Skip to main content
NCBI home page
Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 2010 Mar 12;59(6):933–942. doi: 10.1007/s00262-010-0819-z

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

Yinting Chen 1, Kaihong Huang 1,✉, Xuexian Li 1, Xiangan Lin 1, Zhaohua Zhu 1, Ying Wu 1
PMCID: PMC11030574  PMID: 20224978

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 construction of a novel cancer-targeted agent for cancer diagnosis 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 characteristics 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 equilibrium dissociation constant (K D). 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 Western 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 K D of this scFv was calculated to be 7.85 ± 0.93 × 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

Introduction

CD44 is a family of cell surface adhesion molecules and is known as the principal receptor of hyaluronate (HA), 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 alternatively 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 carcinoma [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 metastatic 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 genetically engineered antibodies have enabled the generation of the single-chain variable fragment (scFv) format of antibodies. 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 isolated by phage display technology [18, 19]. This technology permits displaying antibodies with high affinity against target antigens on the surface of bacteriophages after several 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 specificity 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 Institute. All cell lines were cultured and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone) supplemented with 10% fetal bovine serum (HyClone), 4 mM l-glutamine, 100 unit/ml penicillin, and 100 μg/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 6× His tag (Abcam) and fluorescein isothiocyanate (FITC)-conjugated mouse mAb against 6× 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 (Invitrogen). Complementary DNA (cDNA) was synthesized from 1 μg total RNA using RevertAid first-strand cDNA synthesis kit (Fermentas). Resulting first-strand cDNA was used as a template for amplification of VH and VL (including Vκ and Vλ) 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 5′ overhang. VH-backward and VL-forward, respectively, contained parts of the (Gly4Ser)3 linker motif at 5′ overhang which were overlapped with each other. The NotI restriction site (GCGGCCGC) was joined to VL-backward at 5′ 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 cytometric analysis. For affinity selection (biopanning), primary phage library after amplification (4 × 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-buffered 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 × 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 amplified and cloned into the expression vector pET-30b (+) (Novagen) with 6× 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 proteins were expressed from Escherichia coli growing in shake flasks at 37°C for 4 h under the induction of isopropyl-β-d-thiogalactoside (IPTG) (1 mM final concentration). 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 × 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 dithiothreitol (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 filtrated, 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 gastric epithelial cells) and A375 (human malignant melanoma 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 μg/ml) and immunofluorescence stained using FITC-conjugated mouse mAb against 6× His tag (Abcam, 10 μg/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 μg of total SGC-7901 cell proteins were subjected to 8% SDS-polyacrylamide 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 μg/ml), respectively, overnight at 4°C. Antibodies were detected by adding horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:5,000) for mAb, or mouse mAb against 6× 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 × 106 cells/ml) in PBS. After blocking, cells were incubated with anti-human CD44v6 mAb (Bender, 10 μg/ml) or anti-CD44v6 scFv (10 μg/ml) for 30 min at room temperature. After two rounds of washing, FITC-conjugated anti-mouse IgG for mAb (10 μg/ml) or FITC-conjugated mouse mAb against 6× His tag (Abcam, 10 μg/ml) for scFv was added to each sample and incubated 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 (K D) of anti-CD44v6 scFv was determined using a flow cytometry-based assay as previously described [22]. SGC-7901 cells were harvested from tissue culture flasks. 2 × 105 cells were incubated separately with anti-CD44v6 scFv in gradient dilutions from 0.0625 × 10−6–1.0 × 10−6 M at 4°C until equilibrium was reached. Detection of bound scFv was performed by incubation with FITC-conjugated mouse mAb against 6× His tag (Abcam, 10 μg/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 K D by Lineweaver–Burk method. Experiments were repeated three times, and the average K D 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–Vκ gene (Fig. 1a, b) was 10,00–1,100 bp, and VH–linker–Vλ 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 × 103 pfu/ml and that of amplified library was 4 × 109 pfu/ml (data not shown), which was sufficient for selection.

Fig. 1.

Fig. 1

Open in a new tab

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–Vκ format (a and b) and VH–linker–Vλ 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 Vκ-backward or Vλ-backward primers

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 immunomagnetic beads affinity purification. Based on three repetitive biopanning reactions with human CD44v6 antigen bound to a solid phase, one anti-human CD44v6 scFv was screened from the human phage-displayed scFv library. Obvious enrichment phenomenon was revealed in the repetitive biopanning procedures. Titer from the first round of biopanning was 1.7 × 106 pfu/ml, while titer from the second and the third rounds of biopanning was 1.3 × 108 and 2.3 × 1010 pfu/ml, respectively (data not shown).

Fig. 5.

Fig. 5

Open in a new tab

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 6× 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)

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 characteristics, 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.

Fig. 2.

Fig. 2

Open in a new tab

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 6× 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

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 μg/ml and detected by incubation with FITC-conjugated mouse mAb against 6× 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.

Fig. 3.

Fig. 3

Open in a new tab

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 6× His tag (Abcam). Nuclei were counterstained with Hoechst 33342, and anti-CD44v6 scFv and nuclei images were merged. As shown in the figure, green fluorescence only appeared in SGC-7901 (color figure online)

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. 4.

Fig. 4

Open in a new tab

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 μg of total SGC-7901 cell proteins and 0.18 μg 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)

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 determine the K D 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 6× His tag. Fluorescence intensity was measured by flow cytometry. The K D of the interaction between the scFv and CD44v6 was then determined by Lineweaver–Burk kinetic analysis (Fig. 6). The calculated K D of anti-CD44v6 scFv was found to be 7.85 ± 0.93 × 10−8 M. As shown in Fig. 6, the scFv bound with high efficiency to SGC-7901 cells starting at 0.125 × 10−6 M (2.5 μg/ml) and continuously increasing until 0.5 × 10−6 M (10 μg/ml).

Fig. 6.

Fig. 6

Open in a new tab

Determination of anti-CD44v6 scFv K D value by Lineweaver–Burk method. CD44v6 expressing SGC-7901 cells were the target for binding experiment presented. SGC-7901 cells were incubated with anti-CD44v6 scFv at concentrations from 0.0625 × 10−6–1.0 × 10−6 M. Then, cells were stained with FITC-conjugated secondary antibody, and fluorescence intensity was measured by flow cytometry. This experiment was done independently for three times as indicated by different symbols. The average K D value of anti-CD44v6 scFv was 7.85 ± 0.93 × 10−8 M

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 efficient, 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 following characteristics: (1) high specificity and affinity to the target antigen; (2) no immunogenicity in patients; (3) small 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 therapy, 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 malignant melanoma cell line A375. K D value of anti-CD44v6 scFv is determined to be 7.85 ± 0.93 × 10−8 M by flow cytometry-based assay. This K D 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 constructed 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 valuable 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.

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-β-d-thiogalactoside

References

  • 1.Underhill C. CD44: the hyaluronan receptor. J Cell Sci. 1992;103:293–298. doi: 10.1242/jcs.103.2.293. [DOI] [PubMed] [Google Scholar]
  • 2.Picker LJ, Nakache M, Butcher EC. Monoclonal antibodies to human lymphocyte homing receptors define a novel class of adhesion molecules on diverse cell types. J Cell Biol. 1989;109:927–937. doi: 10.1083/jcb.109.2.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bourguignon LY. Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression. Semin Cancer Biol. 2008;18:251–259. doi: 10.1016/j.semcancer.2008.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Lesley J, Hyman R, Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol. 1993;54:271–335. doi: 10.1016/S0065-2776(08)60537-4. [DOI] [PubMed] [Google Scholar]
  • 5.Jalkanen S, Bargatze RF, de los Toyos J, Butcher EC. Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85–95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J Cell Biol. 1987;105:983–990. doi: 10.1083/jcb.105.2.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Günthert U, Hofmann M, Rudy W, et al. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell. 1991;65:13–24. doi: 10.1016/0092-8674(91)90403-L. [DOI] [PubMed] [Google Scholar]
  • 7.Screaton GR, Bell MV, Jackson DG, et al. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci USA. 1992;89:12160–12164. doi: 10.1073/pnas.89.24.12160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fox SB, Fawcett J, Jackson DG, et al. Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms. Cancer Res. 1994;54:4539–4546. [PubMed] [Google Scholar]
  • 9.Günthert U. CD44: a multitude of isoforms with diverse functions. Curr Top Microbiol Immunol. 1993;184:47–63. doi: 10.1007/978-3-642-78253-4_4. [DOI] [PubMed] [Google Scholar]
  • 10.Mackay CR, Terpe HJ, Stauder R, et al. Expression and modulation of CD44 variant isoforms in humans. J Cell Biol. 1994;124:71–82. doi: 10.1083/jcb.124.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sneath RJ, Mangham DC. The normal structure and function of CD44 and its role in neoplasia. Mol Pathol. 1998;51:191–200. doi: 10.1136/mp.51.4.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Goodison S, Urquidi V, Tarin D. CD44 cell adhesion molecules. Mol Pathol. 1999;52:189–196. doi: 10.1136/mp.52.4.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rudzki Z, Jothy S. CD44 and the adhesion of neoplastic cells. Mol Pathol. 1997;50:57–71. doi: 10.1136/mp.50.2.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Endo K, Terada T. Protein expression of CD44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J Hepatol. 2000;32:78–84. doi: 10.1016/S0168-8278(00)80192-0. [DOI] [PubMed] [Google Scholar]
  • 15.Ponta H, Sherman L, Herrlich PA. CD44: from adhesion molecules to signaling regulators. Nat Rev Mol Cell Biol. 2003;4:33–45. doi: 10.1038/nrm1004. [DOI] [PubMed] [Google Scholar]
  • 16.Heider KH, Kuthan H, Stehle G, Munzert G. CD44v6: a target for antibody-based cancer therapy. Cancer Immunol Immunother. 2004;53:567–579. doi: 10.1007/s00262-003-0494-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bird RE, Hardman KD, Jacobson JW, et al. Single-chain antigen-binding proteins. Science. 1988;242:423–426. doi: 10.1126/science.3140379. [DOI] [PubMed] [Google Scholar]
  • 18.Hoogenboom HR, de Bruïne AP, Hufton SE, et al. Antibody phage display technology and its applications. Immunotechnology. 1998;4:1–20. doi: 10.1016/S1380-2933(98)00007-4. [DOI] [PubMed] [Google Scholar]
  • 19.Griffiths AD, Duncan AR. Strategies for selection of antibodies by phage display. Curr Opin Biotechnol. 1998;9:102–108. doi: 10.1016/S0958-1669(98)80092-X. [DOI] [PubMed] [Google Scholar]
  • 20.Gram H, Marconi LA, Barbas CF, 3rd, et al. In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library. Proc Natl Acad Sci USA. 1992;89:3576–3580. doi: 10.1073/pnas.89.8.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990;348:552–554. doi: 10.1038/348552a0. [DOI] [PubMed] [Google Scholar]
  • 22.Benedict CA, MacKrell AJ, Anderson WF. Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay. J Immunol Methods. 1997;201:223–231. doi: 10.1016/S0022-1759(96)00227-X. [DOI] [PubMed] [Google Scholar]
  • 23.Moshfeghi AA, Peyman GA. Micro- and nanoparticulates. Adv Drug Deliv Rev. 2005;57:2047–2052. doi: 10.1016/j.addr.2005.09.006. [DOI] [PubMed] [Google Scholar]
  • 24.Hong RL, Lee WJ, Shun CT, Chu JS, Chen YC. Expression of CD44 and its clinical implication in diffuse-type and intestinal-type gastric adenocarcinomas. Oncology. 1995;52:334–339. doi: 10.1159/000227485. [DOI] [PubMed] [Google Scholar]
  • 25.Heider KH, Dämmrich J, Skroch-Angel P, et al. Differential expression of CD44 splice variants in intestinal- and diffuse-type human gastric carcinomas and normal gastric mucosa. Cancer Res. 1993;53:4197–4203. [PubMed] [Google Scholar]
  • 26.Müller W, Schneiders A, Heider KH, et al. Expression and prognostic value of the CD44 splicing variants v5 and v6 in gastric cancer. J Pathol. 1997;183:222–227. doi: 10.1002/(SICI)1096-9896(199710)183:2<222::AID-PATH923>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  • 27.Marks C, Marks JD. Phage libraries—a new route to clinically useful antibodies. N Engl J Med. 1996;335:730–733. doi: 10.1056/NEJM199609053351008. [DOI] [PubMed] [Google Scholar]
  • 28.Adams GP, Schier R, McCall AM, et al. High affinity restricts the localization and tumor penetration of single-chain Fv antibody molecules. Cancer Res. 2001;61:4750–4755. [PubMed] [Google Scholar]
  • 29.Muyldermans S, Lauwereys M. Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies. J Mol Recognit. 1999;12:131–140. doi: 10.1002/(SICI)1099-1352(199903/04)12:2<131::AID-JMR454>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  • 30.Cai X, Garen A. Comparison of fusion phage libraries displaying VH or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules. Proc Natl Acad Sci USA. 1997;94:9261–9266. doi: 10.1073/pnas.94.17.9261. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cancer Immunology, Immunotherapy : CII are provided here courtesy of Springer

RESOURCES