Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro

ORIGINAL ARTICLE

Generation of a stable anti-human CD44v6 scFv and analysisof its cancer-targeting ability in vitro

Yinting Chen • Kaihong Huang • Xuexian Li •

Xiangan Lin • Zhaohua Zhu • Ying Wu

Received: 23 June 2009 / Accepted: 8 January 2010 / Published online: 12 March 2010

� Springer-Verlag 2010

Abstract CD44v6 is a cancer-associated antigen that

mainly expresses in a subset of adenocarcinomas. Therefore,

in this study, anti-human CD44v6 single-chain variable

fragment (scFv) has been selected and characterized because

it is the first step of primary importance towards the con-

struction of a novel cancer-targeted agent for cancer diag-

nosis and therapy. In our study, anti-human CD44v6 scFv

was selected from a human phage-displayed scFv library

based on its ability to bind in vitro to CD44v6 antigen.

Subsequently, immunofluorescent staining and Western blot

analyses were performed to measure the binding character-

istics of this scFv. In addition, flow cytometric analysis was

done to verify its cancer-targeting ability in vitro. And a flow

cytometry-based assay was used to determine its equilib-

rium dissociation constant (KD). Finally, one functional anti-

CD44v6 scFv was selected and characterized. Nucleotide

sequencing verified that it was an incomplete scFv gene but

had a variable heavy chain (VH) alone. However, anti-

CD44v6 scFv demonstrated cell-binding and antigen-

binding activities by immunofluorescent staining and Wes-

tern blot analyses. Furthermore, flow cytometric analysis

proved that this scFv specifically targeted CD44v6-

expressing cancer cells other than CD44v6 non-expressing

normal cells or tumor cells in vitro. The KD of this scFv was

calculated to be 7.85 ± 0.93 9 10-8 M. In summary, the

selected human scFv against CD44v6 has specific binding

activity and favorable binding affinity despite lacking a

variable light chain (VL). Moreover, it can effectively and

specifically target CD44v6-expressing cancer cells. All

these characteristics make anti-CD44v6 scFv a promising

agent for cancer detection and anti-cancer therapy.

Keywords CD44 variant 6 � Cancer-targeting �Single-chain antibody fragment � Phage display

Abbreviations

CD44v CD44 variant

CD44s CD44 standard form

scFv Single-chain variable fragment

VH Variable heavy chain

VL Variable light chain

GCa Gastric carcinoma

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

KD Equilibrium dissociation constant

mAb Monoclonal antibody

SOE Splicing overlap extension

IPTG Isopropyl-b-D-thiogalactoside

Introduction

CD44 is a family of cell surface adhesion molecules and is

known as the principal receptor of hyaluronate (HA),

Y. Chen � K. Huang (&) � X. Li � X. Lin � Z. Zhu � Y. Wu

Department of Gastroenterology, The Second Affiliated

Hospital, Sun Yat-sen University, 107 Yanjiang Road West,

510120 Guangzhou, China

e-mail: [email protected]

Y. Chen


X. Li


X. Lin


Z. Zhu


Y. Wu


123

Cancer Immunol Immunother (2010) 59:933–942

DOI 10.1007/s00262-010-0819-z

which is a component of the extracellular matrix [1].

Binding of CD44 to HA mediates cell–cell and cell–matrix

interactions by activating specific signaling pathways [2–4].

Functionally, CD44 is involved in lymphocyte homing,

cell aggregation, adhesion, migration, tumor progression

and metastasis [3–6]. CD44 is often expressed in a variety

of isoforms, and all isoforms are encoded by a single gene

that consists of at least 20 exons, 10 of which are alter-

natively spliced and called variant exons (v1–v10) [7].

CD44 standard form (CD44s), which lacks all variant

exons, is widely distributed in normal tissues and aids in

maintenance of the 3-dimensional tissue/organ structure

[8]. In contrast to CD44s, CD44 variant isoform (CD44v) is

generated by the alternatively splicing of ten variant exons

at a distinct site of the extracellular portion of the CD44s

transcript to give rise to variable extracellular domains [7].

Furthermore, CD44v possesses some unique functional

properties significantly different from those observed in

CD44s [9–11].

CD44 variant isoforms have more restricted expressions

and correlate with the progression of certain types of car-

cinoma [6, 9–11]. CD44v6 is the variant that has been

studied most extensively, since the demonstration that the

transfection of spliced variants CD44v4–v7 was capable of

conferring metastatic potential on cells of a nonmetastatic

rat tumor cell line [6]. CD44 variant isoforms, especially

CD44v6, have been identified as protein markers for met-

astatic behavior in epithelium-derived cancers, such as

hepatocellular, breast, colorectal and gastric cancers [12–15].

All these facts have made CD44v6 become an attractive

factor for the detection of metastasis of epithelium-derived

cancers [16]. Therefore, the application of anti-CD44v6

monoclonal antibody (mAb) to prevent cancer metastasis is

promising.

Recombinant antibodies have become important agents

for diagnosis, prevention and treatment of a wide range of

diseases for their high specificity and affinity to target

antigens. However, clinical application of mAb produced

by the classic hybridoma technique has been hampered

because of its large molecular size (150 kDa) and harmful

immune response in patients. Recent advances in geneti-

cally engineered antibodies have enabled the generation of

the single-chain variable fragment (scFv) format of anti-

bodies. In this format, the variable domains of the heavy

chain (VH) and the light chain (VL) are connected with a

flexible peptide linker [17]. ScFv antibodies specific for a

broad variety of antigens have been most commonly iso-

lated by phage display technology [18, 19]. This technol-

ogy permits displaying antibodies with high affinity against

target antigens on the surface of bacteriophages after sev-

eral rounds of affinity selection (biopanning) [20, 21].

Human scFv antibodies obtained by this technology have

the smallest antibody fragment (25 kDa) that retains

specific binding characteristics without attacking the

patient’s immune system. Moreover, scFv antibodies can

be produced in a large scale by an economic production

method, such as Escherichia coli, thereby adding to their

potential therapeutic value.

In this report, we focused on the significance of CD44v6

as a target antigen for novel antibody-based treatment

modalities. We constructed a human phage-displayed scFv

library and selected anti-human CD44v6 scFv from this

library. Anti-CD44v6 scFv was expressed in Escherichia

coli, purified and refolded. Its binding activity and speci-

ficity to cells and antigen were tested. Further, anti-

CD44v6 scFv was proven to have cancer-targeting ability

to bind CD44v6-expressing cancer cells in vitro. The

characteristics of anti-CD44v6 scFv made it an ideal anti-

cancer agent for antibody-guided therapy in the prevention

and treatment of cancer.

Materials and methods

Cell lines and antibodies

Human gastric carcinoma cell line SGC-7901 was obtained

from Institute of Biochemistry and Cell Biology, Chinese

Academy of Sciences (Shanghai, China). Non-malignant

human gastric epithelial immortalized cell line GES-1 was

purchased from Beijing Institute for Cancer Research

Collection. And human malignant melanoma cell line,

A375, was kindly provided by the Shanghai Cancer Insti-

tute. All cell lines were cultured and maintained in Dul-

becco’s Modified Eagle’s Medium (DMEM, HyClone)

supplemented with 10% fetal bovine serum (HyClone),

4 mM L-glutamine, 100 unit/ml penicillin, and 100 lg/ml

streptomycin. The culture was then incubated in a humid

atmosphere of 5% CO2/95% air at 37�C.

Antibodies mainly used included mouse mAb against

human CD44v6 (Bender), mouse mAb against human

CD44std (Bender), mouse mAb against 69 His tag (Abcam)

and fluorescein isothiocyanate (FITC)-conjugated mouse

mAb against 69 His tag (Abcam). Recombinant human

soluble CD44std protein was purchased from Bender.

Construction of human phage-displayed scFv library

Blood was collected from newborn babies, healthy people

and patients of gastric, colorectal and pancreatic carcinoma

to increase the diversity of library. Then lymphocytes were

separated by density gradient centrifugation, and total RNA

of lymphocytes was isolated by TRIzol Reagent (Invitro-

gen). Complementary DNA (cDNA) was synthesized from

1 lg total RNA using RevertAid first-strand cDNA syn-

thesis kit (Fermentas). Resulting first-strand cDNA was

934 Cancer Immunol Immunother (2010) 59:933–942

123

used as a template for amplification of VH and VL

(including Vj and Vk) gene segments, and primers were

designed based on V-base (http://vbase.mrc-cpe.cam.ac.uk/)

with minor modifications. VH-forward was connected

with the SalI restriction site (GTCGAC) at 50 overhang.

VH-backward and VL-forward, respectively, contained

parts of the (Gly4Ser)3 linker motif at 50 overhang which

were overlapped with each other. The NotI restriction site

(GCGGCCGC) was joined to VL-backward at 50 overhang.

Finally, cDNAs encoding VH–linker and linker–VL were

assembled randomly by splicing overlap extension (SOE)

PCR to yield the full-length form (VH–linker–VL) of scFv

encoding gene flanked by the SalI and NotI restriction sites.

The amplified products from the SOE-PCR components

were cloned into T71-2b phage vector DNA (Novagen).

After in vitro packaging, plaque assay was performed to

determine the number of recombinant phages generated. In

brief, E. coli BLT5403 (Novagen), growing in the log

phase, was infected by phages in serial dilution. Then

molten top agarose (10 g/liter Bacto tryptone, 5 g/liter

yeast extract, 5 g/liter NaCl, 6 g/liter agarose) was added

to the above mixtures, and the contents were poured onto

pre-warmed Luria–Bertani (LB) agar plates. The plates

were incubated for 3–4 h at 37�C before the plaques were

counted. The phage titer, described in plaque forming units

(pfu) per unit volume, was the number of plaques on the

plate times the dilution times 10. The primary phage library

(packaged phage) was amplified prior to biopanning by

liquid lysate method, and the lysate was titered by plaque

assay.

Affinity selection of scFv against CD44v6

Pure human CD44v6 antigen was purified from proteins of

human gastric carcinoma (GCa) cell line SGC-7901 using

Dynabeads M-280 Tosylactivated (Dynal Biotech) and

anti-human CD44v6 mAb (Bender), after the expression of

CD44v6 on SGC-7901 cells was confirmed by flow cyto-

metric analysis. For affinity selection (biopanning), pri-

mary phage library after amplification (4 9 109 pfu) was

incubated overnight at 4�C in ELISA plate coated with

pure human CD44v6 antigen. ELISA plate was then

washed five times with 0.05% Tween 20/Tris–HCl-buf-

fered saline (TBS) (pH 7.4), and bound phages were eluted

by 1%SDS. The collected phages were titered by the

above-mentioned plaque assay. Meanwhile, the culture was

used to reinfect E. coli BLT5403 growing in the log phase

and incubated with shaking at 37�C. When lysis was

observed, the culture was centrifuged at 8,000 9 g for

10 min, and the supernatant containing selected and

amplified phages was stored at 4�C for the next round of

biopanning. This biopanning procedure was performed for

three rounds. The selected phages in the third round of

biopanning were titered, and individual clones were

obtained for further analysis.

DNA sequencing of the selected scFv

Individual clones of the third round biopanning were

scraped to yield a sufficient amount of phage DNA for PCR

amplification using T7Select Up and T7Select Down

primers. Both DNA strands of the PCR products were

sequenced (Sangon, Shanghai). The nucleotide sequences

were analyzed by online tool IgBlast (http://www.ncbi.

nlm.nih.gov/igblast/). Determination of framework regions

(FWRs), complementarity determining regions (CDRs) and

immunoglobulin families was performed according to the

Kabat and Chothia databases.

Expression, purification and refolding of scFv

The encoding gene of anti-CD44v6 scFv was PCR ampli-

fied and cloned into the expression vector pET-30b (?)

(Novagen) with 69 His tag engineered at the C- and

N-terminus of the peptide to facilitate purification. E. coli

BL21(DE3)plysS (Promega) was infected with the

recombinant plasmid. Then anti-CD44v6 scFv fusion pro-

teins were expressed from Escherichia coli growing in

shake flasks at 37�C for 4 h under the induction of iso-

propyl-b-D-thiogalactoside (IPTG) (1 mM final concentra-

tion). To isolate protein from cellular extracts, cells were

collected by centrifugation and disrupted by BugBuster

Protein Extraction Reagent (Novagen). The pellet which

contained scFv proteins in the form of inclusion body was

saved by centrifugation at 16,000 9 g for 20 min at 4�C.

The resulting proteins were then applied to a Ni-MAC

Cartridge (Novagen) under denaturing conditions (6 M

urea). Recombinant proteins were eluted by increasing

concentrations of imidazole in the presence of 6 M urea.

The eluted proteins were then refolded by convenient

dialysis. Briefly, the samples were first dialyzed against the

buffer containing 20 mM Tris–HCl and 0.1 mM dithio-

threitol (DTT) over a period of 6–12 h at 4�C. Afterwards,

the buffer was changed to 20 mM Tris–HCl by dialysis for

6–12 h at 4�C. Finally, the protein preparations were fil-

trated, sterilized and stored at -80�C. The collected protein

fractions were then analyzed using sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) and

Western blot.

Detection of scFv binding activity to cell and antigen

Binding activity of scFv to CD44v6-expressing SGC-7901

cells was assessed by immunofluorescent staining. SGC-

7901 (human gastric cancer cells), GES-1 (normal human

Cancer Immunol Immunother (2010) 59:933–942 935

123

http://vbase.mrc-cpe.cam.ac.uk/

http://www.ncbi.nlm.nih.gov/igblast/

http://www.ncbi.nlm.nih.gov/igblast/

gastric epithelial cells) and A375 (human malignant mel-

anoma cells) were grown, respectively, in 24-well plates

and fixed with 4% formaldehyde/phosphate-buffered saline

(PBS) (pH 7.4). After nonspecific sites were blocked, each

type of cell was incubated with anti-CD44v6 scFv (10 lg/ml)

and immunofluorescence stained using FITC-conjugated

mouse mAb against 69 His tag (Abcam, 10 lg/ml). Nuclei

were stained with Hoechst 33342. Photographs were taken

under fluorescent microscopy.

Specific antigen binding of anti-CD44v6 scFv was

evaluated in comparison to anti-human CD44v6 mAb

(Bender) by Western blot analysis. About 40 lg of total

SGC-7901 cell proteins were subjected to 8% SDS-poly-

acrylamide mini-gels. After electrophoresis, the proteins

were transferred to polyvinylidene difluoride (PVDF)

membranes, and then blocked with 5% non-fat dry milk in

0.1% Tween 20/TBS (pH 7.4) for 2 h at room temperature.

Then protein membranes were incubated with anti-human

CD44v6 mAb (Bender, 1:1,000) or anti-CD44v6 scFv

(1 lg/ml), respectively, overnight at 4�C. Antibodies were

detected by adding horseradish peroxidase (HRP)-conju-

gated anti-mouse IgG (1:5,000) for mAb, or mouse

mAb against 69 His tag (Abcam, 1:2,000) followed by

HRP-conjugated anti-mouse IgG (1:5,000) as the third

antibody for scFv. Signals were detected by enhanced

chemiluminescence.

To further examine if the scFv binds specifically to the

variant isoform 6 rather than standard form of CD44, total

proteins of SGC-7901 and recombinant human soluble

CD44std protein (Bender) were analyzed by Western blot

using anti-human CD44v6 mAb, anti-CD44v6 scFv or anti-

human CD44std mAb (Bender, 1:1,000), respectively, as

primary antibody. Detailed process was carried out as the

above-mentioned.

Identification of scFv cancer-targeting ability in vitro

To identify the cancer-targeting ability of anti-CD44v6

scFv in vitro, flow cytometric analysis was performed on

CD44v6-expressing SGC-7901 (human gastric cancer

cells), along with CD44v6 non-expressing GES-1 (normal

human gastric epithelial cells) and A375 (human malignant

melanoma cells). Each cell line was detected by both anti-

human CD44v6 mAb and anti-CD44v6 scFv. All cells

were separately removed from culture flasks, washed and

resuspended (1 9 106 cells/ml) in PBS. After blocking,

cells were incubated with anti-human CD44v6 mAb

(Bender, 10 lg/ml) or anti-CD44v6 scFv (10 lg/ml) for

30 min at room temperature. After two rounds of washing,

FITC-conjugated anti-mouse IgG for mAb (10 lg/ml) or

FITC-conjugated mouse mAb against 69 His tag (Abcam,

10 lg/ml) for scFv was added to each sample and incu-

bated for 30 min at room temperature. The cells were then

washed repeatedly, detected and analyzed by flow

cytometry.

Determination of scFv KD value

Equilibrium dissociation constant (KD) of anti-CD44v6

scFv was determined using a flow cytometry-based assay

as previously described [22]. SGC-7901 cells were har-

vested from tissue culture flasks. 2 9 105 cells were

incubated separately with anti-CD44v6 scFv in gradient

dilutions from 0.0625 9 10-6–1.0 9 10-6 M at 4�C until

equilibrium was reached. Detection of bound scFv was

performed by incubation with FITC-conjugated mouse

mAb against 69 His tag (Abcam, 10 lg/ml). Then relative

fluorescence intensity of stained cells was analyzed by flow

cytometer to detect cell-bound antibody. The inverse of the

fluorescence intensity was plotted as a function of the

inverse of the scFv concentration to determine KD by

Lineweaver–Burk method. Experiments were repeated

three times, and the average KD values were reported as

mean ± standard error of mean. Values and graphical

analysis were generated using Sigma Plot 10.0.

Results

Construction, selection and sequencing of anti-CD44v6

scFv

Human VH and VL gene segments were PCR amplified and

assembled by linker primers and SOE-PCR to yield the

full-length form of scFv encoding gene. The amplified

products from the SOE-PCR components were about 800–

1,100 bp as shown in agarose gel (1.5%) electrophoresis

(Fig. 1). According to the marker, VH–linker–Vj gene

(Fig. 1a, b) was 10,00–1,100 bp, and VH–linker–Vk gene

(Fig. 1c, d) was 800–900 bp. The assembled scFv encoding

gene flanked by SalI and NotI restriction sites were

digested and cloned into T71-2b phage vector DNA. The

titer of primary human scFv library was 1 9 103 pfu/ml

and that of amplified library was 4 9 109 pfu/ml (data not

shown), which was sufficient for selection.

To select anti-CD44v6 scFv from human scFv library,

pure human CD44v6 antigen was obtained from CD44v6-

expressing cells. Human GCa cell line SGC-7901 was the

candidate. CD44v6 was highly expressed on SGC-7901

cells, and expression rate was over 95% according to flow

cytometric analysis (Fig. 5a). Pure human CD44v6 antigen

was isolated from proteins of SGC-7901 cells by immu-

nomagnetic beads affinity purification. Based on three

repetitive biopanning reactions with human CD44v6 anti-

gen bound to a solid phase, one anti-human CD44v6 scFv

was screened from the human phage-displayed scFv


123

library. Obvious enrichment phenomenon was revealed in

the repetitive biopanning procedures. Titer from the first

round of biopanning was 1.7 9 106 pfu/ml, while titer

from the second and the third rounds of biopanning was

1.3 9 108 and 2.3 9 1010 pfu/ml, respectively (data not

shown).

The nucleotide and deduced amino acid sequences of the

selected anti-CD44v6 scFv encoding gene were analyzed.

The whole nucleotide sequence, including restriction sites,

was 767 bp and was composed of the VH gene segment

(390 bp), the (Gly4Ser)3 linker (45 bp) and the VL gene

segment (318 bp). However, because of termination

codons appearing at the end of the VH gene segment, the

scFv protein expression was terminated halfway. As a

result, the scFv was not a full-length form but contained VH

domain alone (Fig. 1e). The positions of CDRs and FWRs

of the scFv were identified by using the Kabat and Chothia

numbering schemes (http://www.bioinf.org.uk/abs). Based

on sequence homology search in the European Molecular

Biology Laboratory and GenBank databases, the VH region

belonged to the Kabat human heavy chain subgroup HV1.

SDS-PAGE and Western blot analyses of scFv

Although the scFv was not a full-length form but had VH

domain alone, the scFv could be utilized for anti-cancer

agent based on the premise that the scFv had specific

binding activity and favorable binding affinity. To detect

whether the scFv with VH domain alone had such charac-

teristics, the anti-CD44v6 scFv encoding gene was

restriction digested with SalI and NotI and cloned into

pET-30b (?). Anti-CD44v6 scFv fusion proteins were

expressed in E. coli BL21(DE3)plysS cells by IPTG

induction. The different protein fractions collected from

IPTG-induced cells, together with affinity-purified scFv

proteins and total proteins of non-induced cells, were

separated by 12% SDS-polyacrylamide mini-gels and then

either stained with Coomassie brilliant blue or transferred

to PVDF membranes after electrophoresis. Anti-CD44v6

scFv fusion proteins were found in the form of inclusion

body (Fig. 2). The predicted molecular mass of the scFv

was 20 kDa, which was confirmed in the SDS-PAGE and

Western blot analyses.

Binding characteristics of anti-CD44v6 scFv

To identify whether the anti-CD44v6 scFv recognizes

specifically SGC-7901 (human gastric cancer cells) but not

GES-1 (normal human gastric epithelial cells) or A375

(human malignant melanoma cells), immunofluorescent

staining was performed. Each cell line was incubated with

anti-CD44v6 scFv at 10 lg/ml and detected by incubation

Fig. 1 Gel electrophoresis and sequencing of the full-length scFv

encoding gene. Gene segments encoding the variable heavy chain

(VH) and variable light chain (VL) were assembled by linker primers

and splicing overlap extension (SOE) PCR to yield the full-length

(VH–linker–VL) scFv encoding gene. The amplified products from the

SOE-PCR components were resolved in 1.5% agarose gel and stained

with colloidal gold. Furthermore, both DNA strands were sequenced,

and amino acid sequence of VH was deduced. The full-length scFv

construct contained VH–linker–Vj format (a and b) and VH–linker–

Vk format (c and d). Indicated are framework regions (FWR) and

complementarity determining regions (CDR) according to the Kabat

and Chothia numbering schemes (e). Lane marker 100 bp DNA

ladder; lanes 1–9 the amplified VH–linker–VL products using nine

different VH-forward primers and Vj-backward or Vk-backward

primers


123

http://www.bioinf.org.uk/abs

with FITC-conjugated mouse mAb against 69 His tag.

Nuclei were stained in blue with Hoechst 33342. As shown

in Fig. 3, strong green fluorescence appeared in the cellular

membrane of SGC-7901, whereas no green fluorescence

was shown in GES-1 or A375. It demonstrated highly

efficient binding of the scFv to the surface of SGC-7901.

This scFv, meanwhile, reacted to SGC-7901 but not to

GES-1 or A375.

Subsequently, Western blot was used to confirm the

specificity of the scFv against human CD44v6. When total

proteins of SGC-7901 were subjected, a specific band at

about 82 kDa was both detected using anti-human CD44v6

mAb and the scFv as primary antibodies (Fig. 4a, b).

Although there were two other weak bands at 62 and

100 kDa detected in Fig. 4b, the 82 kDa band was the most

strong. Furthermore, Fig. 4c showed that SGC-7901

expressed CD44v6 as well as CD44std, as total proteins of

SGC-7901 were both stained with anti-human CD44v6

mAb and anti-human CD44std mAb with CD44std protein

(Bender) as a positive control. Meanwhile, CD44v6 and

CD44std expressed in SGC-7901 were nearly the same

molecular weight of 82 kDa. Moreover, anti-human

CD44v6 mAb and the scFv both detected the same band of

82 kDa when total proteins of SGC-7901 were subjected.

While CD44std protein (Bender) was loaded, neither anti-

human CD44v6 mAb nor the scFv recognized CD44std

with anti-human CD44std mAb as a positive control

(Fig. 4c). Therefore, these Western blot results indicated

that the scFv specifically recognized the same antigen as

anti-human CD44v6 mAb and did not react with CD44std.

Fig. 2 Analysis of the scFv expression by SDS-PAGE and Western

blot. Non-induced and IPTG-induced cells were harvested and

subjected to 12% discontinuous SDS-PAGE. Bacterial proteins were

stained with Coomassie brilliant blue (a) or transferred to PVDF

membranes for Western blot analysis using mouse mAb against 69

His tag (Abcam, 1:2,000) and HRP-conjugated secondary antibody

(1:5,000) (b). Lane marker low molecular weight protein markers;

lane 1 total proteins of non-induced cells; lane 2 total proteins of

IPTG-induced cells; lane 3 soluble proteins of IPTG-induced cells;

lane 4 inclusion bodies of IPTG-induced cells; lane 5 affinity-purified

scFv

Fig. 3 Cell-binding assay of

anti-CD44v6 scFv. CD44v6-

expressing SGC-7901 (human

gastric cancer cells), along with

CD44v6 non-expressing GES-1

(normal human gastric epithelial

cells) and A375 (human

malignant melanoma cells)

were, respectively, fixed and

incubated with anti-CD44v6

scFv. Then, the scFv was

detected using FITC-conjugated

mouse mAb against 69 His tag

(Abcam). Nuclei were

counterstained with Hoechst

33342, and anti-CD44v6 scFv

and nuclei images were merged.

As shown in the figure, greenfluorescence only appeared in

SGC-7901 (color figure online)


123

Cancer-targeting ability of anti-CD44v6 scFv in vitro

To find out the different expression rate of CD44v6 on

SGC-7901 (human gastric cancer cells), GES-1 (normal

human gastric epithelial cells) and A375 (human malignant

melanoma cells), flow cytometric analysis was done. As

shown in Fig. 5, CD44v6 was highly expressed on SGC-

7901 cells, and expression rate was 96.97% (Fig. 5a),

while there was hardly any expression of CD44v6 on

GES-1 or A375 cells (Fig. 5c, e). Accordingly, anti-

CD44v6 scFv had interaction with 97.67% of SGC-7901

cells (Fig. 5b) versus 0.15% of GES-1 cells (Fig. 5d) and

0.12% of A375 cells (Fig. 5f), which was similar to the

CD44v6 expression rate in SGC-7901, GES-1 and A375

cells, respectively.

Binding affinity of anti-CD44v6 scFv

A flow cytometry-based assay was performed to deter-

mine the KD value of the scFv binding to CD44v6-

expressing SGC-7901 cells. SGC-7901 cells were

incubated with anti-CD44v6 scFv in gradient dilutions,

and bound scFv was detected by FITC-conjugated mouse

mAb against 69 His tag. Fluorescence intensity was

measured by flow cytometry. The KD of the interaction

between the scFv and CD44v6 was then determined by

Lineweaver–Burk kinetic analysis (Fig. 6). The calculated

KD of anti-CD44v6 scFv was found to be 7.85 ±

0.93 9 10-8 M. As shown in Fig. 6, the scFv bound with

high efficiency to SGC-7901 cells starting at 0.125 9

10-6 M (2.5 lg/ml) and continuously increasing until

0.5 9 10-6 M (10 lg/ml).

Discussion

Cancer-targeted therapy requires that foreign materials,

including gene and drugs, be transferred to the targeted

tissues. And the main objective is the development of effi-

cient, non-toxic carriers that can encapsulate and deliver

foreign materials into specific cell types such as cancerous

cells. Antibodies have great potential to be applied to anti-

cancer therapy or used as targeted ligands for antibody-

mediated diagnostic and therapeutic agents, respecting their

high specificity and affinity to target antigens. Recently,

nanoparticles conjugated with monoclonal antibodies have

attracted much attention. These targeted nanoparticles can

target malignant tumors with high specificity and affinity

while reducing side-effects [23]. Ever since the invention of

monoclonal antibodies, many antibody-based therapeutics

have been used in basic or clinical research for the treatment

of cancer. Cancer-targeting antibodies should be against the

appropriate target antigen that is of high expression in

tumor tissues and almost no expression in normal tissues.

Various cell surface markers and/or receptors, including

those associated with cancer, have been explored as

potential targets. CD44v6 is an ideal target antigen that

displays a favorable pattern of expression according to

present studies. CD44v6 is mainly expressed in a subset of

adenocarcinomas, such as gastric, breast, colorectal and

hepatocellular cancers [12–15]. Especially in gastric cancer,

CD44v6 appears to be an ideal target antigen for its high

and homogeneous expression in most patients [24–26].

Antibodies for clinical application should fulfill the fol-

lowing characteristics: (1) high specificity and affinity to the

target antigen; (2) no immunogenicity in patients; (3) small

Fig. 4 Western blot analysis for specific antigen binding of anti-

CD44v6 scFv. Total proteins of SGC-7901 were analyzed by SDS-

PAGE in a 8% gel and transferred to the PVDF membrane for

Western blot analysis in a separate detection using anti-human

CD44v6 mAb (Bender) (a) or anti-CD44v6 scFv (b). Arrow indicated

a band at about 82 kDa shown in both a and b. Furthermore, to prove

that the scFv recognizes CD44v6 rather than CD44s, approximately

40 lg of total SGC-7901 cell proteins and 0.18 lg of recombinant

human soluble CD44std protein (Bender) were detected using anti-

human CD44v6 mAb, anti-CD44v6 scFv or anti-human CD44std

mAb as primary antibody, respectively. c Showed that CD44v6 and

CD44std were both expressed in SGC-7901 at the same molecular

weight of 82 kDa, and the scFv specifically recognized the same

antigen as anti-human CD44v6 mAb but did not react with CD44std.

Lane M broad range molecular weight protein markers; lane 1 SGC-

7901; lane 2 recombinant human soluble CD44std protein (Bender)


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molecular size and favorable tissue penetration; (4) validity

in a large amount by an economic production system.

Genetically engineered antibodies, such as scFv isolated

from phage display library, fulfill all these qualities and

have advantages over intact monoclonal antibodies.

To obtain a human scFv against CD44v6 for further

research on antibody-mediated cancer diagnosis and ther-

apy, we have conducted this current study. Our results

suggest that phage display libraries can effectively be used

to generate human scFvs with therapeutic potential. Here,

we have selected one anti-human CD44v6 scFv from a

human phage-displayed scFv library based on its ability to

bind in vitro to CD44v6 antigen. The selected scFv has

been sequenced, and it has demonstrated that it is not full-

length (VH–linker–VL), but has the VH domain alone.

However, scFv with VH or VL domain alone can also

strongly recognize antigens versus VH and VL pairs [27].

We have found that the selected scFv has high specificity

and affinity to CD44v6. This scFv maintains its specific

cell-binding and antigen-binding activity as shown in

immunofluorescent staining, flow cytometric and Western

blot analyses. Moreover, anti-CD44v6 scFv has cancer-

targeting activity to epithelium-derived cancer cells in

vitro, as it can specifically bind to human gastric carcinoma

cell line SGC-7901 but not to CD44v6 non-expressing

normal human gastric epithelial cell line GES-1 or human

Fig. 5 Flow cytometric

analysis for cancer-targeting

activity of anti-CD44v6 scFv in

vitro. SGC-7901, GES-1 and

A375 cells were separately

incubated with mouse anti-

human CD44v6 mAb (Bender)

or anti-CD44v6 scFv, and then

accordingly stained with FITC-

conjugated anti-mouse IgG for

mAb or FITC-conjugated

mouse mAb against 69 His tag

(Abcam) for scFv and analyzed.

The fluorescence on cells

stained with anti-human

CD44v6 mAb or anti-CD44v6

scFv was shown. The

expression rate of human

CD44v6 in SGC-7901 was

96.97% (a), while GES-1 was

0.07% (c) and A375 was 0.06%

(e). Similarly, the binding rate

of the scFv antibody in SGC-

7901 was 97.67% (b), while

GES-1 was 0.15% (d) and A375

was 0.12% (f)


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malignant melanoma cell line A375. KD value of anti-

CD44v6 scFv is determined to be 7.85 ± 0.93 9 10-8 M

by flow cytometry-based assay. This KD value is in the

optimum range of affinity constant from 10-7–10-11 M for

quantitative tumor retention in cancer therapy [28]. These

findings strengthen the fact that the presence of both VH

and VL domains is not necessary for the construction of an

effective antigen-binding unit. Similar observations appear

in the construction of a phage display library expressing VH

domains only [29, 30]. In addition, scFv with VH or VL

domain alone has even smaller molecular size (20 kDa),

resulting in better penetration through blood vessels to

tumors.

Furthermore, as shown in immunofluorescent staining

and flow cytometric analyses, anti-CD44v6 scFv is capable

of targeting epithelium-derived cancer cells that expressed

CD44v6, while there is no binding to CD44v6 non-

expressing normal epithelium cells or non-epithelium-

derived tumor cells. The in vitro binding of anti-CD44v6

scFv to adenocarcinoma cell line shows promise for

CD44v6 targeting cancer in vivo. And further studies are

currently underway to test the cancer-targeting potentials

of anti-CD44v6 scFv in vivo.

In conclusion, we have presented a simple and highly

efficient model for the generation of human scFvs with

potential clinical applications. In our study, one anti-human

CD44v6 scFv (VH domain alone) is selected from a con-

structed human phage-displayed scFv library. This scFv

has high specificity and affinity to CD44v6 antigen.

Furthermore, it can specifically recognize and bind to

CD44v6-expressing cancer cells. These results confirm that

anti-human CD44v6 scFv is a promising anti-cancer agent,

especially for epithelium-derived cancers, with high

expressions of CD44v6. Furthermore, anti-human CD44v6

scFv can also be attached to nanoparticle vector and

modified as antibody–nanoparticle fusion vehicles to

transport foreign materials to the targeted tissues for cancer

diagnosis and therapeutics. This is the aspect in which

future efforts should be focused on.

Acknowledgments We thank Xia Yang and Zhumin Xu for valu-

able discussions. We also thank Jing Wei for technical assistance in

flow cytometric analysis. National Natural Science Foundation of

China, Grant No. 30670951; National Natural Science Foundation of

Guangdong Province, China, Grant No. 6021322 are acknowledged.

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Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro

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