Preparation Of Functional Monoclonal Antibodies Against Human CD28 Molecules And Study Of Their Biological Characteristics

An optimal T cell response is achieved by a combination of signals delivered through the antigen-specific T cell receptor (TCR) engagement of the peptide-major histocompatibility complex (MHC) on antigen presenting cells(APCs) and accessory signals provided by the interaction of receptors on T cells with their ligands on APCs and is referred to as costimulation.B7 and CD28 families are regarded as radical costimulatory molecules. B7:CD28 signaling is essential for priming resting T cells. Including B7.1(CD80) and B7.2(CD86), there are currently six members of the B7 family of costimulatory ligands,such as PD-L1.PD-L2 ,GL50 ar d B7-H3. All of these molecules are type I transmembrane proteins with two or four conserved extracellular immuno-globulin-like V and C domains and exhibit approximately 20% homology to one another.In contrast to CD80 and CD86, the novel B7 molecules are more broadly expressed on the surface of macrophages,dendritic cells and other APCs. CD28 is a molecular weight 44KD type I transmembrane glycc protein which is encoded by a gene locating at 2q33 and is a membrane differentiation ar tigen expressed on peripheral blood T lymphocytes (about 95% CD4+ T cells and 50% CD8+ T cells) , thymocytes,human multiple myeloma cell lines U266 and Jurkat and partly NK cells.CD28 molecule belongs to immunoglobulin superfamily (IGSF) with similar structure composed of IgV-like,IgC-like,transmembrane and short bu: highly conserved cytoplasm regions.MYPPPY damain,a conserved six-peptide motif,located in CDR3 of IgV-like region of CD28.The interaction of CD28 and its ligand B7-1/B7-2 molecules can promotes T cells activation, proliferation,cytokine secretion and formation of immunological synapse.Enhancing costimualtory sigials is available for immunotherapy of cancer.Paradoxically,blocking the signal results in the immune tolerance specific to Tcells and contributes to precaution and treatment of autoimmune,hypersensitivity and allogenetic graft rejection.But the role soluble CD28 molecule palys in immune response and immune regulation is still unclear. Therefore,it is very important to prepare functional monoclonal antibodies against human CD28 molecule and to study their biological effects. l.Preparation and characterization of five monoclonal antibodies against CD281.1 Establishment of hybridoma cells linesAfter pretreatment with mitomycin(MMC),human multiple myeloma(MM) cell line,U266 highly expressing on CD28 molecules (positive rate over 98% detected by immunofluorescence) was used to immunize BALE 'C mice.According to the hybridoma technique,the immunized mouse spleencytes were fu $ed with mouse myeloma cells(SP2/0) using polyethylene glycol(PEG),and then were cultuied in HAT selection culture medium. Both cell lines U266 and a mouse malignant lymphoma cell line transfected human CD28 gene named,CD28-T were used as dual positive screening cell lines while a mouse malignant lymphoma cell line and human malignant B lymphoma cell lines Daudi which didn't express CD28 molecules served as negative ccntrol.Through repeatedly cloning and screening,five hybridomas (2D5,2F5,3B6,3F8 and 8G8) secreting anti-CD28 mAb continuously and steadily were eventually obtained. The five hybridomas grew well afer long-term culturing and storage in liquid nitrogen.Fast-strip analysis showed that subclass of 2D5 was IgG2a while those of others belonged to mouse IgGl.1.2 Production and purification of the mAb and their determination of titer BALB/C mice were primed with pristane.Ascites were induced by intraperitonealinjection of well-grown hybridomas and acquired from the fifth to tenth day. Affinity chromatography was used to purify mAbs from bath ascites and culture supernatants. Indirect immunofluorescence assay suggested that ihe titer of ascites to U266 cells was over 1:2000 while that of culture supernatants was o/er l:200.The engagement of purified CD28 mAb to U266 was 0.5ug/5*105 cells1.3 Identification of antigen determinants and recognition mAb for different cells U266 cells were incubated with one of five...