A Human Fab-Based Immunoconjugate Specific for the LMP1 Extracellular Domain Inhibits

Author Manuscript Published OnlineFirst on December 14, 2011; DOI: 10.1158/1535-7163.MCT-11-0725 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A human Fab-based immunoconjugate specific for the LMP1 extracellular domain inhibits

nasopharyngeal carcinoma growth in vitro and in vivo

Chen Renjie 1,2, Zhang Dawei 2, Mao Yuan 3, Zhu Jin 1,4, Ming Hao 5, Wen juan 2, Ma jun 6, Cao qing 2, Lin Hong 1,

Tang Qi 1, Liang jie 1 , Feng, Zhenqing 1,*

1 The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of

Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, China

2 Department of otolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University,

Nanjing, China

3 Department of otolaryngology Head and Neck Surgery, Jiangsu Provincial Official Hospital, Nanjing, China

4 Huadong Medical Institute of Biotechniques, Nanjing, China

5 Department of otolaryngology Head and Neck Surgery, Xuzhou Center Hospital Xuzhou, China

6 Department of otolaryngology Head and Neck Surgery, Yiji Shang Hospital of Wannan Medical College, Anhui,

China

*Corresponding Author’s Information: E-mail: [email protected] Telephone: +86-25-86863100 Fax

numbers: +86-25-86663193 Institution: The Key Laboratory of Cancer Biomarkers, Prevention & Treatment Cancer

Center and The Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University

Address:No.140 Hanzhong road, City: Nanjing State: Jiangsu Province Zip Code:210009 Country: China

Running title: Fab-based immunoconjugate specific for LMP1

Key words: Immunoconjugate, Antibody, Mitomycin C (MMC), Latent membrane protein 1 (LMP1), Nasopharyngeal carcinoma (NPC).

Downloaded from mct.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 14, 2011; DOI: 10.1158/1535-7163.MCT-11-0725 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Nasopharyngeal carcinoma (NPC) is a major cause of cancer-related death in Southeast Asia and in China. Metastasis

and relapse are the primary cause of morbidity and mortality in NPC. Recent evidence suggests that the EBV Latent

Membrane Protein 1 (LMP1) is exclusively expressed in most NPC and is a potential target for bio-therapy. In this study,

we successfully prepared a novel human antibody Fab (HLEAFab) agaisnt LMP1 extracellular domain which was

subsequently conjugated with mitomycin C (MMC), thus forming an immunoconjugate (HLEAFab-MMC). The effects

of HLEAFab-MMC on proliferation and apoptosis in NPC cell lines HNE2/LMP1 and the inhibition rate of growth of

NPC xenografts in nude mouse were examined. The inhibition rate of HNE2/LMP1 cell proliferation was the highest for

HLEAFab-MMC (76%) compared with MMC (31%) and HLEAFab (22%) (P < 0.05) at a concentration of 200 nmol/L

and showed dose-dependent fashion. The apoptosis rate of HNE2/LMP1 cell lines was 13.88% in HLEAFab-MMC

group, 3.04% in MMC group, 2.78% in HLEAFab group and 2.10% in negative control group at the same concentration

respectively. In vivo, the inhibition rate of growth of NPC xenografts in nude mouse was 55.1% in HLEAFab-MMC

group, 26.5% in MMC group and 5.64% in HLEAFab group (P < 0.05). In summary, our findings demonstrate that

HLEAFab-MMC is a unique immunoconjugate with potentials as a novel therapeutic agent in the treatment of

LMP1-expressing NPC.

Introduction

Epstein-Barr virus (EBV) is a herpes virus prevalently associated with a variety of malignant diseases, such as

nasopharyngeal carcinoma (NPC), Burkitt's lymphoma, Hodgkin’s lymphoma and some gastric carcinomas (1). An

association of EBV with NPC has been consistently established by numerous studies. Various prints of EBV have been

found in the tissues of NPC and antibodies against EBV present in NPC patients’ serum. In NPC, most tumor cells are

latently infected and express three latent membrane proteins: latent membrane protein 1 (LMP1), latent membrane

protein 2A (LMP2A), latent membrane protein 2B (LMP2B), and EBV nuclear antigen 1 (EBNA1). Among these, LMP1

and EBNA1 have been detected in NPC biopsies by Western Blotting analysis. LMP1 is considered to be the major EBV

oncoprotein (2). Therefore, both EBNA1 and LMP1 are important tumor-specific markers for NPC. Furthermore, LMP1

positive NPCs are reported to be more progressive than LMP1 negative NPCs and show an increasing tendency of lymph

node metastasis (3). Structurally, LMP1 is an integral membrane protein consisting of a short cytoplasmic N-terminus of

20 amino acids, a trans-membrane domain with six membrane-spanning segments that anchor LMP1 in a patchy

distribution along the plasma membrane, and a long cytoplasmic C-terminus of 200 amino acids (4, 5, 6).

In this study, we chose the extracellular domains of the oncogenic LMP1 as a target of therapy. We had screened a

fully human naïve Fab phage library after panning against extra-membrane domains (EMDs) polypeptide, which consists

of three extra-membrane domains proximal 16 amino acid, by panning on living cells and immobilized LMP1 (7),

followed by characterizing its binding specificity and affinity to native LMP1 as well as the NPC cell line

over-expressing LMP1. Using this strategy, we demonstrated that this human anti-LMP1 EMDs antibody Fab fragment

(HLEAFab) can bind to the extracellular domains of LMP1. We then conjugated this HLEAFab with mitomycin C

(MMC), a chemotherapeutic drug, to generate a potential immunoconjugate agent. This study is the first to clone, express

and functionally validate the HLEAFab targeting the LMP1. The immunoconjugate, which consists of a Fab antibody

fragment derived from a fully human Fab phage library and MMC, killed LMP1-positive NPC cell lines in vitro and

suppressed NPC growth in a nude murine transplantation model.

Materials and Methods

Phage library, helper phage and bacterial strains

A human naive Fab phage library was constructed as previously described (8). Before the first-round panning, the

library was titrated and 2×1013 phage clones were collected for panning. The VCSM13 helper phage and the E.coli strain

Xl1.blue and another E.coli strain, Top 10 F’, for expressing the Fab, were saved and provided by The Key Laboratory of

Cancer Biomarkers, Prevention & Treatment Cancer Center and The Key Laboratory of Antibody Technique of Ministry

of Health, Nanjing Medical University, Nanjing, China. Both strains were tested to preclude any wild-type phage

contamination.

Cell lines and purified extra-membrane domains of LMP1

Two cell lines were used for biopanning and Fab characterization as well as for in vitro bioassays: HNE2 is a

EBV-LMP1-negative human nasopharyngeal carcinoma (NPC) cell line, HNE2/LMP1 is a cell line constantly expressing

LMP1 after the introduction of full-length LMP1 cDNA into HNE2 cells purchased from Xiang Ya Central Experiment

Laboratory, Hulan, China and were authenticated in this laboratory. The initial characterization of HNE2 and

HNE2/LMP1 cells has previously been described (9). Based on PCR and Western blotting analyses for LMP1,

HNE2/LMP1 cells are LMP1 positive whereas HNE2 cells are EBV DNA negative. Both cell lines were cultured in a

RPMI-1640 medium (GIBCO® Invitrogen,USA) with 10% FBS, antibiotics. All of the cell lines are routinely screened

for Mycoplasma species (GenProbe detection kit, Fisher, Itasca, IL). All cells were maintained at 37°C in a humidified

atmosphere of 95% air and 5% CO2. For the experiments, HNE2 and HNE2/LMP1 were used between passages 3 to

7.The extra-membrane domains of LMP1 and GST fusion protein, which was prepared by our research team previously

(7), were affinity purified from Glutathione Sepharose 4B (GE product code:27-4574-01). The eluted LMP1 was verified

by Western Blotting and quantified by BCA protein assay (Bio-Rad).

Bio-panning methods

To isolate phage that specifically bind to LMP1 on the cell surface, subtractive panning was performed on both HNE2

cell line and HNE2/LMP1 cell line, as well as on immobilized LMP1 extracellular domains. 7 rounds of panning were

performed. The 1st, 2nd, 3rd and 6th rounds of panning were performed on the cells, and one million cells were used in

each round. The cells were starved in serum free medium for 12 h, washed twice using pH 7.4 PBS, detached from the

flask with cell dissociation buffer (Invitrogen, Carlsbad, California, USA，Cat:13151-014 ) at 37°C for 10 minutes, and

washed twice in PBS. LMP1 negative HNE2 cells were resuspended and mixed with 1013 phagemids in 1 ml of 1%

BSA-PBS, shaken at 37°C for 30 minutes, and centrifuged at 5000 × g for 3 minutes. The supernatant was collected and

mixed with HNE2/LMP1 and then incubated at 37°C for 1 h. The cells were washed with RPMI-1640 and treated with 3

ml Trypsin-EDTA (Gibco, 25300.054) at 37°C for 5 minutes. The phages were used to infect E.coli. Xl1-blue for amplification and next-round panning. To increase binding specificity as well as affinity to LMP1, the 4th, 5th and 7th rounds of panning were performed on immobilized LMP1, and the amount of coated LMP1 was gradient decreased in each of the three rounds (400ng for 4th round, 200ng for 5th round, 100ng for 7th round).

ELISA screening of LMP1-binding-positive phage clones

Single phage clones from the E.coli Xl1-blue infected by the 7th round of eluted phage were picked up and grown in 1

ml super broth (SB) medium containing 50 mg/ml carbenicillin and 1% glucose, with shaking at 37°C until the

exponential phase. VCSM13 helper phage (1 × 109) was added to each vial. The culture was shaken overnight with 70

μg/ml kanamycin. Fifty microliters of supernatant from each vial was added to each well of 96-well EIA plates coated

with 100ng GST-LMP1 fusion protein that had been pre-blocked with 5% milk blocking buffer (5% milk, 0.5%

Tween-PBS). Purified GST protein was used as negative control. After incubation at RT for 1 h, the plate was washed using 0.5% Tween-PBS; 50 μl of 1:4000 diluted HRP conjugated anti M13 antibody (Amersham code number:

27-942-01) in blocking buffer was added to each well and incubated for another hour, and then the plate was washed again. Fifty microliters of HRP substrate solution (Pierce, Prod# 34021) were added and developed 30 minutes before

stopping by addition of 1M H2SO4, and the absorbance value at 450 nm was read by Multiskan Spectrum Microplate

(Themo Instruments Inc.). All ELISA assays were repeated at least three times.

Expression and purification of a soluble HLEAFab fragment

The recombinant HLEAFab was expressed in the E. coli Top 10 F strain. Briefly, the overnight culture of a single

clone was reinoculated at 1:100 in SB medium with 50 mg/ml carbenicillin until the OD600 reached 1.0 induced by

1mM IPTG in the presence of 4% sucrose at 25°C; and harvested 24 hours later. Western blotting was performed on both

bacteria lysate and sonicated supernatant to determine if the HLEAFab was expressed after IPTG induction, and with a

purified human Fab as positive control. The soluble HLEAFab was purified from the periplasm of the bacteria using

Protein L (GenScript Corporation Cat. No. L00239) affinity purification.

Cell ELISA

Nasopharyngeal carcinoma cell line (HNE2 and HNE2/LMP1) were cultured in complete medium in 96-well flat

bottom plates (Corning) to form a subconfluent monolayer and further incubated overnight in RPMI-1640 medium

containing 10% FBS. The cells were washed 3 times for 5 minutes each with PBS, pH 7.4 and cells were fixed by

treatment with 4% paraformaldehyde for 10 minutes at 4°C. The endogenous peroxidase activity was blocked with 3%

H2O2 in distilled water for 7 minutes at RT followed by 3 × 5 minutes washes in PBS, pH 7.4. Non-specific binding was blocked with 3% bovine serum albumin (BSA) for 1 h at RT. Cells were washed once in PBS and incubated with purified

HLEAFab diluted in 1% BSA for 1 h at RT. The unrelated antibody Fab fragment was used as the negative control

antibody. After 3 × 10 minutes washes in PBS, HRP conjugated anti human IgG(Fab)’ (Sigma，Cat: A0293) was added

for 1 h at RT. The cells were washed again in PBS. Following this, TMB substrate solution, and incubated for 20 minutes

at RT. The color reaction was stopped by adding 1 M H2SO4 and the intensity was read at 450 nm in a Multiskan

Spectrum Microplate (Thermo Labsystems, USA) (10,11,12).

Fluorescence-activated cell sorting (FACS) analysis

HNE2 and HNE2/LMP1 cells were blocked using 1% BSA-PBS at 4°C for 30 minutes, then incubated with 100 μg/ml

HLEAFab at 4°C for 45 minutes, and stained using 1:50 diluted FITC labeled human anti-Fab IgG (Sigma，Cat: F5512)

at 4°C for 20 minutes. The unrelated antibody Fab fragment was used as the negative control antibody. Fluorescence

intensity was analyzed with Cellquest software (Becton Dickinson Bioscience). The cells incubated only with secondary

antibody were analyzed as controls.

Fluorescence staining

HNE2/LMP1 cells were grown in 6 well strips of cell culture plates for staining. At 80% confluence, cells were fixed

using equal volumes of acetone and methanol, blocked by 5% milk PBS at 37°C for 1 h, and coincubated with 50 μg/ml

HLEAFab at 37°C for 1 h before 1:50 FITC labeled human anti-Fab IgG (Sigma,Cat: F5512) in the dark. As mentioned

above, the fluorescence staining experiment was done using cells from different passage numbers and repeated at least

three times.

Conjugation of MMC to HLEAFab

The principle used to conjugate small molecule anti-cancer drug MMC (Zhejiang Hisun Pharmaceutical Co., Ltd.

China) to HLEAFab involved the activation of the amino group with N-succinimidyl 3-(2-pyridyldithio) propionate

(SPDP, Pierce, Rockford, IL), followed by disulfide exchange with iminothiolane-modified HLEAFab (Fig.1) (13). For

activation, MMC was reacted with SPDP at 4°C for 10 h and stored in small aliquots at -80°C until further use.

HLEAFab in PBS was reduced by mixing with 2-iminothiolane for 1 h under nitrogen. The reduced antibody was

purified by size-exclusion chromatography using Desalting Column (Pierce, Rockford, IL) pre-equilibrated with PBS.

The conjugation of activated MMC with thiolated HLEAFab (MW: ～50K) was done at RT for 1 h under nitrogen using

a 1:1 molar ratio. Excess free MMC-SPDP were removed using Desalting Column (Pierce, Rockford, IL)

pre-equilibrated with PBS. HLEAFab–MMC products were filter-sterilized using 0.2 μm supor membrane (Pall, Ann

Arbor, MI). The dosage of the HLEAFab–MMC conjugate for cytotoxicity and in vivo experiments was calculated

according to the total amount of MMC added. The conjugations of MMC with the unrelated Fab fragment were done the same way as described above.

MTT assay for anti-proliferation of HNE2/LMP1 cells by HLEAFab–MMC

MTT assay (14) was performed on HNE2/LMP1 and HNE2 cells on a 96-well plate. A total of 5 × 103 cells were

dispensed into each well and cultured for 12 h. Following removal of cultured media, fresh medium containing MMC,

naked antibody or immunoconjugates (25-200 nmol/L) was added to each well for an additional 48 h incubation.

Subsequently, the culture medium was removed, and 100 μl of MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-

liumbromide) (1 mg/ml in RPMI1640) was added to each well. After 5 h of incubation with MTT at 37°C in 5% CO2

incubator, the supernatant was removed, and 150 μl of dimethylsulfoxide (DMSO) was added to each well followed by

shaking at 150 rpm for 5 minutes. Absorbance at 490 nm was determined spectrophotometrically. The cells without any

antibody treatment served as the negative control. MTT assay was repeated at least three times.

Induction of apoptosis of HNE2/LMP1 cells by HLEAFab–MMC

In order to investigate the apoptotic effects of HLEAFab–MMC on NPC cancer cells, Apo-Direct Kit (BD pharmingen

556381) was employed for detection and quantitation of apoptosis at a single cell level. Briefly, HNE2/LMP1 cells were

cultured in RPMI-1640 medium at 37°C in a 5% CO2 incubator for 12 h until all cells are attached to microwells.

Following HLEAFab–MMC (200 nmol/L) was added for co-incubation of 48 h. As the negative control, MMC,

HLEAFab or PBS of the same concentration was used for the same incubation period. At the end of incubation, the

attached cells were removed from tissue culture wells by Cell dissociation buffer (Invitrogen, USA). Apoptosis of treated

HNE2/LMP1 cells was determined quantitatively by TUNEL assay with the instructions provided by Apo-Direct Kit

(BD pharmingen 556381). Morphological changes of HNE2 and HNE2/LMP1 cells after treatment with

HLEAFab-MMC under fluorescence microscope by living cells\apoptotic cells\ necrotic cells Identification Kit (Nanjing

KeyGen Biotechnology Development Co., Ltd,China). As mentioned above, the TUNEL assay was repeated at least

three times.

Treatment of HNE2/LMP1 xenografts with HLEAFab-MMC

Female 4-week-old Balb/c nude mice with a body weight of approximately 20 g were purchased from ShangHai

SLAC Laboratory Animal CO. LTD (Shanghai, China), and kept under specific pathogen-free conditions. For

HNE2/LMP1 xenograft model, mice injected with 2×106/ml HNE2/LMP1 cells subcutaneously were killed after tumor

diameter of each of the four nude mice was about 1 cm; the rapidly growing tumors were excised, cut aseptically into 2

mm3 pieces and inoculated subcutaneously into the right flank of experimental mice. After inoculation, tumor sizes were

measured every five days with vernier calipers, volumes were calculated by the formula: (smaller diameter)2 × larger

diameter × 0.5, and tumors were allowed to grow for 14 days before treatment (15). To test HLEAFab-MMC on tumor

growth inhibition, the mice with HNE2/LMP1 xenograft were given HLEAFab-MMC location injected (16,17) at dose

levels ranging from HLEAFab (8 mg/kg, 1.6 × 10-5 mol/kg) in HLEAFab group, HLEAFab-MMC (8 mg/kg, 1.6 × 10-5

mol/kg) in HLEAFab-MMC group, MMC (0.053 mg/kg, 1.6 × 10-5 mol/kg) in MMC group on day 1, day 4, and day 7

including the unrelated Fab, the immunoconjugate of MMC with the unrelated Fab and normal saline for negative control.

The mice were followed for observation of xenograft body weight changes. At the end of the treatment period, the mice

were killed, and the tumors were excised to determine tumor weight.

Results

Selection of specific LMP1 binding phage and expression and purification of soluble HLEAFab

After seven rounds of panning, 45 single phage clones were randomly picked up and amplified to test for specific

binding to LMP1 EMDs by phage ELISA. A ratio of sample OD450 versus the blank of greater than 2.5 was set as the

standard for selecting positive clones. As shown in Fig.2A, No.36 clone with strongest binding to LMP1 EMDs were

analyzed by DNA sequencing and were all identical (Table). Soluble expression of HLEAFab was induced overnight at

low temperature (25°C). The expressed HLEAFab was found mainly in the periplasmic space of E. coli (Fig.2B).

SDS-PAGE and Coomassie Blue staining showed equal expression of heavy and light chains. The purity was above 95%

after Protein L affinity purification (Fig.2C).

The HLEAFab reacts with the extracellular domains of LMP1 in native conformation

Further analysis of HLEAFab reactivity was performed by comparing the binding ability of the antibody fragments to

LMP1-positive (HNE2/LMP1) and LMP1-negative (HNE2) NPC cells using cell surface ELISA. Shown in Fig.3A, the

purified HLEAFab recognized LMP1 present in HNE2/LMP1 cells. We further evaluated whether the HLEAFab binds to

extracellular domains of LMP1 using a flow cytometric assay. Both HNE2/LMP1 and HNE2 cells were treated with or

without HLEAFab before addition of fluorescence-labeled secondary conjugate. The result in Fig.3B shows that the

population of HLEAFab-treated HNE2/LMP1 cells was clearly separated from non-treated cells by fluorescent intensity,

while no difference was noted between HLEAFab-treated and non-treated HNE2 cells, suggesting the HLEAFab binds to

LMP1-positive cell membranes only. Furthermore, to provide morphological evidence that this HLEAFab binds to the

cell membrane, a cell single-staining experiment was performed, and again, this HLEAFab stained only LMP1-positive

NPC HNE2/LMP1 cells, and not LMP1-negative NPC HNE2 cells (Fig.3C).

Cytotoxicity and proapoptotic activity of immunoconjugates HLEAFab-MMC against LMP1-positive NPC

HNE2/LMP1 cells in vitro

After confirming the binding capability of this HLEAFab, we conjugated it with MMC to investigate its anti-tumor

effect in vitro. Mass spectrophotometer analysis showed that the molar ratio of HLEAFab to MMC was about 1:1

(average molecular weight of the conjugate increased 342 Da in comparison to HLEAFab, and the molecular weight of

MMC is 334.3). The binding affinity of MMC-conjugated HLEAFab to LMP1 did not decrease significantly as

compared to nonconjugated HLEAFab (data not shown), and the stability of MMC-conjugated HLEAFab is reasonable

as it was exposed at RT for days during conjugation reaction. To determine the efficacy of HLEAFab-MMC to

specifically inhibit the growth of HNE2/LMP1 cells, MTT assays were performed. Viable HNE2/LMP1 or HNE2 cells

were treated with different concentrations of HLEAFab-MMC, HLEAFab, MMC, unrelated Fab or unrelated Fab-MMC.

The results showed that HLEAFab-MMC was effective in inhibiting 76％ cell proliferation at 200 nmol/L. However,

MMC and HLEAFab only yielded 31% and 22% inhibition respectively. In fact, the cell inhibition rate decreased as the

concentration of HLEAFab-MMC declined, denoting a dose-dependent response. These data indicated the cell

cytotoxicity seen with HLEAFab-MMC is mediated by LMP1 specifically in a dose-dependent manner (Fig.4A). MMC,

naked antibody or immunoconjugates exhibited very modest or no cytotoxicity at the highest dose tested (200 nmol/L) in

HNE2 cells (Fig4B).By employing TUNEL assay, inductions of apoptosis following treatments of cancer cells with

HLEAFab-MMC were quantitatively determined. As shown in Fig.4C, significant increases in apoptosis of treated

HNE2/LMP1 cells were detected upon the treatment with HLEAFab-MMC (200 nmol/l) as compared to the negative

control and HNE2 cells. The highest apoptosis rate of HNE2/LMP1 cell lines was observed in HLEAFab-MMC group

(13.88%) compared with the MMC group (3.04%), HLEAFab group (2.78%), unrelated Fab (2.34%) or unrelated

Fab-MMC (3.2%) or negative control (2.10%) at the same concentration. Characteristic morphological changes of cell

apoptosis were observed by fluorescence microscopy after HLEAFab-MMC acted on NPC HNE2/LMP1 cell lines.

Normal cells were round, uniform, cells were stained green, the size and the shape were single (Fig.4D1); necrotic cells

have oval-shaped cells stained orange, the size and shape were single (Fig.4D2); early apoptotic cells were green, cell

shape was irregular, like a crescent (Fig.4D3); late apoptotic cell nuclei were orange, the nucleus broke into pieces,

showing bud-like cellular processes (Fig.4D4).

The HLEAFab-MMC immunoconjugate suppresses tumor development in a HNE2/LMP1 xenograft model

Mice models bearing HNE2/LMP1 xenografts were established and were confirmed for their LMP1-overexpressing by

IHC method (Fig.5A). As illustrated in Fig.5B, HNE2/LMP1 xenografts mice treated with HLEAFab-MMC (1.6 × 10-5

mol/kg, MMC-equivalent at 0.053 mg/kg HLEAFab-equivalent at 8 mg/kg) achieved significant regressions from 20

days to 40 days when compared with controls (P < 0.05). Although MMC (0.053 mg/kg) injection was potent to inhibit

tumor growth, the efficacy was much lower than that of HLEAFab-MMC (MMC: 0.053 mg/kg) which suggested an

extra advantage of LMP1-mediated-specific tumor targeting. As shown in Fig.5C, the HLEAFab-MMC

immunoconjugate resulted in an average 55.1% inhibition of HNE2/LMP1 tumor growth in comparison with their

controls (P < 0.05). However, the MMC alone produced 26.5% inhibition, HLEAFab alone 5.64%, unrelated Fab 9.63%

and unrelated Fab-MMC 24.4%. So the HLEAFab-MMC immunoconjugate remarkably enhanced the tumor growth

inhibition. During the entire treatment period, the antineoplastic antibiotic MMC, the antibody and immunoconjugates

administration were nontoxic according observation of weight, survival and animal activities as well as pathological

examination results (Fig.5D). No other complications such as anaphylaxis and skin necrosis were detected.

Discussion

In this study, we successfully prepared the immunoconjugate HLEAFab-MMC against LMP1-positive NPC. This

involved the following procedures: (1) The HNE2/LMP1 cell line, which has high LMP1 expression, and the

LMP1-negative HNE2 cell line were selected for subtractive panning to increase the chances of capturing the phage that

binds to native LMP1. (2) We were able to generate a human HLEAFab fragment that binds to the LMP1-positive NPC

cell lines. (3) We showed that the major immunogenic region of the LMP1 extracellular domain can be used as an

immunoconjugate target to kill LMP1-positive NPC cell lines in vitro, (4) An antibody-based immunoconjugate

HLEAFab-MMC killed LMP1-positive NPC cells in vitro and significantly slowed tumor growth in a mouse

LMP1-positive NPC transplantation model, although HLEAFab or MMC alone was insufficient to generate comparable

effects.

In our study, the repeated panning with living cells and coated LMP1 EMD protein in microtiter plates ensured the

enrichment of specific LMP1 EMD binding phage. After seven rounds of panning, 1 out of 45 randomly selected phage

clones showed evidently positive results (Fig.2). Moreover, the DNA sequencing results (Table) were identical to the

phagemids that gave the strongest binding signal by ELISA, indicating the phage with high specific binding to LMP1

were selected out from the panel. The ability of the expressed HLEAFab to specifically bind to native LMP1 on the

HNE2/LMP1 cell surface was further confirmed by Cell ELISA, FACS, and fluorescence staining analyses (Fig.3).

Antibody-based targeting immunotherapy is a well-established treatment for various tumor types, such as

ErbB2-positive breast cancer (18), Hodgkin lymphoma, acute myeloid leukemia, colon cancer, lung cancer, melanoma,

and some of head and neck squamous cell carcinomas (HNSCC) (19-24). Immunoconjugate therapy has not yet been

applied to NPC, although some antigens have been found since many years ago to expresses at high levels on the surface

of these tumor cells (25, 26). This obstacle was overcome after we discovered that the LMP1 EMD is likely to be as a

therapeutic target of the vast majority of NPC.

The LMP-specific mAbs CS1-4, S12 and other monoclonal antibodies are known to recognize intracellular parts of

LMP1 (27) and LMP Antibody-positive sera stained our LMP-expressing cells in indirect immunofluorescence assays

only when these cells were first permeabilized. These Antibodies and sera, however, were unable to stain these cells

without prior permeabilization, suggesting that these antibodies recognized only intracellular parts of the protein (28).

Therefore these introbodies are not fit for the preparation of antibody-based immunoconjugate unless they are

internalized by the target cells (29). Accordingly, we selected LMP1 extra-membrane domain for a specific target

position as an extra-cellular domain antibody is an ideal carrier of antibody-based immunoconjugate.

An antibody per se can be a drug for immunotherapy. Indeed, several neutralizing antibodies have been approved by

the FDA for clinical treatment of a variety of diseases. Besides, antibody can also be used as a carrier to deliver a broad

range of therapeutics selectively to cancer cells by recognizing a specific tumor marker on the cell surface. The

therapeutics might include nucleic acids, chemotherapy compounds, toxins, radionuclides, antibody, cytokine or

antibody-ligand fusion proteins, and enzyme pro-drugs. Such delivery may reduce or eliminate side effects caused by the

drugs in normal cells. For the purposes of immuno-chemotherapy, an antibody capable of receptor binding is essential,

because, in that process, the antibody can help accumulate the conjugated molecules in the cancer cells (20,30). LMP1

has been reported to be over-expressed in most solid tumors at various levels, making it an ideal target for antibody

directed drug delivery (31,32). In this study, we selected MMC to be conjugated to an anti-LMP1 EMD HLEAFab and

made an immunochemotherapeutic agent. Its biological efficacy was tested in vitro anti-tumor activity via cell

proliferation inhibition and apoptosis assays (Fig.4). The results showed that HLEAFab-MMC was effective in inhibiting

76％ HNE2/LMP1 cell proliferation at 200 nmol/L, in contrast to the inhibitory rate of 31% by MMC , 22% by

HLEAFab. The highest apoptosis rate of HNE2/LMP1 cell lines was observed in HLEAFab-MMC group (13.88%)

compared with the MMC group (3.04%), HLEAFab group (2.78%) or Negative control (2.10%) at the same

concentration. These data suggest that the HLEAFab-MMC conjugate is more potent for treating LMP1-positive cancers

and has less side effects relative to those seen with MMC alone.

The anti-tumor effect of this immunoconjugate was also assessed in vivo (Fig.5). As illustrated in Fig.5, 8 mice models

with xenograft tumor were treated with HLEAFab-MMC (MMC-equivalent 0.053 mg/kg, HLEAFab-equivalent at 8

mg/kg) and achieved tumor inhibition and maintained tumor inhibitory effect for more than another 20 days. Although

MMC injection was potent to inhibit tumor growth, the efficacy was much lower than that of HLEAFab-MMC, which

suggested the great advantage of HLEAFab-mediated-specific tumor targeting. To circumvent the relatively significant

errors made by directly measuring tumor size, tumors were removed and weighed after the mice were sacrificed at the

end of experiment (day 40) . The average tumor weight of HLEAFab-MMC treatment group was much smaller than

those from other groups. The tumors shrank to 55.1% comparing with the control group (N.S. injected). In contrast, the

MMC alone treated tumors only shrank to 26.5%. These results clearly demonstrated that HLEAFab–MMC can

specifically inhibit tumor growth in vivo. HLEAFab-MMC significantly inhibits Balb/c nude mice transplantation tumor

growth of human HNE2/LMP1 nasopharyngeal carcinoma in vivo.

Actually, different type of nasopharyngeal carcinomas and different cells in the same tumor tissue are not always the

same level of LMP1-expessing. Only 20%-65% nasopharyngeal carcinomas express LMP1 that can be detected by

western blot or immunohistochemical analysis and another (25,33). Although the results presented here appear promising,

the different level of LMP1-expressing in EBV-related carcinomas, the different biological efficacy of this Fab-based

immunoconjugate. In order to strengthen the anti-tumor biological efficacy of HLEAFab-MMC to the low

LMP1-expressing nasopharyngeal carcinoma, our next strategy is that the antibody will conjugate liposomes or polymer

nanoparticales encapsulating more MMC to elevate potential efficiency and decrease clearance rate. The conjugation can

kill not only the cancer cells of low-expressing LMP1 by targeted efficacy but also the cancer cells of LMP1 negative

through a “bystander” effect. This phenomenon was also reported by Kovtun and his colleagues (34). On the whole, the

evaluation of LMP1 expression in individuals is required before this therapeutic strategy is applied in NPC treatment.

In summary, our findings indicate that the LMP1 extra-membrane domain is a specific marker of human NPC that can

be recognized by a HLEAFab-based immunoconjugate (HLEAFab-MMC). The immunoconjugate, the first targeting

agent based on a fully human antibody Fab fragment described so far, is effective both in vitro and in vivo against NPC

cells and might represent a promising chemotherapeutics that merits further pre-clinical studies.

Acknowledgment

We thank Dr. Yongjun, J for helpful discussions. We also thank AbMax Biotechnology Co., Ltd. for their excellent

technical assistance. We thank Xuan C for helpful image processing. We acknowledge Mr. Patrick Karle and Prof.

Gordon James Leitch from America for polishing our English.

Grant Support

The project was supported by Natural Science Foundation of Jiangsu Province (No.BK2008481), Technology Support

Program of Jiangsu (No.BE2009152), a grant from State Major Basic Research Development Program of

China (973 Project, No. 2010CB933902) and Natural Science Foundation of Nanjing Medical University (No.

NY2005D2D13).

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Table: HLEAFab variable region DNA and amino acid sequence

VL ( immunoglobulin kappa light chain VLJ region ) VH ( immunoglobulin heavy chain variable region )

GAGGTGCAGCTGGTAGAGTCTGGTGGAGGCTTGGTCC GAGCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGC AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC ATCTGTAGGAGACAGAGTCGCCATCACTTGCCGGGCAA TGGATTCACCTTCAGTAGCTATTGGATGAGCTGGGTCC GTCAGAGCATTAGCAGCCATTTAAATTGGTATCAGCAG GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCA AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGC DNA ACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGAC ATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTG sequence TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC CAAGAACTCACTGTATCTGCGAATGAACAGCCTGAGAG AGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAA CCGAGGACACGGCTGTGTATTACTGTGCGAGAGCCTG CAGAGTTACAGTACCCCGTACACTTTTGGCCAGGGGAC GGGGTATAGCAGCTACTACTTTGACTACTGGGGCCAG CAAGCTGGAGATCAAA GGCACCCTGGTCACCGTCTCCCCT

Amino acid EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVR ELQMTQSPSSLSASVGDRVAITCRASQSISSHLNWYQQK sequence QAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKN PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP SLYLRMNSLRAEDTAVYYCARAWGYSSYYFDYWGQGTL EDFATYYCQQSYSTPYTFGQGTKLEIK VTVSP

Figure Legends

Fig.1 Conjugation of anti-LMP1 antibody Fab and mitomycin C. Mitomycin C was first conjugated with the SPDP linker

(A), and Fab was reduced with 2-iminothiolane (B). The final immunoconjugate construct was shown in (C).

Fig.2 ELISA result of 45 individual phage clones randomly picked up from the eluted phage pool after the 7th round of

bio-panning. The last two columns on the right were negative control phages that served as blanks (A), Western blotting

characterization of HLEAFab fragment expressed in E. coli. Lane 1 was Top10F cell lysate control; lane 2 was 25 μl of cell lysates after sonication; lane 3 was 10 μl of cell sedimentation after sonication; lane 4 was 25 μl of culture medium after cells culture. Mouse anti-human Fab HRP conjugate was used at 1:1000 dilution (B). Purified HLEAFab fragment was separated on a 10% SDS-PAGE gel and stained with Coomassie Blue. The double bands were heavy chain Fd (up) and light chain κ (down), and light chain κ were more expressed (C).

Fig.3 The HLEAFab binds to the LMP1 extracellular domain in native confirmation. LMP1 positive cells: HNE2/LMP1,

LMP1 negative cells: HNE2, control: human hepatoma BEL-7402 cells and the unrelated antibody Fab fragment was used as the negative control antibody (A). Flow cytometry analysis: the fluorescent intensity was significantly different between LMP1-positive HNE2/LMP1 cells and LMP1-negative HNE2 cells, Background staining (shown as black line) was obtained by the unrelated Fab (B).Fluorescence microscope analysis of HNE2/LMP1 cells and HNE2 cells: C2 was

HLEAFab stained HNE2/LMP1 cells with mouse anti-human Fab FITC conjugate; C3,4 was cell morphology under light microscopy; and C1,3 were HNE2 cell Negative control. (×200, Olympus Digital Camera)(C)

Fig.4 Inhibitory effect of MMC, naked antibody or immunoconjugates on growth of nasopharyngeal carcinoma

HNE2/LMP1 or HNE2 cell lines. MTT assay to reveal dose-dependent anti-proliferative effects of 25-200 nmol/l of

HLEAFab-MMC (red) (P < 0.05), 25 nmol/l of MMC (yellow) (P < 0.05), except 25-200 nmol/l of HLEAFab (blue),

unrelated Fab (purple) and unrelated Fab-MMC (P > 0.05) for 48 h on the HNE2/LMP1 cells (A). Only 100 nmol/l of

HLEAFab-MMC (red) (P < 0.05) showed anti-proliferative effects on the HNE2 cells. Relative absorbance at 490 nm

was normalized with negative control (NC) without treatment (B). Percent increases in apoptosis in response to

treatments of cultured HNE2/LMP1 (gray) and HNE2 (black) cells with 200 nmol/l of MMC, naked antibody or

immunoconjugates and Control (PBS) for 48 h. Percent increase in apoptosis of cancer cells was revealed statistical

significance at P < 0.05 for HLEAFab-MMC (C). Morphological changes of HNE2 and HNE2/LMP1 cells after

treatment with HLEAFab-MMC conjugate under fluorescence microscope. (×200, Olympus Digital Camera) (D).

Fig.5 Growth inhibition on tumor xenografts by HLEAFab–MMC. Positive expression on LMP1 tumor xenografts was

confirmed by IHC method. (×200, Olympus Digital Camera) (A). The mouse were grouped (8 mouse per group) and

administered in the tumor local with 1.6×10-5 mol/kg HLEAFab, HLEAFab–MMC, MMC, unrelated Fab, unrelated

Fab-MMC or Control (N.S. normal saline) with indicated doses on day 1, day 4, and day 7. Tumor size was measured

one time per five days and tumor volume was calculated according to the equation: tumor size = width2 × length/2, *P <

0.05 compared with control (B). At the end of experiment, the mice bearing tumors were sacrificed and tumors were

removed and weighed, *P < 0.05 compared with control (C). Measurement of body weight throughout the experiments

(D).

A human Fab-based immunoconjugate specific for the LMP1 extracellular domain inhibits nasopharyngeal carcinoma growth in vitro and in vivo

Chen Renjie, Zhang Dawei, Mao Yuan, et al.

Mol Cancer Ther Published OnlineFirst December 14, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-11-0725

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