.

Flow cytometry assays of HN-01A total of 5 � 105 BEL-7402, Huh7, or HL-7702 cell cells were

suspended in PBS and then incubated with 200 nmol/Lisotype antibody (Genscript Biotech Corporation, Catalog No.18K001612) for 15 minutes, followed by 200 nmol/L G7mAb,HN-01, or IgG-NO at 4�C for 1 hour; the control groups wereincubated with 200 nmol/L isotype antibody. All the cells werecollected and washed three times with PBS. After washing, thecells were stained with FITC conjugated goat anti-mouse IgG(Nanjing Sunbio Technology Co., Catalog No.: L330) in the darkfor 30minutes and detected by flow cytometry (FACSCalibur; BDBiosciences).

Figure 1.

Schematic of action of HN-01. Illustration of the chemical structureand mechanism of therapy for the anti-CD24 antibody-nitric oxideconjugate HN-01.

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Internalization assayA total of 5� 105 BEL-7402 orHuh7 cells were collected in PBS

and then incubated with G7mAb or HN-01 (200 nmol/L eachantibody) on ice for 1 hour, and after incubation, the cells werewashed to remove the unbound antibodies. A group of cellsremained on ice to stop the internalization, and the remainderof the cells was incubated at 37�C for different periods oftime from 8 minutes to 2 hour. Next, all those cells were fixedin 2% paraformaldehyde for 20 minutes and then stainedwith goat anti-mouse IgG, FITC conjugated (Nanjing SunbioTechnology Co.). The mean fluorescent intensity (MFI) wasanalyzed by flow cytometry and the receptor–antibody complexinternalization was calculated as percent MFI loss at 37�C relativeto that on ice.

To observe the dynamic process of internalization, wetested labeled antibodies and cells at different time pointsby a confocal microscope. Cy5 Conjugation Kit (Fast; Abcam,Catalog No.: ab188288) was used to conjugate the HN-01 toCy5 (HN01-Cy5). HL-7702 or Huh7 cells were plated in thecell culture dish. After 24 hours, the cells were covered with300 nmol/L DAPI stain solution (ThermoFisher Scientific,Catalog No. D1306), incubated for 5 minutes, and thenwashed. Cy5-labeled HN-01 were added into cells, incubatedat 37�C for 5 minutes or 1 hour and imaged by a confocalmicroscope.

Measurement of intracellular NO4-Amino-5-(methylamino)-20,70-difluorofluorescein diacetate

(DAF-FM DA; Beyotime) can react with NO in cells to produce afluorescent compound, so we used it as a NO fluorescent probe.Huh7 or BEL-7402 cells were plated in the 96-well plates.When cells reached 85% confluence, they were cocultured with5 mmol/L DAF-FMDA (37�C, 20minutes), washed with PBS, andincubated with HN-01 (100 nmol/L), HL-2 (300 nmol/L), orG7mAb (100 nmol/L) for 8 hours. Flow cytometer was used todetect NO production.

Antibody-dependent cellular cytotoxicityThe Non-Radioactive Cytotoxicity Assay (Promega, Catalog

No.: G1780) was performed to detect the antibody-dependentcellular cytotoxicity (ADCC). PBMCs served as effector cells,BEL-7402 or Huh7 cells were cocultured with various amountsof effector cells in the presence or absence of HL-2, HN-01,or G7mAb (4 hours, 37�C). After centrifugation, 50 mLof the supernatant from the coculture medium was analyzedfor LDH release according to the manufacturer's protocol.The following equation was used to calculate the percentageof cytotoxicity: % Cytotoxicity ¼ 100% � ([Experimental –

Effector Spontaneous – Target Spontaneous]/[Target Maximum– Target Spontaneous]).

Proliferation of hepatocellular carcinoma cellsProliferation and viability of BEL-7402,Huh7, orHL-7702 cells

treated with ANC were detected using an MTT assay (VybrantMTT Cell Viability Assay; ThermoFisher Scientific). Briefly, thecells were plated at 4 � 103/well into 96-well plates overnight.After overnight culture, the cells were washed and then treatedwith different concentrations of HN-01, G7mAb, or HL-2 for72 hours. An MTT assay was performed and the inhibition ratewas reported as inhibition % ¼ (1 � untreated controlcells%) � 100%.

Apoptosis assayBEL-7402, Huh7, or HL-7702 cells were incubated with HN-01

(100or 500nmol/L),HL-2 (300or 1500nmol/L) orG7mAb (100or 500 nmol/L) at 37�C for 48 hours. They were then stained withAnnexin V-FITC and propidium iodide (PI; Sangon Biotech) anddetected by flow cytometer. The percentage of early apoptoticcells (Annexin Vþ/PI�) and the percentage of late apoptoticcells (Annexin Vþ/PIþ) were summed as the total percentage ofapoptotic cells.

Measurement of protein-bound 3-nitrotyrosineNitration can react with protein tyrosine residues or free tyro-

sine nitration to form a stable metabolite of 3-nitrotyrosine(3-NT). The nitration can thus be determined by the detectionof 3-NT. JSK was a cancer drug candidate, which belongs to thediazeniumdiolate class. It can react with glutathione to generatetwomoles ofNOat physiological pH,which has some similaritiesto HL-2 (29, 30). So, JSK was used as the positive control in thisexperiment. BEL-7402, Huh7, or HL-7702 cells were incubatedwith different antibodies or compounds (G7mAb 500 nmol/L,HN-01 500 nmol/L, HL-2 1500 nmol/L, JSK 1500 nmol/L) at37�C for 8 hours. Then, cells were fixed with paraformaldehyde(4%, 20 minutes) and Triton X-100 permeabilized (0.1%,15 minutes). 2 mg/mL anti-3-nitrotyrosine antibody (Abcam,Catalog No. ab110282) was incubated with cells overnight at4�C. Goat anti-mouse IgG, FITC conjugated (Nanjing SunbioTechnology Co., Catalog No. L3302) was added into cells for 1hour. Cells were covered with 300 nmol/L DAPI stain solution(ThermoFisher Scientific, Catalog No. D1306), incubated for 5minutes, and then washed. 3-NT was detected with a laser scan-ning confocal microscope (LSCM). An FV10-ASW 3.0 Viewer wasused to calculate the MFI.

Antitumor efficacy of HN-01 in vivoA hepatocellular carcinoma xenograft model was established

by subcutaneously injecting Huh7 or BEL-7402 cells (1 � 107)into right flanks of BALB/c nude mice (Yangzhou UniversityComparative Medicine Centre Yangzhou, China). When the aver-age tumor volume reached 100 mm3, tumor-bearing mice weredivided randomly into seven groups (six mice/group) and treatedwith HN-01(L) (2.5 mg/kg), HN-01(M) (5 mg/kg), HN-01(H)(10 mg/kg), G7mAb (10 mg/kg), HL-2 (0.25 mg/kg),G7mAbþHL-2, or saline intravenously every 3 days. Each in vivoexperiment was repeated three times. Tumor growth was mea-sured using a digital caliper periodically and survival data ofmice was recorded. And the tumor volume was calculated asV ¼ (length � width2)/2. All mice were sacrificed on day 33 forthe collection of their tumors.

Western blotting for cytochrome cBEL-7402 tumor tissue from mice was minced on ice in lysis

buffer that included phosphatase inhibitors, protease inhibitors,and 100 mmol/L PMSF, and tissues were homogenized to collectthemitochondrial proteins. The protein samples were detected byWestern blotting. Anti-cytochrome c (Cyt c) antibody (ab133504)was used as the first antibody. Images were taken using theChemiDoc XRS system (Bio-Rad).

Statistical analysisThe data were calculated by GraphPad Prism software and

expressed as means � SD. The level of significance was estimated

Antibody-Nitric Oxide Conjugate Suppresses Hepatic Carcinoma

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using the Student t test, and P values of 0.05 or less wereconsidered to be statistically significant.

ResultsChemistry

Target compounds 1�7 were synthesized (Fig. 2A). Diaze-niumdiolates, which is an important class of NO donor, canliberate two molecules of NO spontaneously at physiologic con-ditions with a range of half-lives from a few seconds to severalminutes. Nevertheless, the O2-derived diazeniumdiolates gener-ated stable precursors that were cleaved enzymatically in tumorcells to produce a diazeniumdiolate anion and, subsequently, torelease NO in situ, which exhibited potent and selective antipro-liferative activity. The 1,6-elimination-based linker is often usedin self-immolative prodrug designs, and the triggers at the paraposition of the benzyl group supply various strategies to liberatethe active drug from the prodrug molecule, which include dia-zeniumdiolates O2-anion moieties. Disulfide linkage, which istolerant in blood plasma and sensitive in a tumor microenviron-ment, has been usedwidely as a potential strategy in the discoveryof drugs that target tumors. In this regard, we used 1,2-bis(4-methylphenyl) disulfane as a redox-triggered linker to conjugateN,N-diethylamino)diazen-1-ium-1,2-diolate with an antibody.The disulfide linkages were broken in the high GSH tumor

microenvironment, and the diazeniumdiolates subsequentlyreleased large amounts of NO in situ (Fig. 2B).

Preparation and identification of HN-01The free thiol group of the partially reduced antibody reacted

with the maleimide of HL-2, which generated HN-01. Then, HN-01 was confirmed by SDS-PAGE electrophoresis (Fig. 3A). TheSDS-PAGE analysis showed that G7mAb andHN-01 were similarwith the molecular weight of 150KD, because the molecularweight ofHL-2 is only 588Da, and the purity of G7mAbdecreasedslightly after being reduced by TCEP.

Detection of DARHICwas used to analyze the drug-loaded species ofHN-01. The

addition of hydrophobic HL-2 to the G7mAb increased its hydro-phobicity. Elution with a gradient of high concentration of a saltsolution and an increasing organic modifier changed the columnretention, which resulted in drug-loaded species with the leasthydrophobic, unconjugated form to be eluted first and the mosthydrophobic, 4-drug form to be eluted last (Fig. 3B). Based on theHIC results, the percentage in the peak area represented therelative proportion of a particular drug-loaded format, andG7mAb conjugated with two drug-loaded forms was the maincomponent. The average DAR was 3.327, which was calculatedfrom the formula DAR ¼ S(weighted peak area)/100.

Figure 2.

The synthetic and degradationroute of HN-01. A, The syntheticroute for HN-01. Reagents andconditions: a,NaHCO3, H2O,20minutes; b, NBS, benzene, roomtemperature, 6 hours, thenNBS/AIBN, benzene/reflux, 80�C,12 hours; c, diazeniumdiolates, DMF,N2, 0�C, 4 hours; d, compound 2,acetone/DMF (2:1), N2, 40�C, 3.5hours; and e, G7mAb, DMSO, TCEP,room temperature, 2 hours anddegradation route of HN-01. B, GSHtriggered NO release from anti-CD24 antibody-nitric oxideconjugate HN-01.

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In vitro selectivity of HN-01A significantly overexpressed CD 24 was detected by western

blot assay in Huh7 and BEL-7402 cell lines (Supplementary Fig.S1), and the cells were then pre-incubated with G7mAb, HN-01, or IgG-NO and stained with a fluorescent antibody. Theselective binding capacity of HN-01 to the CD24 overexpressingtumor cell lines or a normal liver cell line was performed byflow cytometry (Fig. 3C). The specific percentages of binding ofHN-01 to BEL-7402 (77.0%) and Huh7 (90.8%) wereobtained, but there was no binding with normal liver cellHL-7702. In addition, isotype control IgG-NO showed nobinding activity to BEL-7402/Huh7 cells (less than 4%). Thepercentages of binding of HN-01 was nearly the same withnaked G7mAb, which indicated that HN-01 retained the highspecific binding capacity of G7mAb to hepatic carcinomacell lines.

Internalization assayTo examine whether HN-01 induced the CD24 receptors

on the member surface to mediate internalization, we treatedBEL-7402 or Huh7 cells with G7mAb or HN-01 and thendetected the cell surface level of the antibody by flow cyto-metry. HN-01 or G7mAb elicited a high level of receptor-

mediated internalization, and the speed of internalizationwas faster in Huh7 than in BEL-7402 (Fig. 4A,i and ii).Conjugation with HL-2 was less significant to the internali-zation capacity that was induced by G7mAb. To furtherillustrate that the dynamic process of internalization wasinduced by HN-01, we used a confocal microscope. In thebeginning, ANC was gathered around the edge of the cellmembrane. After 1 hour of incubation, ANC internalizedinto the Huh7 cells and abundant HN-01 was located in thecytoplasm of the CD24-overexpressed tumor cells (Fig. 4B,i).However, ANC showed no internalization into the humannormal liver cell HL-7702 (Fig. 4B,ii).

NO releaseThe intracellular NO release by HN-01 in Huh7 or BEL-7402

cells was detected byDAF-FMDA (31). Comparedwith theMFI ofthe control group (125.5k) and G7mAb group (127.5k), MFI ofHN-01 was 313.0k and was higher than the HL-2 group (298.0k)in Huh7 cells. Similar results were seen in BEL-7402 cells. HN-01produced more NO than HL-2 in Huh7 and BEL-7402 cells. Insharp contrast, almost no NO release was detected in Huh7 andBEL-7402 cells treated with G7mAb, because it was not linkedwith HL-2 (Fig. 4C and D).

Figure 3.

Characterization of HN-01.A, Analysis of gradient SDS-PAGE(4%–12%) that shows one majorband of HN-01 under nonreducingconditions. Lane 1, G7mAb; lane 2,HN-01; lane 3, marker. B, HICanalysis of G7mAb (i) and HN-01 (ii)on a butyl-NPR column yielded fourdominant peaks that correspondedto antibodies that contained two,four, six, and eight drug molecules.DAR ratio was two, four, six, andeight. Illustration of ANCs withdifferent drug load distributions isshown on the right side. C,Analyzing the specific bindingaffinity of HN-01 with native CD24that was expressed on Huh7/BEL-7402 cells. HN-01 maintained highaffinity with CD24-positive Huh7and BEL-7402 cells; thepercentages of binding were 90.8%and 77.0%, respectively. However,both showed no binding with thehuman normal liver cells HL-7702.The isotype control IgG-NO showedno binding with BEL-7402/Huh7cells (less than 4%).






Antibody-dependent cellular cytotoxicityTo detect the ability of the Fc fragment of HN-01 to induce lysis

of effector cell-mediated target cells, an ADCC study on BEL-7402or Huh7 cells was performed, as described previously (32).Approximately 12.26% and 11.16% of the BEL-7402 and Huh7cells, respectively, were lysed when exposed to an effector cell:target cell (E:T) ratio of 30:1 with 100 mg/mL HN-01. When theE:T ratio was 100:1, HN-01 triggered 21.75% lysis of BEL-7402and 26.81% of Huh7 cells. Moreover, the lysis rate of G7mAbgroups was higher than the corresponding groups of HN-01(Fig. 5A). Overall, data indicated that HN-01 retained theFc-mediated ADCC effect.

HN-01 inhibited proliferationThe selective antiproliferative activity of HN-01 was evaluated

based on CD24-positive cell lines and normal liver cell linesby a 72-hour MTT assay. HL-2 (3,000 nmol/L) and G7mAb(1,000 nmol/L) showed a slight inhibition of Huh7 cell lines(38.2% and 24.8%, respectively). However, HN-01 exhibitedsignificant antiproliferative activity on Huh7 in a dose- andtime-dependent manner (60.4%; Fig. 5B,i). The results were

consistent with that of BEL-7402 cells (Fig. 5B,ii). HL-2 exhibitedindiscriminate anti-proliferative activity on HL-7702 (inhibition%:34.5% in 1,000 nmol/L; Fig. 5B,iii). In contrast, HN-01 didnot affect the proliferation of normal cell line HL-7702,which suggested that HN01 exhibited selective cytotoxicityon target cells. Meanwhile, compared with control group,few colonies were observed following treatment with 50nmol/L HN-01 for a 7-day colony formation experiment(Supplementary Fig. S2).

HN-01 promoted apoptosisTo explore the reason for inhibition of proliferation on

BEL-7402/Huh7/HL-7702 cells, apoptosis assay was performed.Basically, the cells were treated with different concentrations ofHN-01, G7mAb, or HL-2 for 48 hours and examined by flowcytometry (Fig. 5C and D). At 500 nmol/L, the apoptosis rates ofHN-01 treated Huh7 and BEL-7402 cells were 21.43% and25.81%, respectively, whichwas significantly increased comparedwith the control group. As a contrast, no obvious apoptosis wasfound in HN-01 treated HL-7702 cells, whereas significantlyincreased apoptosis was detected in HL-2 treated HL-7702 cells

Figure 4.

Internalization and NO release invitro.A, Tumor cell internalizationassay. HN-01 and G7mAbwereinternalized into Huh7 cells in <30minutes, and the percentages ofinternalization stabilized at 60minutes. HN-01 and G7mAbinternalized into BEL-7402 cells in<60minutes, and the percentagesof internalization stabilized at 90minutes. B, Laser confocalfluorescence microscopy images ofHuh7 or HL-7702 cells incubatedwith the HN01-Cy5 and DAPI in 5minutes and 1 hour. C and D, Assayof NO release. NO released in Huh7or BEL-7402 cells was determinedby DAF-FM DA. The relative MFI ofDAF-FM DA indicates theconcentration of NO released byeach group. The data shown hereare representative of three differentexperiments, and data arepresented as the mean� SD(n¼ 3). �� , P < 0.01; �� , P < 0.001;ns, no significance.

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(Supplementary Fig. S3). These results proved that HL-2 non-targeted apoptosis in normal cells. With antibody conjugation,HN01 improved the targeted toxicity of NO donor part.

HN-01 nitrated mitochondrial proteins of hepatocellularcarcinoma cells

NO can react with reactive oxygen species to form reactivenitrogen species, which can lead to nitration of the tyrosineresidue at position 3 in mitochondrial protein Cyt c, forming a

nitrated Cyt c (3-NT Cyt c; refs. 33–36). 3-NT was used as anextensive measure of the content of nitrated protein (34). HN-01released a relatively large amount of NO and exerted an effectiveanti-proliferative effect. Compared with the MFI of the G7mAb-treated group (137.0 � 29.8) and HL-2 group (393.5.0 � 98.8),HN-01 treatment generated relatively high levels of fluorescence(700.7 � 138.8), which meant that the HN-01 group generatedrelatively high levels of mitochondrial 3-NT in Huh7 cells. Thepositive control JSK treatment led to higher levels of 3-NT than

Figure 5.

The antihepatic carcinoma effect of HN-01 in vitro. A, HN-01 enhanced cytotoxicity of PBMCs. HN-01 maintained the ADCC in vitro. Huh7 or BEL-7402 cells wereused as target cells, and PBMCswere used as effect cells. Cell lysis remained essentially unchanged after treatment with HN-01 compared with G7mAb. B, Thecell viability was assessed by MTT assay at 72 hours after treatment with HN-01, G7mAb, or HL-2 (each at 0.8–1000 nmol/L). Cell death induced by HN-01,G7mAb, or HL-2 was plotted relative to the viability of untreated controls set at 100%. Data are given as mean� SD (n¼ 3). C,i and D,i, HN-01 induced apoptosisof Huh7 and BEL-7402 cells. Huh7 or BEL-7402 cells were treated separately with G7mAb (100 or 500 nmol/L), HL-2(300 or 1500 nmol/L), and HN-01 (100 or500 nmol/L) for 48 hours and then analyzed by flow cytometry after staining with Annexin V-FITC and PI. The percentage of cells in each quadrant is indicated.C,ii andD,ii, Quantitative analysis of apoptosis assay. Data are presented as the mean� SD (n¼ 3). � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ns, no significance.






that of HN-01 (Fig. 6A,i), which was similar in BEL-7402 cells(Fig. 6A,ii). Consistent data were presented in BEL-7402 cells(Fig. 6A,ii).However, nomitochondrial 3-NTwas detected inHN-01-treated HL-7702 cells, although significantly increased mito-chondrial 3-NT was generated in HL-2 and JSK-treated HL-7702cells. Overall, the data proved that without targeted delivery, thesmall-molecules like HL-2 and JSK led to nonspecific toxicity.

Antitumor efficacy of HN-01 in tumor-bearing miceBEL-7402 cells were inoculated in the armpit of BALB/c nude

mice subcutaneously, and tumor volumes were measured duringthe treatment. Tumor growth in the high dose HN-01-treated

group was significantly more inhibited than in the control group(P < 0.0001) and the G7mAb group (P¼ 0.0054; Fig. 6B,i). At theend of the treatment, the growth of tumors was inhibited inHN-01(H) group (81.15%). Middle doses and low doses ofHN-01 reduced tumor burdens by 71.4% and 48.96%, respec-tively, althoughG7mAbþHL-2 groups (36.65%)didnot display aprominent anti-tumor effect in the tumor-bearing model(Fig. 6B). To further illustrate the antitumor efficacy in vivo, wecomparedHN-01 treatment groupswith its nakedG7mAb groupsand HL-2 groups, which reduced tumor burdens by 33.43% and19.5% at comparable doses, respectively. These results suggestedthat HN-01 targeted tumor cells and released NO after being

Figure 6.

The antihepatic carcinoma effect of HN-01 in vivo and the preliminary approach to mechanism.A,Measurement of protein-bound 3-NT. Confocal microscopyimages of the protein-bound 3-NT in Huh7/BEL-7402/HL-7702 cells with treatments of G7mAb (500 nmol/L), HN-01 (500 nmol/L), HL-2 (1500 nmol/L), and JSK(1500 nmol/L). An FV10-ASW 3.0 viewer was used to calculate the MFI. B, The anti-HCC efficacy of HN-01 in BEL-7402 xenografted mice. B,i, Tumor growthcurves of each group under different treatments. Treatment of tumor-bearing mice with low-dose HN-01 (2.5 mg/kg), marked as HN-01(L); middle-dose HN-01(5 mg/kg), marked as HN-01(M); high-dose HN-01 (10 mg/kg), marked as HN-01(H); G7mAb (10 mg/kg); HL-2 (0.25 mg/kg); and G7mAbþHL-2; or saline, and thetumor volume was measured when the average tumor volume reached 100mm3. B,ii, Tumor inhibition rates of different groups. Data are given as the mean� SD(n¼ 6). B,iii, The survival of BEL-7402-bearing mice after a 60-day tumor challenge in each group. HN-01 group had better survival compared with G7mAb orHL-2. C,Western blotting for Cyt c. Proteins were extracted from the tumors of different groups of mice. Cyt c levels were measured byWestern blot assay.Lane 1, HN-01(H); lane 2, HN-01 (M); lane 3, HN-01 (L); lane 4, Control; lane 5, HN-01 (H); lane 6, HN-01 (L); lane 7, G7mAbþNO-Donor; lane 8, NO-Donor; lane 9,G7mAb. Data were given as the mean� SD (n¼ 6). � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ns, no significance.

Sun et al.





internalized, and it exhibited better antitumor efficacy comparedwith all control groups. Furthermore, BEL-7402 cells bearingmicetreated with HN-01 showed longer survival than those treatedwith G7mAb and HL-2. These results suggested that HN-01showed better antitumor efficacy compared with G7mAb andHL-2 (Fig. 6B). In Huh7 cells bearing mouse model, HN-01 alsowas superior to the treatment with G7mAb and HL-2. (Supple-mentary Fig. S4).

Meanwhile, the immunohistochemistry staining was used todetect the effect of HN-01 on the cellular proliferation markerKi67 and apoptosis marker cleaved caspase-3 (CC-3). After treat-ment with HN-01, a significant increase in CC-3 and a decrease inKi67 levels were observed, which indicated that ANC inhibitedproliferation of and induced apoptosis in tumor cells (Supple-mentary Fig. S5). The near-infrared (NIR) imaging results indi-cated that HN-01 effectively targeted CD24þ HCC cells in vivo.(Supplementary Fig. S6A).

Assay of cytochrome c in tumor tissuesThe effect of HN-01 on the levels of Cyt c in tumor tissues was

analyzed by Western blot analysis. The relative levels of Cyt c inthe tumor tissues of mice that had been treated with HN-01 weredose-dependent, and they were significantly higher than that inthe control group, G7mAb group, G7mAb cotreatment withHL-2group and HL-2 group (Fig. 6C). The results showed that NOreleased by HN-01 may be the cause of the nitration, whichexerted strong inhibitory activity on HCC cells in vivo.

DiscussionAs a gaseous signaling molecule, NO plays a significant role in

regulating the functions of cardiovascular, neurological, andimmune systems (37).Meanwhile, the functions of NO in tumor-igenesis anddevelopment have gained increasing attention. Avail-able evidence suggests that relatively high levels of NO couldact as a cytotoxic and apoptosis-inducing agent against tumorcells (6, 7, 37–39). A number of GSTp-activatable O2- (sulfony-lethyl derived) diazeniumdiolates were generated as the NOdonors (40, 41). Even though these compounds had a goodtumor-suppressing effect, their side effects were simultaneousand unavoidable due to nontargeted delivery and various bio-logical functions (42). This problem is a significant bottleneckthat blocks their future use. There is an urgent need for a targeteddrug delivery system to address these clinical limitations.

Historically, solid tumors are treated mostly through chemo-therapy or radiotherapy.However, these nontargeting therapeuticoptions are usually associatedwithmany adverse effects. In recentyears, monoclonal antibody drugs have been developed in thefield of cancer therapy. The therapeutic antibody is representativeof a novel targeted drug with high target recognition, affinity, andinternalization rate. However, many monoclonal antibodies facethe problem of limited targeting ability and limited therapeuticefficacy. To better focus the targeted function of antibodies, we useantibodies as biological missiles specifically to deliver cytotoxinsto tumor cells with lower toxicity and a higher therapeuticwindow. Antibody drug conjugates are representative of noveltargeted drug delivery systems that use an antibody as a vehicle,and they have become one of the best alternatives in cancerimmunotherapy (43, 44). CD24 is a membrane protein that islocalized in lipid membrane raft domains (24, 45, 46). It is also areceptor that canmediate antibody internalization (46, 47).CD24

is known as a hepatocellular carcinoma stem cell marker and isupregulated in chemoresistant residual HCC (24). Previously, wedeveloped the anti-CD24 antibody G7mAb, which selectivelytargeted HCC in vitro and in vivo (25, 48). What's more, previousstudies showed that NO sensitize HCC to chemotherapeuticcompounds by nitrosylation of critical thiols in DNA repairenzymes. The increased expression of p53 gene familymembers played an important role in the induction of extrinsicand intrinsic pathways to cell death induced by chemotherapy inHCC (49).

Combining the antitumor effects ofNOdonors andG7mAbonhepatic carcinoma, we generated the antibody-nitric oxide con-jugate HN-01 using a thioether bond to combine NO-donatingHL-2 to G7mAb. By the specific targeting of G7mAb, the toxinmolecules were enriched on the surface of tumor cells, whichimproved the therapeutic index of toxic molecules and reducedthe toxic and side effects on normal tissues.

One hundredmilligram of G7mAbwas prepared throughmiceceliac inoculation. We conjugated the NO-donating HL-2 thatcontained a disulfide bond and maleimide with G7mAb togenerate HN-01, and we obtained 16.8 mg ADC with 56.0%recoveries of G7mAb. An average DAR 3.327 was determined byHIC, which was consistent with the expected coupling of two tofour toxins per antibody (Fig. 3B). The specific binding of HN-01to CD24þ Huh7 and BEL-7402 cells was analyzed by flowcytometry (Fig. 3C). Except the in vitro targeting, our results alsoshowed thatHN-01 specifically targeted toHCCxenografts in vivo.Moreover, the binding of HN-01 to tumor was blocked by freeHN-01. That suggested thatHN-01 has specificity to CD24þHCC,which might result in higher antitumor efficacy with fewer sideeffects (Supplementary Fig. S6).

The endocytosis and endocytic activity of HN-01 to target cellswere examined using flow cytometry and a LSCM. HN-01 hadequivalent endocytosis with its parental G7mAb, and both ofthem were internalized into target cells effectively. The effectorcell-mediated cytotoxicity that was induced by the Fc fragmentwas detected using human PBMCs as effector cells and Huh7 orBEL-7402 cells as target cells. HN-01 had similar specificity and anADCC effect similar to that of naked G7mAb (Fig. 5A). Moreover,theMTT assay suggested thatHN-01 exhibited significantly higherantiproliferative effects in a dose-dependent manner on targetcells compared with G7mAb or HL-2 alone (Fig. 5B). Further-more, the FCM-based apoptotic assay suggested that the cytotox-icity was partially due to proapoptotic activity of the NO donor(Fig. 5C and D). In vitro studies showed that HN-01 agreed withthe essential requirements of ADC, and the antitumor activity ofHN-01 increased significantly, which was consistent with theexpected results.

Finally, BEL-7402-bearing and Huh7-bearing nude mice mod-els were established to evaluate the antitumor activity of HN-01in vivo, and tumor volume of HCC-bearing nude mice wasmeasured after treatment with HN-01. As anticipated, HN-01inhibited the growth of xenografted tumors significantly anddurable and significantly prolonged the survival of tumor-bearingmice (Fig. 6B). In addition, the decrease of Ki67 and increase ofCC3 (Supplementary Fig. S5) indicated that HN-01-mediatedtumor suppression was associated with inhibition of tumor cellproliferation and increased cell apoptosis. NO inhibited cellmitochondrial respiration and caused Cyt c release into thecytosol. The caspase activation cascade was triggered when Cytc was released into the cytosol and led to cell death. Following






HN-01 treatment, the expression of Cyt c in tumor tissues wasmonitored by Western blot analysis. The results demonstratedthat HN-01 increased the amount of Cyt c in the cytoplasm oftumor cells (Fig. 6C), which proved that NO played a role in theinhibition of themitochondrial respiratory chain and induced therelease of Cyt c into cytoplasm.What'smore, the appropriate half-life of HN-01 (Supplementary Fig. S6B) and low toxicity (Sup-plementary Fig. S7) laid the foundation for further developmentof the drug.

In this study, we created the first generation of ANC, which is anew tumor-targeted NO delivery system, which we named HN-01. Basically, HN-01 maintained specific binding ability, theADCC effect, and endocytosis efficiency. In addition, HN-01showedmore effective antitumor activity than either component,G7mAb or HL-2, in vivo. In conclusion, HN-01 combined fullytargeted affinity of antibodies with a NO donor to achieve super-ior efficiency, which provides a new approach for the treatment ofCD24þ malignant tumors.

Finally, the antibody-nitric oxide conjugate design, which usedG7mAb coupled with HL-2, expands the concept of ADCs to thepoint that they are no longer limited to highly toxic, small-molecule drugs. Instead, gas drugs such as NO can be targetedand delivered accurately and, therefore, they can be used fortherapeutic purposes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Z. Ma, X. Zhang, M. Wang, Z. Huang, J. ZhangDevelopment of methodology: F. Sun, Y. Wang, X. Luo, Z. Ma, Y. Xu, T. LvAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Sun, Y. Wang, Y. Xu, X. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Sun, Y. Wang, X. Luo, Z. Ma, Y. Xu, X. Zhang, T. Lv,Z. HuangWriting, review, and/or revision of the manuscript: F. Sun, Y. Wang, Y. Xu,Z. Huang, J. ZhangStudy supervision: Y. Zhang, M. Wang, Z. Huang, J. Zhang

AcknowledgmentsThis study was supported by grants from the National Natural Science

Foundation of China (Grant Nos. 81473125, 81822041, 81673305, and81773573); Natural Science Foundation of Jiangsu Province (BK20161459);Jiangsu Province Funds for Distinguished Young Scientists (Grant No.BK20160033); Jiangsu Province Qinglan Project (2014); and "Double First-Class" University project (CPU2018PZQ12 and CPU2018GY14). We thankThomas A. Gavin, Professor Emeritus, Cornell University, for help with editingthis paper. We thank Prof. Hua He, China Pharmaceutical University, fortechnical assistance of pharmacokinetics. We thank Prof. Yueqing Gu, ChinaPharmaceutical University, for technical assistance of near-infrared imaging.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 10, 2018; revised February 4, 2019; accepted March 22,2019; published first March 27, 2019.

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2019;79:3395-3405. Published OnlineFirst March 27, 2019.Cancer Res Fumou Sun, Yang Wang, Xiaojun Luo, et al. Potently Suppresses Hepatic Carcinoma

Nitric Oxide Conjugate Selectively and−Anti-CD24 Antibody

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Anti-CD24 Antibody Nitric Oxide Conjugate Selectively and ...However, a targeted delivery system for NO donors has remained elusive, creating a bottleneck that limits its druggability