ABSTRACT
OBJECTIVE: To investigate the effects of anti CD3 monoclonal antibody (Ant-CD3 McAb) on immune function in cynomolgus monkeys, using the traditional immunotoxicity assessment methods and T cell dependent antibody response (TDAR) test, microarray, etc, and further explore the mechanism of Ant-CD3 McAb. METHODS: Eighteen Cynomolgus monkeys were individually dosed with saline, 0.5 and 2.5 mg·kg-1Ant-CD3 McAb for 7 d. The clinical observation, body weight, hematological test, antibody detection, TDAR test, lymphocyte subsets, bone marrow, histopathological examination and whole blood gene expression profile analysis were evaluated. RESULTS: Ant-CD3 McAb significantly inhibited the immune response to keyhole limpet hemocyanin (KLH) in cynomolgus monkey, and caused the decrease of peripheral white blood cell counts and CD3+ cells, the decrease of lymphocytes in thymus cortex. Microarray test showed that the gene function of differently expressed genes mainly involved in cell proliferation, immune response, cytokine secretion, apoptosis etc. CONCLUSION: Ant-CD3 McAb could induce obvious immunosuppression effects in cynomolgus monkeys. Detection of gene expression profile of whole peripheral blood is feasible in immunotoxicity evaluation. The apoptosis induction and proliferation inhibition of T lymphocytes might be the main cause of the decreased T lymphocytes. IL-8 might play an important role in the mechanism of Ant-CD3 McAb.
ABSTRACT
Objective:To amplify and sequence of the variable region genes of anti CD3 McAb.Methods:The V H?V L genes were amplified by RT PCR from total RNA that were extracted from WuT3 hybridoma.Recombinant cloning vector was constructed and sequenced after the enzyme digestion.Results:It showed that V H gene consisted of 363 bp encoding amine acid residues,belongs to mouse heavy chain subgroup IIB;V L gene consisted of 330 bp encoding amine acid residues,belongs to mouse ? light chain subgroup III.Comparing with Kabat database,the V H?V L genes were in agreement with the characterization of DNA sequences present in the mouse Ig V H?V L regions respectively.Conclusion:The success of cloning of the V H?V L genes of WuT3 McAb lay a good foundation for the construction and expression of chimeric antibody.
ABSTRACT
Objective To induce human mononuclear cell of cord blood into CD 3 activating killing (CD 3AK) cells with anti-CD 3 monoclonal antibody (CD 3McAb) and recombinant human interleukin-2 (rhIL-2), so that their proliferative activity, activity of killing action, phenotypes and level of secretory cytokines can be observed dynamically. Methods The increase of the number of cells was counted by Tapan-blue staining. The killing action can be measured by using methyl -thiazolyl-tetrazolium-array. The phenotypes of cells were analysed by using indirect immunofluorescence assay. The levels of IL-6, interferon-? (IFN-?) and tumor necrotic factor-? (TNF-?)in culture supernatants were analysed by using enzyme-linked irnmunosorbent assay(ELISA). Results The increase of the number of CD 3AK cells from cord blood was the highest amounting to 78.56 times in the second week. The killing action reached the peak on day 12, and all target cells (malignant cell lines) could be killed significantly. The heterogeneous phenotypes of CD 3AK cells showed that the number of cells with CD 3+, CD 8+, CD 25+, CD 38+, CD 16+ and CD 56+ increased significantly on day 7,14 compared with those of pre-culture (P