Construction And Screening Of The Human Phage Antibody Libraries From A Patient With Malignant Melanoma

Malignant melanoma (MM) is among the malignancies that are still on the increase worldwide. The principle treatment for MM is excision surgery. Surgery can be curative if it is performed at an early stage. However, MM is resistnt to standard radiotherapy and chemotherapy, and there is no effective treatment for metasstatic disease. Consequently, experimental forms of treatment such as immunotherapy have been studied extensively in MM, which is effective in decreasing metastasis of MM, postponing recrudescence, increasing survival rate and has some progress clinically. Immunotherapy includes cancer vaccine therapy, cytokine therapy, adoptive immunocyte therapy, antibody therapy, and so on.Antibody targeted therapy is used early in the 1980s, in which antibodies are conjugated to chemotherapy drugs, toxins, radionuclide for targeted therapy, or to enzyme for precursor drug therapy.However, antibody targeted therapy is still studied and has not been used widely in clinic. Specific antigen of MM is very limited, so is specific antibody. Researchers apply themself to antibodies of MM with good specificity and high affinity. Phage antibody library technology can overcome the complex course to gain human mAb by hybridoma technology and we can screen many specific antibody. The studies about genetically engineered antibodies improve targeted therapy of MM, and some mouse mAbs are used in diagnose and immunotherapy of MM. Mouse mAbs are limited by its immunogen, so genetically engineered antibodies show a good future.We discovered a patient with melanoma survived for twenty-two years, in which MM transfer to skin, liver and thyroid gland. Metastasis MM has regressed except in liver. The study is to construct a human phage antibody library from a patient with melanoma, and to screen specific antibodies to look for better ways for therapy of MM.Total RNA is extracted from the peripheral blood mononuclear cells (PBMCs) of the patient with melanoma survived for twenty-two years, and the heavy chain Fd fragments and K light chains were amplified by RT-PCR. The amplified products were about 700bp and cloned into phagemid vector to construct K chain library and heavy chain library, which were incised by endonucleases Sad + Xba I or SpeI+XhoI. The library members can be accounted by clones in gelose flat. The phagemid vectors from K chain library were incised by endonucleases Spel+ Xhol and big segments which were about 4.1kb were collected , and the phagemid vectors from heavy chain library were incised by endonucleases Spel+Xhol and small segments which were about 0.7kb were collected. Two segments were conjugated by T4DNA ligase to construct Fab antibody library. The library was panned with two kinds of melanoma cells for 4 rounds. Clones in NO.3 or 4 round were selected to express phage antibodies, and cell ELISA was used to examine the specificity of antibodies.The results show that the K chain library contained 1 107 different clones and the recombine rate was 90% , while the heavy chain library contained 3.2 108 different clones and the recombine rate was 80%. The antibody library constructed with Kchain and heavy chain genes contained 1.2 108 different clones and the recombine rate was 75%. Two MM cell lines LiBr and 1585 were used to screen the phage antibody library and 4 clones were obtained for specific phage antibodies against LiBr MM cells after 4 rounds of panning.The successful preparation of specific antibodies against MM cells by phage display technology paves the way for further study and application.