| Background and objectiveNowadays,with the sharply increasing morbidity and stable high mortality, tumor has become the greatest threaten for human health and life in all diseases.So far,human has known few of the nature of the tumor,generally regarded as a complicated disease resulted from multiple factor and progressed by kinds of pathogenesis.Comprehensive treatment is the main cancer treatment including surgery,radiotherapy,chemotherapy,biological therapy and other means of treatment. Surgery is an important treatment for early stage cancer,but it can not solute all of the problems because tumor is a kind of systemic disease.Chemotherapy and radiotherapy are the main anti-cancer methods,especially for advanced tumors. Because the two therapies lack specificity,most patients with these always have poor quality of life and often stop treatment because of its side effects.In addition,the progressive reduction of sensitivity on tumor cells to chemotherapy or radiotherapy and the attendant high relapse rate also obstructed the two treatments to become the ultimate weapon to cure tumor.In recent years,molecular targeting tumor therapy is the most energic and appealing field and is a genuine tumor-specific therapy. Monoclonal antibodies are the basis and highlights of the field. Monoclonal antibodies are produced commercially by gene engineering and labeled tumor toxic substances such as 131I.Then the monoclonal antibodies bind with specific cancer antigen and labeled tumor toxic substances kill tumor cells.It is called bio-targeted therapy or radioimmunotherapy with advantages of good targeting and few side effects.Over the years,researchers prepared several monoclonal antibodies targeting cell membrane antigens to treat cancer by radioimmunotherapy,but there were several problems:①Common tumor antigen has not been found.Neither tumor specific antigen nor tumor-associated antigen is expressed on all kinds of tumor.So, in order to treat different kind of tumor we must prepare different monoclonal antibody.②Because of the heterogeneity of tumor,tumor-associated antigen always was expressed on the membrane of partial tumor cells only.when the monoclonal antibody was injected into body,only small quantity of antibody and tumor toxic substances accumulated in tumor tissue.③Antigen modulation.An antigen expressed on tumor cell membrane may be replaced by another antigen after a period of time.Therefore,monoclonal antibody targeting tumor cell membrane antigen is not the best one for solid tumor and it is important to research new anti-cancer therapy for solid tumor.Although the similar character between normal cells and tumor cells make us feel difficult to decide the final treatment,researchers have found that there is evident difference between normal cells and tumor cells.In normal tissues,only few of cells dead slowly and will be phagocytized by immune system quickly.In the process of death,normal cells showed karyorrhexis but not abnormal membrane permeability.In contrast to normal tissues,while a high proportion of neoplastic cells in a malignant tumor are actively proliferating,there is also a significant proportion of tumor cells in various stages of cell degeneration and cell death and these cells with abnormal membrane permeability lost membrane integrity.Moreover,the proportion of dead and dying cells in a fast-growing tumor population is much higher than in normal healthy tissue,with 50%or more of the tumor cell progeny rapidly succumbing to degeneration or necrosis.The loss of membrane integrity that accompanies cellular degeneration permits macromolecules,including antibodies,to freely enter the cell cytoplasm.Based upon these observations,it was hypothesized that tumors containing dead or degenerating malignant cells may be readily distinguished from normal tissues by their ability to bind monoclonal antibodies directed against insoluble antigens within these abnormally permeable cells and monoclonal antibodies labeled with tumor toxic substance to intracellular antigens,which are integral structural components and are retained by degenerating cells,may be used to target a wide range of human malignancies and kill cancer cells.It is called tumor necrosis therapy(TNT),a new treatment method of tumor.chTNT is a chimeric Tumor Necrosis Therapy(TNT) antibody developed by Peregrine to target solid tumors.The efficacy of it has been described in experimental animal tumor models and man.However,as a chimeric antibody,chTNT has some disadvantages.The humanization of murine monoclonal antibody is a complicated technology.While greatly reducing the risk of immunological rejection,it still have the potential to elicit a human anti-mouse antibody(HAMA) response due to the murine content of the CDR especially when linked to other immune regulatory molecules that can increase their antigenicity.So,human monoclonal antibody is better for tumor necrosis therapy on clinical than chimeric monoclonal antibody.With the development of bio-technology,it is inevitable that the production methodes of monoclonal antibody change from murine,chimeric to human.Nowadays,with the growing maturity of the human monoclonal antibody preparation technology,such as Antibody library technology and human-human hybridoma technology,it is essential for clinical tumor necrosis therapy to prepare human monoclonal antibody against insoluble nuclear antigens in the necrotic regions of tumor.Fab fragment contain heavy chain Fd gene and integritylight chain gene.The molecular weight of Fab fragment is about 1/3 of whole IgG while It has antibody activity.It is better suited for use in therapeutic applications because of its small molecular weight,low immunogenicity and relatively strong organ penetration ability.This study wants to construct a human Fab fragment phage antibody library and panning human Fab fragment against double-stranded DNA by combine antibody library technology and phage display technology.Our study also is hopeful to lay the foundation for further radioimmunotherapy.Methods1) Total RNA was isolated from peripheral blood lymphocytes of four patients with IgG titers in the serum to antinuclear antibody were above 1:10000 by indirect immunofluorescence(IIF).Then,cDNA library was procured from total RNA with designed oligo-(dT) nucleotide primers by Reverse transcription technology.2) Human immunoglobulinκ/λlight chains and heavy chains Fd genes were amplified by polymerase chain reaction(PCR).3) Theκ/λlight chains were first cloned into pComb3Hss vector to construct a human recombinant light chain library.4) The heavy chain genes were subsequently inserted into the corresponding sites ofκ/λ-pComb3Hss plasmid to generate a combinatorialκ/λ-Fd-pComb3Hss plasmid.5) Phage Display:The plasmid transformed into E.coli.XL1-Blue,then E.coli. XL1-Blue was infected by helper phage M13KO7.A random combinatorial library was expressed on the surface of filamentous phage. 6) The reconstructed humanized antinuclear antibody Fab library was enriched by four rounds panning.Plasmid DNA isolated from positive clone was cut off gⅢgenes.After ligated itself,the recombinant plasmid transformed E.coli.XL1-Blue, then XL1-Blue was induced by IPTG to product soluble human antinuclear antibody Fab.Finally,soluble human antinuclear antibody Fab was identified.7) Preparation of affinity chromatography column for the purification of human IgG Fab fragment.8) Purification of human IgG Fab fragment by prepared chromatography column.9) Identification of purified human IgG Fab fragment by western blotting and indirect immunofluorescenceResults1) With designed oligo-(dT) nucleotide primers,cDNA library was gained successfully by Reverse transcription technology from total RNA of peripheral blood mononuclear cells.2) Human immunoglobulinκlight chain and heavy chain Fd genes(MW 660bp) were obtained by PCR successfully.The constructed light chains library size is 2×104 and combinatorial Fab immunoglobulin library is 4×104 members.3) We gained a phage antibody library by phage display technology based on combine antibody library successfully.The titer of the phage antibody library was 2×109 pfu/ml.4) The eluted phages were enriched above 200-fold after the forth round panning. Two unique clones were isolated from the human Fab fragment library.It was proved the recombinant plasmid had been cut off gⅢgenes and ligated by itself through electrophoresis after digested by XhoⅠ.5) The results of indirect ELISA indicated all of two clones of Fab had specific to bind with dsDNA.There was a good correlation between relative binding affinity to a homogeneous with Hep2 and monkey hepar tissues.Fabs exhibiting well binding at a concentration were clearly Crithidia kinetoplast positive.6) We prepared affinity chromatography column for the purification of human IgG Fab fragment.7) We gained purified human IgG Fab fragment by prepared affinity chromatography column.The result of western blotting showed molecular weight of the protein is 45KD and it can be captured by anti-human IgG Fab.The anti-ds-DNA activity of purified protein(500μg/ml) was above 1:10000 by indirect immunofluorescence.ConclusionWe gained cDNA library successfully by Reverse transcription technology from total RNA of peripheral blood mononuclear cells of four patients with IgG titers in the serum to antinuclear antibody were above 1:10000 by indirect immunofluorescence.A human antinuclear antibody Fab fragment library was constructed based on it. Subsequently,human Fab fragment phage antibody library against double-stranded DNA was constructed successfully by phage display technology.Through solid phase adsorption of antigen screening method,two unique clones were isolated from the human Fab fragment library.We finished the expression and preliminary purification of soluble Fab fragment.The results of western blotting and indirect immunofluorescence indicated the prepared protein was human IgG Fab and has antibody activity against double-stranded DNA.
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