CD176 single-chain variable antibody fragment inhibits the adhesion of cancer cells to endothelial cells and hepatocytes

Abstract

CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate epitope (glycotope) functionally involved in blood spread and liver metastasis of cancer cells by mediating the adhesion of cancer cells to endothelial cells and hepatocytes, respectively. CD176 could be a promising target for antitumor immunotherapy. We applied B lymphocytes obtained from mice immunized with CD176 antigen and constructed a phage display library. A positive clone of CD176 single-chain variable antibody fragment (scFv) was successfully screened from this library. The CD176 scFv was expressed in Escherichia coli and purified. The purified scFv can bind to the natural CD176 expressed on the surface of cancer cells. Furthermore, the CD176 scFv inhibits the adhesion of CD176⁺ cancer cells to endothelial cells and hepatocytes. This CD176 scFv provides a basis for future development of recombinant CD176-specific antibodies that can be used in therapeutic application.

Keywords

CD176 / Thomsen-Friedenreich antigen / scFv / cancer / therapy / adhesion / metastasis

Introduction

Cancer is one of the primary causes of death worldwide. Anti-tumor antibody drugs are proven effective treatments for cancer patients. The development of single-chain variable antibody fragment (scFv) provided an approach for obtaining a therapeutic antibody. The scFv consists of variable regions of heavy chain (VH) and light chain (VL) and is the smallest unit of immunoglobulin with antigen-binding activity. The scFv appears to a great potential for in vivo diagnostics and therapeutic approaches, because the scFv is small molecular size and has lower retention time in non-target tissue, more rapid blood clearance, better tissue penetration [ 1]. More importantly, the scFv is among the most prevalent means in antibody engineering because of its low level of immunogenicity. For example, murine antibody can be successfully humanized using a murine scFv. However, because of more rapid blood clearance and lower interaction energy, the scFv exhibits limitations as a therapeutic antibody. To increase the avidity of scFv and to enhance therapeutic effects of antibody drugs, the scFv is often modified into a variety of forms, such as diabody, triabody, tetrabody, and bi-specific antibody. When anti-tumor antibody drugs are developed, the scFv genetically fuses with human Fc region in most cases, thereby allowing interaction with the patient’s immune system [ 2]. However, whether monovalent scFv, multivalent scFv, or humanized antibodies are used as therapeutic antibodies, acquisition of the scFv toward novel targets is one of the key steps for developing novel antibody drugs.

Thomsen-Friedenreich antigen (TF, Galb1-3GalNAca-1-Ser/Thr) is an oncofetal antigen [ 3]. TF was assigned as CD176 during the 7th Conference on Human Leucocyte Differentiation Antigens [ 4]. In normal and benign adult tissue, CD176 is often masked by terminal glycosylation, but it is exposed during tumorigenesis as a tumor-associated antigen [ 5]. Approximately, 70%–80% of carcinomas carry CD176 on their cell surface [ 3]. CD176 is also expressed on some cancer stem cells [ 6]. Importantly, CD176 is functionally involved in blood spreading and liver metastasis of cancer cells because CD176 plays an important role in docking cancer cells onto vascular wall and liver by specifically interacting with receptors expressed on endothelium and hepatocytes, respectively [ 7, 8]. Additionally, we observed that anti-CD176 antibody induces the apoptosis of leukemic cells [ 9, 10]. In our previous study, the CD176 antibody treatment could effectively prolong the survival time of CD176+ leukemia mice and inhibit the growth and spread of CD176+ cancer cells in bone marrow, spleen, liver, and lung [ 11]. These data demonstrated that CD176 antibody-based immunotherapy may have good clinical prospect.

Phage display is a widely used method to generate specific recombinant antibody fragments [ 12, 13]. In phage display, a scFv fragment can be displayed on the phage surfaces as a functional protein that retains an active antigen binding domain capability [ 14]. Phage libraries are used most often in the form of scFv [ 15]. Many phage libraries have been developed with different recombinant antibody fragments [ 16, 17]. In this study, we constructed a phage display library to screen a positive scFv that can bind to CD176. Thereafter, this scFv was expressed in Escherichia coli cells. Subsequently, we tested the biological characteristics of the CD176 scFv. These studies provide a basis for the future development of therapeutic drugs.

Materials and methods

Cell lines

SW480 (colorectal adenocarcinoma), SGC7901 (gastric carcinoma), and Caco-2 (colorectal adenocarcinoma) were cultured in Petri dishes containing Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO₂ at 37 °C. Acute myelogenous leukemia (KG-1) and immortalized human normal hepatocyte (HL-7702) were maintained in RPMI 1640 (GIBCO Invitrogen) containing 10% FBS. Human umbilical vein endothelium cell (HUVEC) was cultured in DMEM supplemented with 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement (ECGS), and 10% FBS.

Reagents

Asialoglycophorin A (aGP) and TF (CD176)-polyacrylamide (TF-PAA) were purchased from International Laboratory Ltd. (San Francisco, CA, USA) and Glyco Tech Corp. (Gaithersburg, MD, USA), respectively. E. coli, the suppressor strain TG1, and Dynabeads, were obtained from GIBCO Invitrogen. Helper phage M13KO7 and anti-M13 antibody were bought from GE Healthcare Life Sciences China (Beijing, China). CD176 monoclonal antibody, A78-G/A7 [ 18], was obtained from Glycotope, Berlin, Germany. Horse reddish peroxidase (HRP)-labeled anti-His tag antibody and Fluorescein Isothiocyanate (FITC)-labeled anti-His tag antibody were purchased from Abcam (Cambridge, UK).

Preparation of phage display library

Eight-week-old to ten-week-old BALB/c mice (Experimental Animal Center, Kunming Medical University, China) were housed at the Animal Facility, Kunming Institute of Zoology, Chinese Academy of Sciences. Mice were immunized three times by 10 mg aGP mixed with Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). After detecting CD176 antibody in the serum by solid-phase enzyme-linked immunosorbent assay (ELISA) using aGP and TF-PAA, spleen was obtained from immunized mice. Total RNA was isolated from spleen lymphocytes, the cDNA was synthesized by reverse transcription (RT)-polymerase chain reaction (PCR) with oligo-dT_12-18 as the primer according to the manufacturer’s instructions. All experiments were performed in accordance with the regulation for animal experimentation and were approved by authorities.

Construction of phage display library and panning

VH and VL genes were amplified by PCR and linked using a (G₄S)₃ linker as scFv. The scFv gene was cloned into pCANTAB-5E; both of them were digested by Not I and Sfi I restriction enzymes. The primers A–F, which were used in this vector construction, were described in Table S1. The vector was transformed into TG1 competent cells. The cells were seeded onto plates containing solid Luria-Bertani (LB) medium supplemented with 100 mg/ml ampicillin and 2% (w/v) glucose, and cultured at 37 °C overnight. The CD176 phage display library representing over 10⁹ independent mouse scFvs was subjected to four rounds of panning by dynabeads as described previously [ 19, 20].

Collection of phage display scFv and ELISA assay

Based on the successful construction of the naïve library, we used aGP and TF-PAA as the target antigen to isolate the antigen binders. About 1 × 10¹⁰ TG1 cells from the library stock were grown in 2 × Yeast Extract-Tryptone (2 × YT) medium containing 100 mg/ml ampicillin and 1% (w/v) glucose at 37 °C. After 2 h of culture, the cells were infected with 1 × 10¹² M13KO7 helper phages for 30 min at 30 °C. The infected cells were harvested and re-suspended into 2 × YT medium supplemented with 100 mg/ml ampicillin and 70 mg/ml kanamycin. Then, the cells were incubated overnight at 30 °C and 220 r/min. The phages were precipitated from culture supernatant with PEG 8000/NaCl and re-suspended in sterile phosphate buffered saline (PBS) [ 21]. About 2 × 10¹¹ phage particles were used for each bio-panning against aGP and TF-PAA (200 mg/ml) coated on 96-well plates. Antigens were coated on 96-well polystyrene microplates at 4 °C overnight. Uncoated antigens were washed off by PBS buffer and then blocked with 2% bovine serum albumin (BSA). The plates were incubated with collected phage particles or M13KO7 helper phage for 2 h at room temperature. CD176 mAb (IgM) [ 18] (biotin labeled) was used as a positive control. Subsequently, HRP-labeled anti-M13 antibody and avidin were added as a second antibody, followed by a color reaction developed with o-phenylenediamine dihydrochloride. After stopping the reaction with 2.5 mol/L sulfuric acid, the optical density value (OD) was determined using a microplate reader (BioRad, Hercules, CA, USA) at 490 nm. Then, the identified clones were collected for DNA sequencing.

Construction of the expressed vector and expression of the scFv in E. coli

The CD176 scFv gene was amplified by PCR from phagemids encoding respective scFv genes as templates. The primers G and H used for the vector construction were listed in Table S1. The scFv gene was ligated into NcoI-XhoI-digested pET32a(+) vector to construct pET-scFv expression vectors. E. coli DH5a and BL21 (DE3) were used as host cells for plasmid preparation and expression of scFv protein, respectively. The scFv sequence was confirmed by DNA sequencing.

The constructed plasmids were transformed into BL-21 E. coli bacterial cells, and a single colony was inoculated into a 50 ml LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, and 1% NaCl) supplemented with 100 mg/ml ampicillin, and cultured overnight at 37 °C with shaking at 250 r/min. The next day, the cultured medium was added into 1000 ml medium with 100 mg/ml ampicillin. The bacterial cells were grown in a shaking incubator at 37 °C until the OD reached 0.6 at 600 nm. The protein expression was induced by adding isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol/L. The bacterial cells were kept in a shaking incubator for 4 h at 30 °C and then spun by centrifugation at 5000 r/min for 15 min at 4 °C. The supernatant was removed, and the pellet was re-suspended in 5 ml PBS. The cells were lysed by sonication. Centrifugation was performed to remove detergent-insoluble materials at 10 000 r/min for 10 min at 4 °C. The cell lysates were determined by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as described previously [ 22]. After electrophoresis, the gel was stained with Coomassie brilliant blue on a slowly rotating platform for 2 h at room temperature. The gel was then destained in the methanol/acetic acid solution.

Protein purification from the bacterial cell lysates

The soluble proteins were purified through a 2 ml Ni-Sepharose^TM Fast-Flow resin (GE healthcare, Cat#18-1000-71) followed by elution of bound protein with the same solution buffer containing 500 mmol/L imidazole. Imidazole was eliminated by dialysis in 50 mmol/L TrisHCl for 24 h at 4 °C.

Ion exchange chromatography (IEC) was used for further purification. The scFv at 1 ml was diluted with 3 ml of 0.1 mol/L PBS at pH 6.0 and mixed with 20 ml of proteinase inhibitor mixture. The diluted scFv was applied to a Sephadex G-50 (superfine, 2.6 × 100 cm; Amersham Biosciences, Piscataway, NJ, USA) gel filtration column equilibrated with 0.1 mol/L PBS. The elution was performed with the same buffer, and fractions of 3 ml were collected. The absorbance of the eluate was monitored at 280 nm. The elution protein was digested by enterokinase to eliminate the fusion Trx·Tag™ thioredoxin protein. The purified proteins were analyzed with SDS-PAGE.

Flow cytometric analysis

The binding characteristic of the anti-CD176 scFv was assessed by fluorescence activated cell sorting (FACs). KG1 cells (a CD176⁺ cell line) were incubated with the scFv, and then treated with anti-His-FITC. After the staining, cells were analyzed using a FACS Caliber flow cytometer (BD Biosciences, Rockville, MD, USA).

Immunofluorescence staining

The CD176⁺ cell lines (KG1, SW480, Caco-2, and SGC7901) were prepared on glass slides. The cells were fixed in ice acetone and blocked by 2% BSA. Subsequently, the cells were incubated with the purified scFv for 1.5 h at room temperature. CD176 mAb (IgM) [ 18] was used as a positive control. The FITC-labeled anti-His antibody and goat anti-mouse IgM were used as second antibodies. The cell nuclei were stained with 4',6-diamidino-2-phenylindole. After mounting, staining results were observed with a laser scanning confocal microscopy.

Adhesion assay

HUVEC was grown to confluency in 96-well plate. SW480 cells (1 × 10⁴), which were transferred by using green fluorescent protein (GFP) gene using lentivirus, were added into the wells mixed with the scFv or BSA, and incubated at 37 °C. After 2, 5, and 12 h of incubation, the cells were washed. The result was observed under a fluorescence microscope.

We also performed a luciferase assay to determine the extent of the cellular adhesion that was inhibited by this scFv. Luciferase gene had been transferred into SW480 and SGC7901 cells through retrovirus-mediated transfer method. Subsequently, the cells were added into a 96-well plate in which HUVEC and HL-7702 were grown to confluency. Cells were incubated for 12 h at 37 °C. The plates were washed by PBS, and the luciferase activities were measured using Dual-Luciferase Reporter System (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions.

Results

The CD176 phage display library was constructed and screened

To generate a large and highly diverse scFv library, spleen cells were isolated from mice immunized by aGP. Total RNA was extracted. The cDNA was synthesized by employing both random and oligo-dt primers. The VH and VL of immunoglobulin were amplified by PCR at different conditions to maximally preserve the initial diversity.

A library containing about 1×10⁹ clones was constructed. After four rounds of panning with dynabeads, more than 500 individual clones were grown, and recombinant phages were tested for binding to aGP and TF-PAA by ELISA. In ELISA, the titer of recombinant phages was standardized to 5.6×10¹⁰ phage particles per well. The parent phage (M13KO7) was used as a negative control. A mouse anti-CD176 mAb was used as a positive control. A positive clone was selected and named as the clone 34. By comparing DNA sequence of clone 34 with other clones, we found that it was a new clone. ELISA analysis showed that this phage antibody strongly bound to CD176 (Fig. 1).

The CD176 scFv was expressed and purified

The CD176 scFv (scFv-His) gene was sub-cloned into PET-32a which was designed for cloning and highly expressing the peptide fused with the 109aa Trx·Tag™ thioredoxin protein. The CD176 scFv was expressed in E. coli, BL21 (DE3) pLysS cells. The level of the CD176 scFv expression was first assessed in the smaller scale culture using BL21 (DE3) pLysS. Following IPTG induction, the overexpressed protein was detected in a band with an expected size of approximately 50 kDa (scFv-His tag plus thioredoxin) on the SDS-PAGE gel (Fig. 2A).

Thereafter, the scFv-His was expressed in the larger scale culture and purified by nickel-affinity purification. The purified protein was digested by enterokinase to remove the fusion protein, and the purified scFv-His antibody was obtained (30 kDa, Fig. 2B). To obtain a pure antibody, the Sephadex G-50 gel filtration was performed, and the highest protein fraction was collected (Fig. 2C). After filtration, a single band was detected by SDS-PAGE (Fig. 2D).

High performance liquid chromatography (HPLC) was used for the next purification. The result of SDS-PAGE also demonstrated that the pure antibody was obtained (data not shown).

The CD176 scFv bound to the natural CD176 antigen

The binding of the purified scFv was identified by flow cytometric analysis. The scFv-His antibody could bind to the surface of KG1 cells that expressed CD176 (Fig. 3A). In addition, the immunofluorescence staining also demonstrated that the scFv reacted with CD176, which was expressed on the surface of KG1, SW480, SGC7901, and Caco-2 cells (Fig. 3B). In immunohistology, the scFv also stained cancer cells, which were positive for the CD176 mAb (data not shown). These results illustrated that the purified scFv-His antibody could bind to the natural CD176 antigen.

The CD176 scFv inhibited the adhesion of CD176⁺ cancer cells to endothelial cells and hepatocytes

CD176 could mediate the binding of CD176⁺ cancer cells to endothelial cells and hepatocytes; CD176 was functionally involved in the blood spreading of cancer cells and the liver metastasis process of tumors, respectively. We wanted to determine whether the scFv could inhibit the adhesion of CD176⁺ cancer cell to endothelial cells and hepatocytes. Through adhesion assay, we found that adhesion of cancer cells to HUVEC were strongly inhibited by the treatment of the CD176 scFv compared with the control at different incubation times (Fig. 3C). In the luciferase assay, the CD176 scFv significantly affected the adhesion of SW480 and SGC7901 to HUVEC and HL-7702, respectively (Fig. 3D and 3E). The CD176 scFv had potential to inhibit cancer cell adhesion mediated by CD176.

Discussion

The immunotherapy had been accepted as an effective treatment for cancer in clinical medicine [ 23]. Antibody drugs play the most important roles in cancer immunotherapy. Increasing numbers of antibody drugs have been explored for cancer immunotherapy in clinical trials [ 24]. However, although some antibody drugs are applied for tumor therapy and these antibody drugs have good therapeutic effects, most cancer patients have a poor prognosis because effective treatment is lacking. Therefore, novel antibody drugs need to be developed. The development of antibody drugs has become one of the most important research areas in cancer treatment.

CD176 is a tumor-associated antigen specifically expressed on the cellular surface of many cancers [ 3]. CD176 is also expressed in some cancer stem cells [ 6]. More importantly, CD176 acts as a functional molecule involved in the blood spread, liver metastasis, and apoptosis of cancer cells [ 7– 9]. Through systematic studies, we believed that CD176 is a potential target for cancer immunotherapy, and the CD176 antibody drug may be an effective treatment for clinical application. The recombinant antibody technology has revolutionized the development of antibody and is also one of the most important methods for producing antibody drugs. However, developing antibodies against carbohydrate antigens is difficult because of low immunogenicity [ 25, 26]. CD176 has a small carbohydrate structure. Thus, screening of CD176 scFv is more difficult than screening of a scFv against a protein. In this study, we used B cells obtained from mice immunized with the CD176 antigen and constructed a phage display library. After a long period of screening, a positive clone of CD176 scFv was successfully selected from this library. Thereafter, the CD176 scFv was expressed in E. coli. The CD176 scFv specifically binds to CD176 and does not react with other glycotopes (unpublished data). Moreover, the CD176 scFv can also bind to the natural CD176 expressed on the surface of cancer cells. We observed that the CD176 scFv could inhibit the adhesion of CD176⁺ gastric and colorectal cancer cells to endothelial cells and hepatocytes. Although several CD176 (TF) scFvs have been obtained [ 27, 28], the effect of the CD176 scFv, which blocks the adhesions cancer cells to endothelial cells and hepatocytes, is reported for the first time. The adhesion of cancer cells to endothelial cells and hepatocytes mediated by CD176 is one of the key steps involved in the process of blood spread and liver metastasis of CD176⁺ cancers, respectively [ 7, 8]. The blood spread and liver metastasis of cancer cells are very significant factors in the clinical prognosis of cancer patients. The CD176 scFv and CD176 antibody treatments, which block the adhesions of cancer cells to endothelial cells and hepatocytes, may reduce blood spread and liver metastasis of cancer cells. We emphasized the following in this study: (1) gastrointestinal cancers are one of the common malignancies and leading causes of cancer-related death; (2) 60%–80% of gastrointestinal cancers expressed CD176 [ 3, 8, 29]; and (3) liver metastasis is one of the major hazards of gastrointestinal cancers. Therefore, we think that CD176 antibody drug may be a potential treatment for cancers, particularly for gastrointestinal cancers. The CD176 antibody drug may have a good clinical prospect. Although several laboratories developed CD176 (TF) scFv [ 27, 28], no CD176 antibody drug is used in clinical practice. A CD176 antibody drug should be developed. We are continuing this work in our laboratory.

In conclusion, a positive clone of CD176 scFv was successfully screened from a phage display library, and the CD176 scFv was expressed in E. coli. The purified scFv can bind to natural CD176 expressed on the surface of cancer cells and inhibit the adhesion of CD176⁺ cancer cells to endothelial cells and hepatocytes. CD176 scFv provides a basis for the future development of recombinant antibodies specific for CD176 toward therapeutic application.

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