Original Article

Construction and Selection of Human Fab AntibodyPhage Display Library of Liver Cancer

Xuan Shui, Jian Huang, Yue-Hui Li, Ping-Li Xie, and Guan-Cheng Li

The aim of this study was to construct the fully humanized anti-hepatoma Fab fragment phage libraries andselect antibodies against hepatoma specifically. PBMCs of liver cancer patients were immunized in vitro withHpeG2 cells and were then transformed by Epstein-Barr virus (EBV). After total RNA was extracted, the heavychain Fd and k=l light chain were amplified by RT-PCR and cloned into the vector pComb3 to construct thelibraries of Fab fragments. The libraries were then panned by HpeG2 cells. By means of ELISA and immu-nochemistry, the Fab phage antibodies binding with hepatoma were selected and identified. The Fd and lightchain PCR products were subsequently inserted into pComb3, and the volume of Fab libraries reached1.7�107. The libraries were enriched about 138-fold by three cycles of panning. 540 phage clones were pickedrandomly. Using cell ELISA and immunohistochemistry with cultured cells, one clone Fab phage antibody,which had binding activity with hepatoma, was picked out. Fully humanized anti-hepatoma Fab antibodyphage display libraries were constructed. One phage clone was selected and confirmed to specifically bind tohepatoma cells. The selected Fab antibody may be further developed and applied to clinical diagnosis andtherapy.

Introduction

Liver cancer is one of the most malignant tumors inChina, and its rate of incidence is very high. The major

treatment of liver cancer is surgery. But most patients lose thechance for surgery because the course of disease is already atan advanced stage when the cancer is diagnosed. So it hasbecome clear that a better prognosis of liver cancer is relatedto early diagnosis and treatment.(1,2)

One focus of cancer immunology is on the isolation ofantibodies that react selectively with human tumor cells,(3–6)

because the antibodies could have important application forantigen detection and therapeutics. The specificity combinedwith low toxicity makes them a promising pharmaceuticalcommodity.(7) Therapeutic use of antibodies is limited bymethodological constraints. The established approach hasbeen able to generate large panels of monoclonal antibodies(MAb) from mice immunized with human tumor cells and toscreen the antibodies for reactivity against the tumor.(8) De-spite the enormous efforts put into this approach, few anti-bodies that react preferentially with human tumors andalmost none that react specifically with one type of tumorhave been reported. Additionally, the heterologous proteinsare often immunogenic for humans, preventing their thera-peutic use.(9)

Further attempts to isolate more specific and high affinityantibodies against human tumors will require improved

methods of generating and selecting antibodies. The con-struction and selection of antibody combinatorial librariesdisplayed on filamentous phage surfaces became a viablealternative to this approach.(10) In this technique, the reper-toire of V genes of one or more individuals is amplified withprimers covering all V gene families, giving rise to humanantibodies. The library is generated by a random combinationof variable light (VL) and variable heavy (VH) chain genesproduced as antigen binding (Fab) or single chain variable(scFv) antibody fragments.(11) Theoretically, each clone codesfor a specific antigen-binding site, corresponding to a naturalrepertoire, increased by an artificial domain combination thatextrapolates the individual repertoire. This panning proce-dure mimics the B cell clonal selection system in vitro byspecifically enriching phage particles that display antibodieswith desired specificity.(12)

Several human antibody combinatorial libraries displayedon filamentous phage surface have been built by variousgroups, from either naıve(13–15) or immunized=infected(16,17)

repertoires. This system has been applied to select antibodiesagainst different antigens, including melanoma,(18) colorec-tal,(19) and prostate(20) cancer proteins. It can resolve theproblems of generating humanization MAb by the hybridomaapproach. Here we describe the construction of immunephage display libraries expressing Fab antibodies derivedfrom B cells of liver cancer patients and report the initial re-sults of panning the libraries for anti-hepatoma antibodies.

Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan Province, China.

HYBRIDOMAVolume 28, Number 5, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=hyb.2009.0028

341

Materials

Vector, E. coli, and helper phage

The vector (pComb3), about 4029 base pairs (bp), containsampicillin resistance (AmpR) gene. The expression of insert-ing fragments is controlled by the same LacZ promoter on itsupstream sequence. Escherichia coli XL1-Blue contains tetra-cycline resistance (TetR) gene on its gene type of Tn10. Helperphage (VCSM13) contains kanamycin resistance (KanR) genewith valency of 1013 pfu=L. It is amplified in SB culture me-dium and preserved at 48C.

Human cells

Human bone marrow mesenchymal stem cells (HBMSC),human umbilical vein endothelial cells (HUVEC), hepatocyteline (Chang liver), human embryo kidney epithelium cellline (HEK293), liver cancer line (HepG2), colorectal carcinomaline (HRT-18), nasopharyngeal carcinoma line (CNE2), anduterine cervix cancer line (Caski) were conserved in our lab-oratory. Culture medium for all the cell lines was Dulbecco’smodified Eagle’s medium (DMEM) supplemented with100 mL=L fetal calf serum and antibiotics.

Methods

Separation and immunization of PBMCs

The PBMCs (peripheral blood mononuclear cells) were iso-lated from 20 liver cancer patients by density centrifugation.The PBMCs were separated into two parts: one part was im-munized with hepatoma cells and transformed with EBV after6*8 days, and the other was directly transformed with EBV.

Transformation of PBMCs with Epstein-Barr virus

The Epstein-Barr virus (EBV)-containing supernatant wasfiltered through a 0.22 mm sterile filter, stored at �708C. Priorto transformation with EBV, PBMCs were washed twice withHank’s buffer. Cells were infected for 2 h at 378C using 1 mL ofEBV-containing supernatant per 4�106 PBMCs. After infec-tion, the cells were grown in DMEM containing 100 mL=L fetalcalf serum (FCS).

Indirect ELISA

Indirect ELISA assay was used to detect specific antibodiesin the culture supernatant. The hepatoma cells were grown in96-well plates until almost confluent, and fixed with 1.5 mL=Lgluteraldehyde for 10 min at room temperature. The cells wereblocked with 200mL of BSA (10 g=L in PBS) for 2 h at 378C.Culture supernatants were added into wells (100 mL=well)and incubated at 378C for 2 h. After washing, 100 mL goat anti-human IgG labeled with HRP was added (100 mL=well)and incubated at 378C for 2 h. After washing 5 times withPBS=0.05% Tween-20 (PBST), 2’2-azino-di-3-ethyl-benzthiaz-line sulfonate (ABTS) substrate was added, and the opticaldensity at 405 nm was read after 20 min. Then, the positivecells were amplified to 106–107.

Isolation of RNA and amplification of Fd and light chaingenes of antibodies by PCR

Lymphocyte total RNA was prepared from the positivecells, and first-strand cDNAs were synthesized with random

hexamers and poly (A) primers using kits purchased fromInvitrogen (Carlsbad, CA). The coding regions for the heavychain Fd (VH-CH1) and the k and l light chains were sepa-rately amplified by PCR using the primers shown in Table 1.The PCR condition was: 948C for 30 s, 558C for 1 min, 728C for1 min, 35 cycles, followed by final incubation at 728C for10 min. The PCR products were purified by electrophoresis in10 g=L agarose gel and extraction from the gel using the Qiaexkit (Qiagen, Hilden, Germany); the purified DNA were dis-solved in 50mL of Tris=EDTA (TE; 10 mmol=L Tris [pH7.5]=1 mmol=L EDTA) buffer.

Cloning light chain k and l into pComb3

The light chain k and l fragment PCR product were cutwith an excess of the restriction enzymes Sac I and Xba I andabout 600 ng was ligated with 2mg of Sac I=Xba I-linearized

Table 1. The Primers for Amplification

of Human Fab Fragments

30primer of human heavy chain50-CTC GAC ACT AGT TTT GCG CTC AAC

TGT CTT-30CG2A

50-TGT GTG ACT AGT GTC ACC AAG TGGGGT TTT-3

CG3A

50-GCA TGT ACT AGT TTT GTC ACA AGATTT GGG-30

CG1Z

50-GCA TGA ACT AGT TGG GGG ACC ATATTT GGA-30

CG4A

50primer of human heavy chain50-CAG GTG CAG CTC GAG CAG TCT GGG-30 VH1A50-CAG GTG CAG CTG CTC GAG TCT GGG-30 VH1F50-CAG GTG CAG CTA CTC GAG TCG GG-30 VH2F50-GAG GTG CAG CTC GAG GAG TCT GGG-30 VH3A50-GAG GTG CAG CTG CTC GAG TCT GGG-30 VH3F50-CAG GTG CAG CTG CTC GAG TCG GG-30 VH4F50-CAG GTG CAG CTA CTC GAG TGG GG-30 VH6F50-CAG GTA CAG CTC GAG CAG TCA GG-30 VH6A30primer of human l chain50-CGC CGT CTA GAA TTA TGA ACA TTC

TGT AGG-30CL2

50primer of human l chain50-AAT TTT GAG CTC ACT CAG CCC CAC-30 VL150-TCT GCC GAG CTC CAG CCT GCC TCC

GTG-30VL2

50-TCT GTG GAG CTC CAG CCG CCC TCAGTG-30

VL3

50-TCT GAA GAG CTC CAG GAC CCT GTTGTG TCT GTG-30

VL4

50-CAG TCT GAG CTC ACG CAG CCG CCC-30 VL550-CAG ACT GAG CTC ACT CAG GAG CCC-30 VL650-CAG GTT GAG CTC ACT CAA CCG CCC-30 VL750-CAG GCT GAG CTC ACT CAG CCG TCT

TCC-30VL8

30primer of human k chain50-GCG CCG TCT AGA ATT AAC ACT CTC

CCC TGTTGA AGC TCT TTG TGA CGG GCG AAC TCA

G-30Ck1D

50primer of human k chain50-GAC ATC GAG CTC ACC CAG TCT CCA-30 Vk1A50-GAC ATC GAG CTC ACC CAG TCT CC-30 Vk1S50-GAT ATT GAG CTC ACT CAG TCT CCA-30 Vk2A50-GAA ATT GAG CTC ACG CAG TCT CCA-30 Vk3A50-GAA ATT GAG CTC ACA CAG TCT CCA-30 Vk3B

342 SHUI ET AL.

pComb3 vector (isolated by agarose gel electrophoresis) in atotal volume of 150 mL with 166.7 nkat of ligase (Promega,Madison, WI) at 168C for 18 h. The ligation products werepurified by extraction with phenol=chloroform form andprecipitation with ethanol and were dissolved in 20 mL ofwater. The purified DNA was used to transform E. coli XL1-Blue by electroporation.(21) After transformation, culture ali-quots were plated on LB agar=ampicillin (50 mg=L) incubatedat 378C overnight, and the plasmid was extracted. XL1-Bluesamples (10, 1, 0.1 mL) were withdrawn for plating to deter-mine the rate of transformation and the insertion of targetlight chain fragments were detected by Sac I=Xba I digestionfrom the plasmids extracted from several random XL1-Bluemonoclones. The plasmids with light chain insertion werenamed pþL.

Cloning heavy chain Fd into pþL to constructFab antibody libraries

The Fd products of PCR and recombined pþL (isolated byagarose gel electrophoresis) were cut with an excess of therestriction enzymes Xho I and Spe I. The ligation, transfor-mation, and determination of the transformation rate were thesame as described above. The insertion of target Fab frag-ments was detected by digestion with Xho I=Xba I from theplasmids extracted from several random E. coli XL1-Bluemonoclones. After transformation, 5 mL SOC medium wasadded, and the culture was incubated for 1 h at 378C at250 r=min. SB medium (10 mL), containing ampicillin(20 mg=L) and tetracycline (10 mg=L), was added to thetransformed culture. After additional incubation under thesame conditions, 100 mL SB medium, supplemented withampicillin (50 mg=L) and tetracycline (10 mg=L), was added.After 1 h of incubation, as above, 1012 plaque-forming units ofVCSM13 helper phage were added and the culture was sha-ken for 1 h at 378C and 250 r=min. Kanamycin (70 mg=L) wasthen added, and the culture was incubated overnight at 378Cand 250 r=min. Phages were obtained from the culture me-dium by polyethylene glycol=NaCl precipitation and wereresuspended in PBS containing 10 g=L BSA.

Panning the Fab phage display libraries

The Chang liver and HepG2 cells were grown as an attachedmonolayer in 35-cm2 flasks respectively until almost confluent,then fixed with 1.5 mL=L gluteraldehyde for 10 min at roomtemperature, blocked with 2 mL BSA (10 g=L in PBS) for 2 h at378C. For the first panning step, 1011 transforming units wereadded to the Chang liver cells in 2 mL PBS. The culture flaskwas shaken gently for 1 h at room temperature. The unabsorbedphages were transferred to the culture flask containing fixedHepG2 cells and shaken for 1 h under the same conditions. Thenthe cells were washed rapidly 10 times with PBS. The phagebinding with the antigens of HepG2 eluting from the flask wereused to reinfect the XL1-Blue cells. A round of panning wasfinished after superinfection by VCSM13.

Cloning the phage

After three panning steps, the eluted phages were mixedwith E. coli XL1-Blue cells for 30 min at low phage-cell ratio,and the cells were plated on LB agar=ampicillin (50 mg=L)following incubating at 378C overnight. Individual colonies

were inoculated into SB=ampicillin (50 mg=L) medium andgrown overnight with shaking; the medium containing thecloned phage stock was stored at 48C for ELISA assays.

ELISA analysis of Fab displaying

The HepG2 cells were grown in 96-well plates until almostconfluent, fixed with 1.5 mL=L glutaraldehyde, and thenblocked with BSA (10 g=L in PBS). A stock of cloned phagesupplemented with 10 g=L bovine serum albumin was addedto each well and incubated at 378C for 2 h. After washing withPBST buffer, 100mL of peroxidase-conjugated anti-M13 mono-clonal antibody (1:2500 dilution) was added and incubated at378C for 1 h. Finally 100mL of ABTS as peroxidase substrate wasadded and color development was monitored at 405 nm. TheA405 values of positive clones were at least two-fold higher thanthat of negative control. The positive clones were further testedwith other cultured human cells by ELISA assay.

Immunohistochemistry with cultured cells

Human cells were grown as an attached monolayer on thecover glass, the following procedure being almost the sameas that with the ELISA assay. After incubating peroxidase-conjugated anti-M13 polyclonal antibody (1:100) and washing,diaminobenzidine (DAB) was added and reacted about 3 min.Then the reaction was stopped by water and hematoxylin wasadded successively to counterstain for 1 min. The cells weredehydrated with alcohol and mounted with neutral gummi.

Immunohistochemistry with tissue of normalliver and hepatoma

The positive clones were also verified by means of immu-nohistochemistry with tissues. The slides of normal liver tis-sue and hepatoma were deparaffinized using dimethylbenzene and then hydrated by alcohol. The following stepswere the same as described above.

Sequence analysis

The plasmids of positive clones selected by ELISA andimmunohistochemistry were extracted and followed by DNA

FIG. 1. EBV-transformed B lymphocytes.

LIVER CANCER Fab ANTIBODY 343

sequencing. The sequencing reactions were prepared withspecific primer PL (50-ATT GAA TTC AGG AGG AA-30) forVL genes and PH (50-TGA AAT ACC TAT TGC CTA-30) forVH genes.

Results

Transformation of PBMCs with EBV

After transformation with EBV, the morphology of twogroup PBMCs was similar. Initially, considerable numbers ofPBMCs were dead. After 2 weeks, a few cell clones occurred inthe hepatoma cell–immunized group, mostly around the well,and grew gradually in mass (Fig. 1). Four weeks later, the cellswere collected into 25-cm2 plastic bottles for amplification.After 4 to 6 weeks, the cells were collected and used for thefollowing experiment.

Detection of specific antibody

Detection of indirect ELISA showed that 4 of 20 liver cancerpatients’ B cells, immunized by hepatoma cells and trans-formed by EBV, could produce antibodies against liver can-cer. Those patients who were not immunized by hepatomacells were negative.

Amplifying the fragments of Fd and light chaingenes of antibodies by PCR

The total RNA of the four liver cancer patients’ B cellstransformed by EBV were isolated. The integrity of RNA wasshown by electrophoresis in 1% agarose gel. Their purity(A260=A280) exceeded 1.8 (Fig. 2). For amplifying Fd and lightchain, 50 primers and 30 primers were used, respectively,which contained some sites of the restriction enzymes corre-sponding to the vector pComb3. Thirteen types of lightchain genes and 28 types of Fd genes were obtained by PCR.The PCR products of Fd (Fig. 3) and k=l (Fig. 4) were about660 bp.

Construction and identification of Fab generecombinants

The recombinants of pþFab were constructed by cloningFd and light chain fragments successively into vectorpComb3. After transforming XL1-Blue, the Fab genes librarieswere formed and the quantity of the transformants reached1.7�107. Following extracting the plasmids from 10 randomXL1-Blue monoclones, the insertion of target Fab fragmentswas detected by digestion with Xho I and Xba I. All of theseplasmids discharged the fragments about 2.4 Kb (Fig. 5). Sothe practical volume of Fab library was about 1.7�107�100%¼ 1.7�107.

Panning and selection of the Fab phagedisplay libraries

After three rounds of panning, the libraries were enrichedabout 138-fold. Using primacy test by HepG2 cell ELISA, 163clones of phage antibody with positive ELISA reaction werepicked out of 540 clones. The percentage of positive cloneswas 30.2%. When further screened by a panel of cultured cellsELISA, the clone E35 was found to react strongly with HepG2,

FIG. 2. Total RNA from EBV transformed B lymphocytes.

FIG. 3. Cloning immunoglobulin variable region genes ofheavy chain by PCR. Lane M, DL2000; markers 1–10, thePCR products of different human Fd chain.

FIG. 4. Cloning immunoglobulin light chain genes by PCR.Lane M, DL2000 markers; lanes 1–5, VK1d as 30 primer;VK1a, VK1s, VK2a, VK3a, VK3b as 50 primer. Lanes 6–13,CL2 as 30 primer; VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8,VL9 as 50 primer.

FIG. 5. Ratio of Fab fragment insertion. Lane M, lDNA=EcoRIþHind marker; Lanes 1–10, identification of random10 clones by digestion with XbaI=XhoI.

344 SHUI ET AL.

HEK293, but not with other human tumor cell lines. It alsoreacted weakly with normal liver cell line Chang liver(Table 2). The immunohistochemistry results showed thatthere was brown sedimentum on the plasma membrane andcytoplasm of the HepG2, but no obvious color development in

the Chang liver (Fig. 6A and B). E35 reacted specifically withthe hepatoma cells in human hepatoma tissue sections butalmost not at all with the cells in human liver tissue sections(Fig. 6C–F).

Sequence analysis

E35 was further analyzed by DNA sequencing. The se-quence of E35 was identical. The heavy chain gene analysisrevealed 97.1% homology with the human embryonal geneIgVH3-21. There was also 91.6% homology between the lightchain gene and human embryonal gene IgV1-13.

Discussion

Fd parts and light chains (660 bp) of immunoglobulin werePCR-amplified after reverse transcription from the RNAs,which were isolated from PBMCs of liver cancer patients. Weattempted to maximize the library diversity by using 50

primers based on the leader exon and the N terminus of firstframework (FR1) sequences according to their relatively

Table 2. ELISA Results of Selected Fab Fusion

Phage Clones for Binding to Cultured Human

Cell Lines A405(�xx – s)

Cell lines E35 D65 Blank vector

HepG2 1.211� 0.132 1.033� 0.187 0.097� 0.004Chang Liver 0.368� 0.042 0.499� 0.050 0.132� 0.015HEK293 0.945� 0.210 1.398� 0.015 0.047� 0.003HUVEC 0.122� 0.056 0.416� 0.040 0.150� 0.010HBMSC 0.151� 0.023 0.472� 0.031 0.070� 0.037Caski 0.098� 0.008 0.579� 0.102 0.054� 0.006CNE2 0.104� 0.022 0.779� 0.098 0.087� 0.011HRT-18 0.087� 0.005 0.649� 0.128 0.060� 0.012

FIG. 6. Immunocytochemical staining of cultured cells and tissues with E35. (A) HepG2 (100�). (B) Chang liver (100�). (C)Liver cancer tissue with E35.Cells are stained prominently (200�). (D) Liver cancer tissue with E35 (400�). (E) Liver cancertissue with PBS as negative control (200�). (F) Normal liver tissue with E35. Tissue is stained weakly (100�).

LIVER CANCER Fab ANTIBODY 345

constant regions, and 30 primers within the constant regionsduring amplifying the V genes of immunoglobulin Fab. Toincrease the specificity of antibody, we used cell antigens ofhepatoma to sensitize B cell in vitro, because the antibodygenes would generate mutation theoretically.(22,23) To obtain agreat quantity of antibodies, we used EBV to infect B cells. Bythe surface protein gp350=220 of virus combined with thecomplement receptor of cell, EBV penetrated B cells to causethe cell immortalization, and a few of antigenic specificity Bcell could proliferate. Thus, the immunoglobulin genes en-coding antigenic specificity antibodies could be enriched, andthe loss of the specific antibody gene fragments VH=VL in thesuccessive procedure could be reduced. Moreover, it couldimprove the primitive pairing probability of VH=VL.(24)

Our library size was compatible with the reported size fornaive and immunized human phage displayed libraries,which range from 106 to 109.(25,26) It has long been recognizedthat actual library size must correspond to the functionalantibody forms. Thus, the real size is smaller than that foundby counting recombinant colonies, since frameshifts, stopcodons, or deletions can be generated by PCR or can be aproduct of non-productive immunoglobulin rearrangements.Our real libraries formed almost 100% functional clones,originating around 1.7�107 Fab fragments. This order ofmagnitude should permit successful antigen-binding anti-body selection.

The ultimate aim of phage display is the selection of phage,which bind to the target antigen of interest with high affinityfrom a large excess of phage clones that do not bind or do sowith lower affinity. This is achieved by multiple roundsof binding the phage to the target, washing to remove non-bound phage, followed by elution and retrieval of specificallybound phage. It is important to assemble antibody moleculesthat have correctly folded heavy and light chains and thusretain antigen recognition capabilities. Previously it has beendemonstrated that Fd chains (comprising VH and CH1, thevariable region and constant domain 1 of the immunoglobulinheavy chain) and light chains targeted to the periplasm ofE. coli assemble to form functional Fab molecules. If the Fdchain was anchored in the membrane and concomitantlyprovided with secreted light chains, functional Fab moleculescould form on the membrane surface facing the periplasm.The co-expression behind the PelB leader sequence of Fd fusedto cpIII (the major coat protein of M13 phage) and e chainsleads to membrane anchoring of the Fd chain and compart-mentalization of the light chains in the periplasm. These twochains could assemble in situ to allow accumulation of func-tional Fab on the membrane surface, which, by virtue of thecpIII sequences, would be incorporated along the entire lengthof the filamentous phage particles on subsequent infectionwith the helper phage. These phages displaying Fab couldinfect E. coli again. It is important that screening and selectingphage antibodies be performed on native antigen, because thepotentially valuable antibodies possibly were discarded whena monomeric phage display system was used for screening.(27)

So we selected the phage antibody fragments in vitro on non-purified antigens attached to solid supports(28) from intactmammalian HepaG2 cells. In order to prevent the non-specificity reaction, the phage antibody libraries were incu-bated with normal liver cells first.(29) Following rounds ofpanning by adsorption-wash-amplification, the phages com-prising specific antibody molecules could be enriched.(30)

Our study showed that the phage Fab libraries were enrichedabout 138-fold after three rounds of panning, suggesting thatthe liver cancer antigens had selected the phages in the librariesdisplaying Fab. The initial identification of the antibody frag-ment showed that the phage displaying Fab had bindingspecificity with the antigen related to liver cancer. Consideringthat nowadays there are few and hypo-specific antibodies re-lated to liver cancer, the immune Fab antibody phage displaylibraries of hepatoma can be used to select the affinity andspecificity antibodies related to liver cancer. It is important todiagnose and treat liver cancer using these antibodies.

Acknowledgment

This research was supported by the Health DepartmentResearch Foundation of Hunan Province (A2003001).

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Address correspondence to:Dr. Guan-Cheng Li

Cancer Research InstituteXiang-Ya School of Medicine

Central South University88 Xiangya RoadChangsha 410078Hunan Provience

China

E-mail: libsun@public.cs.hn.cn

Received: February 27, 2009Accepted: April 27, 2009

LIVER CANCER Fab ANTIBODY 347