Preparation Of Anti-human Endoglin Single-Chain Antibody And Study On Its Application To Radioimmunoimaging In Nude Mice Bearing Human Ovarian Cancer

Background for research: Endoglin, also named CD105, is a kind of cell membrane glycoproteins and one of markers for endothelial cells proliferation. It shows over-expression in proliferative endotheliocytes and endothelial cells of new vessels in tumor tissues, whereas it exhibits non expression or weak expression in normally maturated vascular endothelial cells. Endoglin is also one of components of transforming growth factor receptor-β(TGF-β) compound and has the functions of regulating the response of endotheliocytes to TGF, promoting the proliferation of endotheliocytes and the formation of blood vessels. It is associated with the angiogenesis of tumors. According to the viewpoint that the growth and metastasis of tumors are dependent on tumor neovascularity, many animal researches have been done showed that the radioimmunoimage and therapeutical effects on transplanted tumors could be implemented by targeting endoglin expressed on tumor new vascular endothelium, demonstrating that Endoglin can be used as an ideal target for imaging in the diagnosis and treatment of in vivo tumors.If we can detect ovarian cancers in early stage and then give correct treatments, the purpose of reduction of the mortality of patients with ovarian cancers can be attained easily. So, it is imperative to establish a special method for diagnosis of ovarian cancers. Ovarian cancers are highly vascularized malignant tumors, the anti-human endoglin antibody, therefore, might play an important role in the diagnosis and treatment of ovarian cancers.There are a lot of reports about monoclonal antibody in the study on the endoglin antibody. However, monoclonal antibody is not very effective in its clinical application because of its disadvantages. For example, monoclonal antibody has a large molecular weight and a weak penetrating power, and due to its Fc fragment, it is likely to bind nonspecifically with the Fc acceptors of normal tissue, most of them are mouse original and easily lead to human anti-mouse antibody response(HAMA). In addition, it is usually miscellaneous and toilsome in preparation. In comparison, a single-chain antibody is smaller in molecular weight, stronger in penetrating power, quicker in clearance in vivo, absence of Fc fragment, lower in immunogenicity, prone to get enough volume through recombination and expression in escherichia coli, apt to be prepared and modified through gene engineering, and its stability and affinity can be increased by the construction of a bivalent or bispecific single-chain antibody. The single-chain antibody is a valuable micromolecule antibody, which can target drugs, toxin and radioactive substance toward a tumor, and therefore, is widely applied to diagnosis and treatment of tumors.Phage surface display is the technology to express exogenous genes by phages. It takes a modified phage as vector, and through which, an objective gene fragment is directionally inserted into a coat protein gene area of phage to express and display an exogenous polypeptide or antibody on the phage surface. Furthermore, the phage with a specific peptide or antibody was enriched by several rounds of absorb-elute-enrich panning, and then determined DNA sequence. Phage surface display, combining together genotype and phenotype, molecular binding activity and the amplification of phage, is a high effective screening system. It has been widely used in the fields of epitope simulation, protein engineering reform, single-chain antibody preparation, drug screening and vaccine development.Based on the above analysis, the present research will use the phage surface display technology to prepare a specific antihuman Endoglin single-chain antibody and study its biologic activity and its effect on targeting the new vascular endothelium of internal tumors in order to lay a foundation for exploring a diagnostic method for early ovarian cancers.Objectives: To prepare an antihuman Endoglin single-chain antibody and detect its biologic activity; To preliminary study on the role of the antihuman Endoglin single-chain antibody in the radioimmunoimaging of tumors using nude mice bearing human ovarian cancers in order to seek a new route for early ovarian cancers diagnosis.Methods and results: The research is divided into 4 parts. Part 1: mRNA was extracted from spleens of mice immunized by extracellular domain of human Endoglin (rhEndoglin). RT-PCR was used to amplify the gene fragments of variable region of heavy chain (VH) and light chain (VL). 1% agarose gel electrophoresis showed that VH was about 340bp and VL was about 325bp as expected; Using splicing overlap extension PCR, single-chain antibody gene (scFv) was constructed by fusion of VH and VL via a linker DNA fragment. 1% agarose gel electrophoresis showed that scFv was about 750bp, conforming to expectation. Then the scFv gene was digested with restriction enzyme SfiⅠand NotⅠ.Part 2: The antihuman Endoglin scFv gene digested by SfiⅠand NotⅠwas cloned into a phagemid vector pCANTAB5E digested by the same restriction enzyme, and then escherichia coli TG1 was transformed and then infected with helper phage M13KO7 to construct an antihuman Endoglin single-chain antibody phage display library with a capacity about 3.98×106; the library was adsorbed, eluted, enriched by panning for 5 rounds and then positive recombinant phage clones with antigen binding capacity were screened and identified with phage ELISA. As a result, 37 positive clones were screened out in one time from 94 randomly selected recombinant phage clones with a detection rate about 39.36%; according to the phage ELISA result, the recombinant phage clones with the strongest antigen affinity were sequenced to gain a scFv DNA; after comparison and analysis, it was found that the scFv DNA has a length of 738bp encoding 246 amino-acid residues. Among them, 122 amino-acid residues were encoded by VH gene and were located at the N-terminal of the single-chain antibody and 109 amino-acid residues were encoded by VL gene and were located at the C-terminal of the single-chain antibody. VH and VL were connected through 15 peptides, which were composed of (Gly4Ser)3. No antibody gene sequence identical with the scFv gene was found through data base querying; the amino acid sequence of the single-chain antibody protein was analyzed with ANTHEPROT V5.0 software and the protein molecular weight was calculated to be 25925.818 Da. There were 2 cysteines respectively in the heavy variable region and the light variable region, which can form 2 disulfide bonds. Their isoelectric point was predicted to be 9.015. In addition, a Prediction Model of the secondary structure and tertiary structure of protein encoded by this gene was gained through automatic modeling at biomedicine sites;Part 3: The recombinant phage correctly sequenced in part 2 infected escherichia coli HB2151 and induced by IPTG to successfully express the soluble single-chain antibody protein with a proximate molecular weight of 29×103, which was identified and analyzed with SDS-PAGE and Western Blot. The proteins are located in the periplasm and culture supermatant with the proteins in the periplasm enjoying the highest expression. The optimal culture conditions were determined to be: temperature 30°C, IPTG concentration 1mmol/L, shaking speed 250rpm, induction culture 5 h; single-chain antibodies were purified from periplasmic extract induced with 1mmol/ IPTG for 5 h with HiTrap Anti-E Tag column affinity chromatography, and then buffer exchanged into neutral phosphate buffer with HiTrap desalting column. Purity of the purified antibodies was identified with SDS-PAGE and it was over 95%; the concentration of single-chain antibodies was determined to be 1.12mg/ml with Bradford method after concentrating with PEG 8000; its functional affinity constant(2.14±0.84)×107M-1 was determined with indirect ELISA; compared with the antigen binding activity of monoclonal anti-rhEndoglin antibody, the antigen binding activity of the single-chain antibody was determined with competive ELISA, and the result showed that the single-chain antibody could bind with rhEndoglin antigenic epitope by competion with monoclonal anti-rhEndoglin antibody, and the completive action was enhanced with the increasing concentration of single-chain antibody; through indirect immunofluorescence experiments, Endoglin expressed on HUVECs cell membrane can be detected by the single-chain antibody and manifested as green color fluorescence spots diffusively distributing on cells membrane;Part 4: SKOV3 human ovarian cancer cells were inoculated subcutaneously into nude mice to establish animal model of nude mice bearing human ovarian cancer, after which, the animal model was verified to be successful with histopathology of transplanted ovarian cancers; the immunohistochemisty result of paraffin sections showed that the single-chain antibody could make the vascular endothelium of transplanted ovarian cancer take positive staining, suggesting that there are cross reactions between the single-chain antibody and the murine vascular endothelium in transplanted ovarian cancer; single-chain antibody labeled with 99mTc was completed with pre-stannous direct labeling and 83% of labeling efficiency was determined by paper chromatography. The single-chain antibodies labeled 99mTc were injected into nude mice bearing tumors through its caudal vein. Under a Spect apparatus, we could observe tumor location image at 1 h post injection and the tumor images was most clear at 3 h post injection; the radioactive count of various tissues were measured with aγradio-immunity counter and ratios between the tumor tissue radioactive count and the non tumor tissue radioactive count were calculated. These ratios reflect the characteristics of the distribution of anti-human Endoglin single-chain antibodies in nude mice bearing tumors, and the highest value was 2.96±1.71.Conclusion: using phage surface display technology, an anti-human endoglin single-chain antibody was successfully prepared; both in vivo and in vitro experiments have confirmed that this single-chain antibody has a strong antigen binding capacity; this single-chain antibody is hopeful to be used as an ovarian cancer vascular endothelium targeting vector and the present research has laid a foundation for studies on diagnostic methods for ovarian cancers in early stage.