Human esophageal carcinoma cell line Eca109 (Cytology Institute of Chinese Medical Academy, Beijing) and HeLa cell line (Shanghai Cytology Institute, China) were cultured at 37 °C in RPMI1640 medium supplemented with 100 mL/L fetal calf serum (Hyclone, USA) in a humidified atmosphere of 50 mL/L CO₂. Normal human esophageal epithelial cell (NHEEC) line was a primary cell line from a 20-wk conception fetus cultured in RPMI1640 with 200 mL/L fetal calf serum.

Primer sequences were created as previously described[4] with some modifications in PCR assembly part (Table 1B). We designed complementary coding sequences for a peptide linker at the 5’-end of J_H forward primers and the 3’-end of human V kappa (or lambda) back primers to optimize the diversity and efficiency of ligation. The primers were synthesized by Sunbiotech Company (Beijing, China) and the sequences are shown in Table 1. Sequences were given using the IUPAC nomenclature of mixed base (R = A or T, K = G or T, Y = C or T, S = G or C, H = A or C or T, N = A or C or G or T).

Metastatic lymph nodes of 5 esophageal cancer patients were collected for total RNA extraction (TRizol, Gibco BRL, UK). First-strand cDNA synthesis was performed in the presence of 40 U RNase inhibitor, 200 U Superscript II transcriptase (Gibco BRL, UK). The sample was finally treated with 2 U RNase H for 30 min at 37 °C and stored at -20 °C until use.

IgG-specific variable heavy (V_H) and light (V_L) chain gene fragments were amplified using Pyrobest PCR system (TarkaRa Biotechnology, Dalian, China) for 30 cycles (at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 1 min), with each forward oligonucleotide primer and one of the back primers (Table 1A). The fragments were isolated from a 15 g/L agarose gel with the QIAex kit (QIAgen, Germany). Then fragments were used as templates for PCR amplification to extend a linker, V_H fragments used human JH-linker primers and human V_H back primers, V_L fragments used linker-human Vκ primers (or linker-human Vλ primers) and human Jκ forward primers (or human Jλ forward primers), respectively.

The amplified V_H-linker and V_L-linker PCR products were combined in a SOE-PCR reaction mixture. First, approximately 100 ng each of VH-linker and V_L-linker was assembled by PCR without primers in which the short regions of complementarities built into the ends of primers drove hybridization of various fragments. An initial denaturation step (at 94 °C for 5 min) was followed by five cycles (at 94 °C for 1 min, at 60 °C for 1 min and at 72 °C for 1.5 min) in the absence of primers. After the outer primers containing restriction sites (Table 1C) were added, 30 cycles (at 94 °C for 30 s, at 60 °C for 1 min and at 72 °C for 1.5 min) were performed. These were gel-purified, digested with SfiI and NotI (TarkaRa Biotechnology, Dalian), cloned into the vector pCANTAB 5E (Amersham Pharmacia Biotech, Sweden) and transformed into electrocompetent E. coli TG1 (Amersham Pharmacia Biotech, Sweden). After electroporation, cells were plated on SOBAG medium (containing 20 g/L glucose and 100 mg/mL ampicillin) in 20 dishes and incubated overnight at 30 °C. The clones were scraped off the plates in 50 mL 2 × YT medium with 100 mL/L glycerol and subsequently stored at -70 °C. Plasmid DNA was prepared from 10 randomly selected clones using Qiagen plasmid minikit (Qiagen, Germany). PCR and a SfiI/Not I double digestion reaction were performed to identify the positive insert clones.

Ampicillin-resistant colonies were scraped into 2 × YT medium and superinfected by M13K07 (Amersham Pharmacia Biotech, Sweden) helper phage. After an overnight induction in 2 × YTAK medium without glucose, the phage preparation was precipitated in 40 g/L PEG/0.5 mol/L NaCl and resuspended in 10 g/L PBS.

To screen the positive phage clones, live Eca109 cell line as the antigen was used for panning. Live NHEEC was used for subtractive panning. The panning procedure was carried out essentially as described previously[5]. After four rounds of panning and three rounds of subtractive panning, the unabsorbed phages were amplified.

To detect the scFv-phage recombinant antibody, cell ELISA was performed. Eca109 cells (5 × 10⁴) as antigens were grown in 96-well plates at 37 °C for 24 h, then fixed with 25 g/L glutaradehyde and blocked with 10 g/L BSA. This was followed by incubation with scFv-phage at 37 °C for 2 h. After washed three times with PBS, HRP/anti-M13 monoclonal conjugate (1:5000) (Amersham Pharmacia Biotech, Sweden) was added into wells with scFv-phage and they were incubated for 1 h at 37 °C. After washed again as above, 1 × ABTS substrate solution was added, and incubated in darkness for 30 min and the reactions were read at 405 nm. PBS was used as negative control. The absorbance reading for the positive was 0.2 or above at least three times higher than that for the negative control.

To produce soluble scFvs, strongly positive recombinant phage clones were used to infect log-phage E. coli.HB2151 (Amersham Pharmacia Biotech, Sweden). Expression of soluble scFv was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol/L and the cultures grown overnight at 30 °C. The induced culture was centrifuged at 1500 r/min for 20 min. Cell pellets were resuspended in 2% of culture volume ice-cold 1 × TES. Subsequently, 3% of culture volume ice-cold 0.2 × TES was added, the mixture was incubated on ice for 30 min to induce a mild osmotic shock. The contents were centrifuged at 12000 g for 10 min. The supernatant, containing the soluble antibodies from the periplasm, was transferred to the clean tubes and stored at -20 °C.

Soluble scFvs from periplasm were purified by affinity chromatography. Anti-E tag antibody (Amersham Pharmacia Biotech, Sweden) was covalently coupled to a protein G column (Amersham Pharmacia Biotech, Sweden) and soluble scFvs were selected by binding to anti-E tag antibody. After washed with 20 mmol/L phosphate buffer, pH7.0, + 0.5 g/L NaN3, scFvs were eluted from the column with 0.1 mol/L glycine-HCl, pH3.0, and neutralized immediately with 1 mol/L Tris/HCl, pH8.2, + 0.5 g/L NaN3. Column fractions were assayed and positive fractions were pooled and stored at -70 °C.The expressed soluble scFv proteins were analyzed by 120 g/L sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and confirmed by Western blotting. Purity and concentration of proteins were determined with Bradford assay.

To detect the activity of soluble scFv, HRP/anti-E tag antibody (Amersham Pharmacia Biotech, Sweden) was used. Eca109 cells, HeLa cells and NHEEC cells (5 × 10⁴) were used as antigens. The cell ELISA assay procedure was performed as described above.

Plasmid DNA was prepared from recombinant clones using the Qiagen plasmid minikit (Qiagen, Germany). Nucleic acid sequencing was carried out on the ABI PRISM 377 DNA sequencer by the method of dideoxynucleotide sequencing. DNA and deduced amino acid sequence were compared with NCBI database.

The presence of VH and VL gene fragments obtained by RT-PCR was confirmed by electrophoresis, with their sizes being approximately 350 bp (Figure 1A). The scFv genewas assembled successfully. Its size was about 750 bp (Figure 1B).

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Figure 1 V_H and V_L and scFv fragments in 20 g/L agarose gel electrophoresis with staining of ethdium bromide (EB). (A) V_H and V_L fragments, (B) scFv fragment, M: DGL 2000 marker.

After the scFv gene repertoires were transformed into E. coli TG1 cells, approximately 9 × 10⁶ ampicillin-resistant clones grew. PCR and SfiI/NotI double digestion reactions showed the positive insert ratio was about 95% (19/20). Rescued by M13K07 helper phage, the recombinant phage antibody library (about 9 × 10¹¹ cfu/mL) was constructed.

Four rounds of panning with Eca109 cells resulted in a 130-fold enrichment of tumor cell binding scFv-phage. After three rounds of subtractive panning with NHEEC cells, individual phage-displayed scFv fragments were tested for reactivity with Eca109 cells by cell ELISA. Of the 95 clones screened, 25 were positive. The highest A_405nm value was found in AD09 clone.

The soluble scFv was stably expressed in E. coli HB2151 transfected with AD09 positive phage clone. In pCANTAB 5E, the pel B signal peptide upstream from the scFv directed the expression to periplasmic compartment. The periplasmic extract of AD09 was run through anti-E tag antibody affinity chromatography column and soluble AD09 was eluted from the column as a single peak (data not shown). The expressed and purified AD09 was loaded on 120 g/L SDS-PAGE and analyzed by Western blot. This protein migrated with a molecular mass approximately 31 ku (Figure 2). The overall yield of soluble AD09 in E. coli flask culture was more than 0.55 mg/L. The purity was about 90%.

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Figure 2 Expression and purification of AD09. A: SDS-PAGE. Lane 1: Markers (Amersham Pharmacia Biotech, Sweden). Lane 2: Product of E. coli HB2151 without scFv gene. Lane 3: Expression of AD09 in E. coli HB2151 with induction of IPTG. Lane 4: Purified AD09 protein. B: Western blot. Lane 1: Markers. Lane 2: Purified AD09 protein.

The immunoreactivity of purified soluble AD09 was determined by ELISA. The result revealed that AD09 was highly specific and could bind to Eca109 cells, but not to HeLa and NHEE cells (Table 2).

Sequencing of six randomly selected antibodies from the positive clones was performed with an ABI PRISM 377 DNA sequencer using pCANTAB5 sequencing primer set (Amersham Pharmacia Biotech, Sweden). The sequence f AD09 clone is shown in Figure 3. Compared using BLAST, both V_H and V_L had sequence similarities to the variable fragments of some known human antibodies. Alignment with the V_H and V_L sequences of Ig, blast analysis of immunoglobulin sequences showed that V_H was the γ chain subgroup IV of human immunoglobulin and V_L was the κ chain subgroup I of human immunoglobulin.

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Figure 3 Sequence of AD09 gene.