The Construction Of Human Single-Chain Variable Fragment Antibodies Of Lung Adenocarcinoma And Its Radioimmunoimaging In Bearing Cancer Nude Mice

Lung cancer is one of the leading causes of death in the world, especially in heavy air pollution cities and increasing number of smokers in our country. The incidence of lung cancer is gradually rising. Experts predict that the number of death could reach 1,000,000 in 2025. However, the cure rate of lung cancer is only 15%, the survival rate of 5 years is only about 30-50%. Surgery, chemotherapy and radiation therapy are main methods of treatments in clinics. But there are still no effective measures for those advanced unresectable tumors and metastatic or recurrent ones. These patients have to accept chemotherapy and radiation therapy, but the effect is not satisfactory. Thus, more attention was focused on targeted therapy of cancer, because targeted therapy can kill cancer cells exclusively, decrease the toxic side effect to normal cells in a maximum. If radioactive nuclide is coupled with the targeted specific antibody, on one hand, the antibody can bind to specific tumor antigen, on the other hand, rays that radioactive nuclide gives off would intensify killing cancer cells, promoting the anti-tumor effect. During the past ten years, targeted therapy of cancer has become an important therapeutic method used extensively in clinical and experimental fields. Immunoimaging and immunotherapy are critical parts of biotherapy and play vital role in diagnosis of cancer, metastasis, fine tumor staging and decision of therapeutic approaches.The key to study targeted therapy depends on the choice and construction of antibody. The first strain of monoclonal antibody was produced by Kohler and Milstein since 1975, the research of monoclonal antibody have experienced three stages including murine antibody humanized reconstruction, micromolecule antibody and antibody library. Especially, the appearence of antibody library promote gene engineering antibody techniques to enter a new stage.To overcome the problem of xenogenicity occurring in repeated clinical usage of murine monoclonal antibodies and the low penetrance of intact antibody in targeted therapy, we constructed a human single-chain antibody library using phage display techniques from lymph nodes of lung adenocarcinoma patients, screened this library and labeled it with radioactive nuclide and made radioimmunoimaging in bearing lung adenocarcinoma nude mice.ObjectiveTo construct human phage single-chain antibody library associated with lung adenocarcinoma and to screen the specific scFv labeled with radioactive nuclide ¹³¹I and make radioimmunoimaging in bearing lung adenocarcinoma nude mice. It will provide an effective approach to treat lung cancer patients.Methods and ResultsPart 1 Construction of phage antibody library:1. Generation of Human scFv Autibody Gene Repertoires:Lymph nodes near the lung adenocarcinoma were used as the B cells sources, the total RNA of these B cells was extracted. The cDNA gene library of VH and VL fragments were amplified by RT-PCR. First, to screen graticule two pairs of primers of the heavy and light fragment separately, then the VH and VL fragments were first amplified from the cDNA. Second, the VH-linker and VL-linker were amplified from the VH and VL, fragments. Last, SOE-PCR was used to connect the VH-linker and VL-linker, and the Sfi I and Not I restriction site were inlet in it. After PCR products were purified from gel, we can get the scFv. The results showed: The total RNA of these B cells has two bands 28S and 18S in agar gel electrophoresis. The pairs of primers for VH fragment were HuJH3FOR and HuVH5aBACK, The pairs of primers for VL fragment were HuJκ4FOR and HuVκ5aBACK. The pairs of primers for the VH-linker were HuJH3Linker and HuVH5aBACK, The pairs of primers for the VL-linker were HuJκ4FOR and Linker-HuVκ5aBACK. The pail's of primers for the Sfi I and Not I restriction site were HuVH5aBACKSfi and HuJκ4FORNot. VH fragment is about 370bp, VL fragment is about 350bp and scFv is about 750bp.2. Ligation of scFv Gene Repertoire into pCANTAB-5EHigh E.coli TG1 cells with high density were prepared, the electro competence is 108cfu/ug pUC18 DNA. The gel purified scFv gene repertoires are overdigested by Sfi I and Not I separately. The ligation mixes of scFv and pCANTAB-5E are transformed into electro competence E.coli TG1 2.2x10⁷ cfu/Bg ampicillin resistant bacteria colonies grow. Random digestion reaction showed that the positive insert ratio was 83.3% (20/24). The results of sequencing showed that variable domain of heavy and light chains were correctly inserted into phagemid vector frame.3. Display of Phagemid antibodyThose ampicillin resistant bacteria colonies were scraped into 2-YT medium and superinfected by M13K07 helper phage. After overnight induction in a medium containing no glucose, we got recombinant scFv phage with a titer of 1012 cfu/ml.Part 2 Screening of phage antibody library:1. Panning the phage antibody library using lung adenocarcinomaThe phage antibody library was panned with fibroblast and lung adenocarcinoma A549 in suspension. After five rounds of panning, ELISA was performed to determine the specificity of the phage antibody. Titer of scFv increased gradually after panning compare with that before panning. The first phage yield is 1.59×10^-6, the fifth phage yield is 2.88×10^-4, the fifth phage yield is as 181 times as the first phage yield.2. Expression of soluble scFvStrong positive recombinant phage clones were used to infect log-phage E.coli HB2151. Expression of soluble scFv was induced by adding isopropy1β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol/L.3. Purification of soluble scFvSoluble scFv from periplasm were purified by affinity chromatography. Anti-E tag antibody was covalently coupled to a protein G column and soluble scFvs were selected by binding to anti-E tag antibody. After washed with phosphate buffer pH 7.0, scFvs were eluted from the column with glycine-HC1, pH 3.0, and neutralized immediately with Tris/HC1, pH 8.2, column fractions were assayed and positive fractions were pooled and stored at -70 0C.4. Cell ELISA assay for immune activity of soluble scFvTo detect the activity of soluble scFv, lung adenocarcinoma A549 A405nm is 0.71±0.05, breast cancer MDA-MB-435 cells A405nm is 0.25±0.08. The result revealed that soluble scFv was highly specific and could bind to lung adenocarcinoma A549 cells, but not to breast cancer MDA-MB-435 cells. The P/N value of 6 phage scFvs reacting to A549 cell is more than 2 times than that of MDA-MB-435. Part 3 Radiolabeling of scFv and its identification:Soluble scFv after screening lung adenocarcinoma A549 cells was labeled with ¹³¹I. After being Purified by Sephadex G200 column chromatography, labeling yield, specific activity and radiochemical purity were tested by paper chromatography. The radiochemical purity of ¹³¹I-scFv at ambient temperature was determined to observe its stability with incubated in fresh human serum at 37℃. Results: Labeling yield of scFv with ¹³¹I was 81.2±5.3% and specific activity 3.1±0.2MBq/μg. The first radioactive peak after being separated and purified with Sephadex G200 column was ¹³¹I-scFv, Radiochemical purity subtly declined after 24h which was more than 90%. The stability was analyzed with incubation in fresh human serum at 37℃, radiochemical purity determined by paper chromatography at 48h (average more than 90%) was slightly lower than that at 1h.Part 4 The distribution of ¹³¹I-scFv in bearing adenocarcinoma nude mice and its SPECT imaging:The results shows: 1. The distribution of ¹³¹I-scFv in bearing adenocarcinoma nude mice: Radioactive ratio of tumor/serum and tumor/muscle of ¹³¹I-scFv gradually increased, reaching its peak (4.06±0.13 and 5.17±0.97 respectively) at 3d. 2. SPECT imaging in bearing adenocarcinoma nude mice: Radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. ConclusionThe human single-chain antibody library of lung adenocarcinoma was constructed using phage display techniques in present study. The scFv was labeled with radioactive nuclide ¹³¹I and radioimmunoimaging is made in bearing lung adenocarcinoma nude mice. Results indicated the scFv from lymph nodes of lung adenocarcinoma patients labeled with radioactive nuclide ¹³¹I had high labeling yield and radiochemical purity, the specific activity could meet the demand of research, and it was very stable with a definite target, moreover, the effect of radioimmunoimaging is satisfactory, which maked it possible to treat cancer with targeted therapy.