Preparation Of The Agonist/antagonist Monoclonal Antibodies Against B7 And CD28 Molecules And Analysis Of Their Effects On Costimulatory Signals Transduction

B7:CD2S ~ Preparation of the agonistlantagonist monoclonal antibodies against B7 and CD28 molecules and analysis of their effects on costimulatory signals transduction Ph.D Candidate Qiu yuhua Supervisor Zhang xueguang Abstract A variety of immune cells and molecules (membrane and soluble) involves in immune response, a complicated and strictly regulated process for organisms. Costimulatory molecules, a category of membrane molecules, perform an essential function owing to their regulative expression, interaction and signal transduction during the process. 67 and CD28 families regarded as radical costimulatory molecules belong to immunoglobulin superfamily (IGSF), including B7-1(CD8O), B7-2(CD86) and CD28, CTLA-4 respectively. B7-1 expressing on antigen presenting cells(APCs) which is engaged with CD28 expressing on T cells mediates necessary signals promoting T cell activation, proliferation and function. Discovery of costimulatory molecules and establishment of costimulatory-signal theory develop some new strategies, methods and biological reagents which have been proved and widely used. Enhancing costimulatory signals is 14 B7:CD28 ~~J/~J J14~f~3T~E available for immunotherapy of cancer. Paradoxically, blocking them results in the immune tolerance specific to I cells and contributes to precaution and treatment of autoimmune, hypersensitivity and allogenetic graft rejection. It is very important to prepare and study agonists and antagonists of costimulatory molecules and investigate their regulatory functions on B7:CD28 signal transduction. I Preparation and Characterization of anti-human B7-1(CD8O) mouse monoclonal antibody(mAb) 1.1 Establishment of hybridoma cell lines As immunogen a multiple myeloma(MM)cell line-XGB7 was used, which was transfected human 67 gene. After mitomycin pretreatment(50 i.1 gil X iO~ cells). BALB/c mice were immunized by the intraperitoneal and multi-spot neck intracutaneous injection. The spleen cells immunized were fused with mouse myeloma cells(SP2/O) by using polyethylene glycol(PEG), then hybridomas were cultured in HAT selection medium. In consequence, a positive colone secreting anti-human B7-1 (CD8O) continuously and steadily was obtained after repeatedly cloning and screening. The hybridomas grew well after long-term culturing and storage in liquid nitrogen. 1.2 Production and purification of mAb The mAb was produced by in-vivo procedures. Ascites can be obtained from 80% mice, 7.9m1 each on average by in-vivo inducing. Affinity chromatography was used to purify mAbs from ascites. The mAb concentration was 2.18mg/mi. 15 B7:CD28 ~ j/~ J~j J~ fri1~Yr~ 1.3 Determination of subclass and titer Fast-strip method analysis indicates that mAb4E5 belongs to mouse IgGl subclass. Flowcytometric analysis showed ascites titer was over 1:1000, while every 5-lOX 106 cells required 1 microgram purified mAb4E5. 1.4 Identification of antigen determinants...