Cloning and Expression of Human CD20 Gene on NIH-3T3 Cell Membrane.

CD20 is a specific antigen expressed on normal and neoplastic B cells exclusively. Recent researches showed that in B cell leukemia, CD20 was over-expressed. Therefore monoclonal antibody (McAb) to CD20 may be of clinical value in diagnosis and treatment of some leukemias and lymphomas. In this study, the full length gene of CD20 cDNA were cloned from total RNA of Raji cells, inserted into an eukaryotic expression vector pcDNA3.1, forming a recombinant plasmid pcDNA3.1/CD20. NIH-3T3 cells were transfected with pcDNA3.1/CD20 and selected with G418 for the transfected cells. Alkaline phosphatase against alkaline phosphatase assay(APAAP) experiments showed that the selected cells could express the human CD20 onto its surface. Balb/c mice were immunized with CD20( ) NIH-3T3 cells once three weeks for 3 shuts. Indirect immunofluorescence experiments were done with the Raji cells and the serum of the immunized mice, and the results showed that the spleen of the immunized mice could be used to prepare the McAb to CD20.

[Expression and Characterization of Single-chain Fv-Fc Fusion Protein against Human P185(erbB2)].

In order to increase therapeutic effects and decrease immunogenicity of mouse McAb, the single-chain Fv (scFv) created by fusing the light and heavy chain variable region genes of anti-human P185(erbB2) McAb was conjugated to the Fc gene of human IgG1 to construct a scFv-Fc fusion gene. The scFv-Fc fusion gene was cloned into the expression vector pCIDN. The scFv-Fc fusion protein was synthesized as secreted two-chain molecule in CHO cells, and purified by affinity chromatography on recombinant protein A. A special 185 kD P185(erbB2) protein was immunoprecipitated by the scFv-Fc fusion protein. The fluorescence-activated cell sorting (FACS) using SK-BR-3 cells as the target indicated that the fusion protein could bind to the extracellular domain of P185(erbB2). The affinity of the scFv-Fc fusion protein, determined by ELISA, was K=7.5x10(-10) (mol/L)(-1). This work laid basis for further studies on the anti-P185(erbB2) scFv-Fc fusion protein.

[Cloning and expression of soluble B lymphocyte stimulator].

AIM: To clone and express soluble B lymphocyte stimulator (sBLyS). METHODS: Total RNA was isolated from peripheral blood mononuclear cells, and used to synthesize cDNA by reverse transcription. sBLyS cDNA was amplified by PCR with specific primers and inserted into a prokaryotic expression vector pET-30a. Recombinant plasmid was transformed into E.coli strain BL21(DE3). sBLyS was expressed in E.coli, purified in vitro, and analyzed with peptide mass fingerprinting and Daudi cell proliferation assay. RESULTS: sBLyS cDNA was cloned. Peptide mass fingerprinting of purified BLyS matched with that of BLyS proteins. Purified sBLyS could stimulate Daudi cell proliferation in vitro. CONCLUSION: sBLyS with biological activity was successfully expressed and purified.

[Construction and expression of chimeric anti-human CD20 monoclonal antibody].

AIM: To construct the eukaryotic expression vector of chimeric anti-human CD20 monoclonal antibody (mAb) and realize its expression. METHODS: The light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR and were cloned to T vector and sequenced. Proteins of mAb 1-28 were separated by reducing SDS-PAGE. Light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. Software Biolynx and pepeseq were used to evaluate the score of probability. Correctness of the light- and heavy-chain DNA sequences was verified by their protein sequences. Genes of V(H) and V(L) were amplified from T vector and cloned into chimeric antibody expression vector (pCMV-V(H) and pCMV-V(L)), generating the expression vectors of chimeric anti-human CD20 mAb (C1-28) including light chain expression vector C1-28L and heavy chain expression vector C1-28H. The two plasmids were co-transfected into 293T cells with Lipofectamine 2000. RT-PCR was used to detect the transcription at mRNA level. C1-28 expression was detected by Sandwich ELISA and Western blot methods. RESULTS: mAb 1-28's genes were successfully cloned and verified by peptide mass fingerprinting. Eukaryotic expression vectors of anti-human CD20 mAb were constructed and expressed in 293T cells with the expression amount reaching 257 mg/L and the molecular weight consistent with that of human IgG. CONCLUSION: These experiments lay solid foundation for further study on the role of chimeric CD20 antibody in the treatment of non-Hodgkin's lymphoma.

A novel neutralizing monoclonal antibody against cell-binding polypeptide of ricin.

One strain of neutralizing monoclonal antibody (MAb) against cell-binding polypeptide of ricin, named 3E1, was generated efficiently. The antibody recognized the linearity epitope of RTB located in a toxin structure domain characterized by Western blotting. The safe period of mice for intraperitoneal injection of 100 microg of antibody was 20 min after intraperitoneal injection of 2 microg of Ricin (10 times LD50). The neutralizing MAb we obtained could be developed into an immunotherapeutic agent to counteract the use of ricin as a terrorist or biological warfare weapon. It might be useful, as well, for antibody-based prophylaxis.

[Experimental study of adoptive immunotherapy using CD3AK cells].

AIM: To analyze the immunological properties and biological activity of a monoclonal antibody (mAb) against CD3 molecule(yCD3), and to observe the tumor-suppressive activity of CD3AK cells in vitro and in vivo. METHODS: FCM was used to test the specificity of yCD3 and the immunological phenotype and cytokine production of CD3AK. 3H-TdR assay was used to measure the transformation of lymphocytes activated by yCD3. LDH assay was used to analyze the cytotoxic activity of CD3AK in vitro. Mice bearing tumors were used to observe the anti-tumor effect of CD3AK cells. RESULTS: yCD3 could bind specifically to T cells. 5 microg yCD3 could competitively inhibit 70% of standard anti-CD3 antibody to bind with CD3 molecules on cell membrane. 8 microg/L of yCD3 stimulated the proliferation of peripheral blood lymphocytes, which could be further boosted by IL-2 or anti-CD28 antibodies. Among activated CD3AK cells, CD3+, CD8+, and CD25+ cells increased. IL-2 and IFN-gamma producing CD3+ cells were also increased, to 3.29- and 2.47- fold, respectively, under the co-stimulation of anti-CD28 antibody. When the ratio of effective cells and target cells was 80:1, 57.54% target cells were killed. As compared with control, the percent of tumor inhibition in CD3AK cells treated tumor-bearing mice was 33.17%, and the inhibition rate of lung metastasis was 39.70%. The CD3AK cells treatment was more effective when combined with LAK cells. CONCLUSION: yCD3 could activate T cells and significantly induce the tumor-suppressive activity of CD3AK cells in vitro and in vivo, which lays a foundation for adoptive immunotherapy against tumors in clinical medicine.

IL-6 inhibits apoptosis of human myeloma cell line XG-7 through activation of JAK/STAT pathway and up-regulation of Mcl-1.

BACKGROUND & OBJECTIVE: IL-6 can protect myeloma cells from apoptosis induced by various stimuli. A series of intracellular molecules might participate in this process. Our aim in this study is to investigate which anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, Mcl-1) and which signal transduction pathway(JAK/STAT, Ras/MAPK, PI-3K/Akt) can mediate the anti-apoptotic effect of IL-6 on a human myeloma cell line XG-7. METHODS: Apoptosis of XG-7 cells was analyzed by flow cytometry with propidium iodide(PI) staining of nuclei. The expression of three Bcl-2 family proteins in XG-7 cells were monitored by immunoblot assay. AG490, PD98059, and LY294002, three specific antagonists for JAK/STAT, Ras/MAPK, and PI-3K/Akt signal transduction pathways respectively, were used to determine which signal transduction pathway was responsible for the effect of IL-6 on XG-7 cells. RESULTS: IL-6 inhibited the apoptosis of XG-7 cells and up-regulated the expression of only one of the three anti-apoptotic Bcl-2 family proteins-Mcl-1. In addition, up-regulation of Mcl-1 expression induced by IL-6 was significantly inhibited in the presence of AG490, while not of PD98059 and LY294002. CONCLUSIONS: IL-6 inhibited apoptosis of XG-7 cells through up-regulation of Mcl-1 and the activation of JAK/STAT rather than Ras/MAPK nor PI-3K/Akt signal transduction pathway.