The influence of Ara-C on anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.</p><p><b>METHODS</b>The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C. The expressions of B7-1 and B7-2 on K562 and K562/AO2 cells were detected by FACS. The cytotoxicity of T-lymphocytes combined with anti-CD3/anU-Pgp plus Ara-C was analyzed by CytoTox 96 nonradioactive method.</p><p><b>RESULTS</b>The expressions of B7-1 and B7-2 on K562 and K562/A02 cells treated by Ara-C was significantly higher than those untreated. The effect/target ratio was from 0.39:1 to 25:1, and the killing rate of activated T cells to anti-drug-resistant leukemia cells was from (16.44 +/- 1.20)% to (60.49 +/- 2.90)%. The killing rates were increased gradually, with both the effect/target ratio and the antibody concentration increasing (P < 0.05).</p><p><b>CONCLUSION</b>Ara-C may be an important adjuvant for improving anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.</p>

Effect of calmodulin antagonist O-4-ethoxyl- butyl-berbamine on the proliferation of human breast cancer cell line MCF-7 and its relevant mechanism / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine (EBB) on proliferation of human breast cancer cell MCF-7 and its possible mechanism.</p><p><b>METHODS</b>MTT assay was used to analyze the effect of EBB on tumor cells growth. Flow cytometry was used to detect its impact on the cell cycle of MCF-7 cells. Immunofluoresce labeling technique and laser scanning confocal microscope were used to reveal the changes of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum in the cells.</p><p><b>RESULTS</b>The IC50 value of EBB in MCF-7 cells was (13.0 +/- 3.7) micromol/L. MCF-7 cells were arrested at S phase after EBB treatment. Meanwhile, depolymerization of the microtubule and microfilament, impairment of the mitochondrion and swelling of endoplasmic reticulum were observed.</p><p><b>CONCLUSION</b>EBB arrests MCF-7 cells at S phase by inhibiting the growth of MCF-7 cells, which may be related to the changes of structures and functions of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum.</p>

Preparation and activity detection of monoclonal antibody against anti-CD3 ScFv / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration.</p><p><b>METHODS</b>McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb.</p><p><b>RESULTS</b>McAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively.</p><p><b>CONCLUSION</b>The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.</p>

Three-step purification of preparative-scale antiCD20 (Fab')2 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a three-step purification method of preparative-scale antiCD20 (Fab')2 using AKTA prime.</p><p><b>METHODS</b>AntiCD20 (Fab')2 was extracted by hyperosmotic solution and then purified by CM sepharose FF, phenyl sepharose FF, and protein G sepharose FF.</p><p><b>RESULTS</b>Around 8 mg anti-CD20 (Fab')2, whose purification was 96.678%, was purified. The antigen-binding activity of antiCD20 (Fab')2 was similar to that of antiCD20 (Fab')2 purified by protein G sepharose FF and S-100.</p><p><b>CONCLUSION</b>The three-step purification method can obtain high-purity preparative-scale antiCD20 (Fab')2 in a simple way.</p>

Design and activity determination of small molecular inhibitors of integrin alphavbeta3 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the design and activity determination of small molecular inhibitors of integrin alphavbeta3 through structure-based virtual screening.</p><p><b>METHODS</b>Based on the crystal structure of integrin ctv33 extracellular segment in complex with an ARG-GLY-ASP ligand, docking procedure against the receptor binding domain was performed on 3D database. Integrin alphavbeta3-mediated cell adhesion assay was performed to assess the adhesion-inhibiting ability of the candidate compounds. Cell migration assay and capillary-structure-like formation inhibition assay were used to estimate the effects of the compounds on integrin alphavbeta3. Analysis of molecular graphics was carried out to deduce a probable binding model of compound with integrin alphavbeta3.</p><p><b>RESULTS</b>From the top 1000 compounds with the best DOCK energy score, 50 compounds were selected for biological assay based on chemical and drug-like diversity. Seven of 50 compounds showed notable inhibition activity on cell adhesion, and two with half-maximum inhibition concentration (IC50) values less than 100 mol/L. The compound with best activity (1-37) showed high inhibitory activity in cell migration assay and capillary-structure-like formation inhibition assay. Molecular graphics analysis indicated that metal ion-dependent adhesion site (MIDAS) might be involved in the compound 1-37-mediated inhibition of ligand binding with integrin alphavbeta3.</p><p><b>CONCLUSIONS</b>Through virtual screening combined with biological assay, a promising lead compound was discovered to inhibit integrin alphavbeta3, which embodies the rational drug design with computation aid and brings a new thought and approach to find novel inhibitors of integrin.</p>

Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7/ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the reversal effect of O-(4-ethoxyl-butyl)-berbamine (EBB) on multidrug resistance (MDR) in MCF-7/ADR cell.</p><p><b>METHODS</b>3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the antitumor effect of EBB and determine the reversal effects of different concentrations ( < or = IC20) of EBB on MCF-7/ADR cell. Flow cytometry was applied to observe the intracellular accumulation of Rh123 and cell cycle in the presence of EBB. The expressions of MDR-related genes mdr 1 and topoisomerase II b (top II b) were evaluated by reverse transcription-polymerase chain reaction.</p><p><b>RESULTS</b>The sensitivity of MCF-7/ADR to adriamycin (ADR) was enhanced up to 50. 40, 89.80, and 14.88 folds after exposure of the cells to 3 micromol/L EBB, 7.5 micromol/L EBB, and 10 micromol/L verapamil (VPL), respectively. After 2 hours of incubation with 6 micromol/L EBB, intracellular Rh123 accumulation in MCF-7/ADR cells was increased to the level comparable to that in MCF-7 cells. When 6 micromol/L EBB was added together with 2 micromol/L ADR, MCF-7/ ADR cells showed to be arrested in the G2/M phase. The declination of mdr 1 gene expression was observed when 6 micromol/L EBB, 12 micromol/L EBB, and 10 micromol/L VPL were added for 48 hours; meanwhile, the expression of top II b mRNA showed no significant change.</p><p><b>CONCLUSION</b>EBB has a strong reversal effect on MDR in MCF-7/ ADR cell, which may be achieved by enhancing the arrestment of MCF-7/ADR cells at G2/M phase and increasing intracellular drug concentration.</p>

Effect of monoclonal antibody against extracellular domain III of vascular endothelial growth factor receptor KDR on proliferation of vascular endothelial cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity.</p><p><b>METHODS</b>Extracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph. Monoclonal antibody against KDR III was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [(3)H]-TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells.</p><p><b>RESULTS</b>McAb Ycom1D3 against KDR III was prepared which bound specifically to both the soluble KDR III and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells.</p><p><b>CONCLUSION</b>McAb Ycom1D3 against KDR III may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.</p>

An anti-P-gp/anti-CD3 bispecific antibody cytotoxic to human multidrug resistant KB cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target.</p><p><b>METHODS</b>The anti-P-gp/anti-CD(3) diabodies were secreted from E. coli strain 16C9 containing the expression plasmid PAYZDCP, grown at 30 degrees C in a shaker flask; the diabodies were purified by affinity chromatography and identified by SDS-PAGE; the effect of the anti-P-gp/anti-CD(3) diabody mediated lysis of P-gp-expressing tumor cells was assayed by (51)Cr release assay in vitro, and by human KB nude mouse xenograft models in vivo.</p><p><b>RESULTS</b>The diabodies were generated by bacteria as a soluble functional form and purified by one-step affinity chromatography with a yield > 4 mg/L culture medium. In (51)Cr release assay, the diabodies targeted human activated T cells to lyse P-gp(+)-KB/MDR cells in a dose-dependent manner. It suggested that the diabody was able to induce an efficient lysis of the target cells by human T cells in vitro. When combined with activated human T cells, the diabody significantly inhibited the growth of KB/MDR, but had no effect on KB xenografts.</p><p><b>CONCLUSION</b>The anti-P-gp/anti-CD(3) bispecific antibody is a potent agent for targeting human T lymphocytes to lyse solid tumor cells overexpressing P-gp in vitro and in vivo.</p>

Effect of calmodulin antagonist EBB on invasion of human fibrosarcoma cell HT1080 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the potential effect of EBB, a calmodulin antagonist, on invasion of human fibrosarcoma cells HT1080.</p><p><b>METHODS</b>The antitumor effect of EBB was assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by Zymogrophy analysis. The mRNA levels, of MMP-2, MMP-9, and tissue inhibitor of metalloproteinases (TIMP)-1 were evaluated by reverse transcriptionpolymerase chain reaction (RT-PCR). Transwell chamber assay was applied to measure the effect of EBB on the invasion of HT1080 cells.</p><p><b>RESULTS</b>Calmodulin antagonist EBB inhibited the proliferation of HT1080 cells with an IC50 of (8.2 +/- 1.2) microg/ml. EBB down-regulated the activities of MMP-2 and MMP-9, and down-regulated the mRNA levels of MMP-2 and MMP-9, while up-regulated the mRNA levels of TIMP-1. The invasive ability of HT1080 cells was decreased to (31.13 +/- 2.265)%, (59.91 +/- 2.566)%, and (71.58 +/- 0.5960)% after exposure of the cells with 2, 5, and 10 microg/ml EBB, respectively.</p><p><b>CONCLUSION</b>Treatment with calmodulin antagonist EBB is effective in suppressing tumor invasion. The possible mechanism is the down-regulation of MMPs.</p>

Cloning and expression of the extracellular domain of 4-1BBL / 生物工程学报

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.

Specific targeting cytotoxicity to resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the specific targeting cytotoxicity to drug-resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody.</p><p><b>METHODS</b>The diabody was purified by affinity chromatography and identified by SDS-PAGE and FACS. The effect of the anti-Pgp/anti-CD3 diabody mediated lysis of Pgp-expressing tumor cells was assayed by human leukemia nude mice xenograft model in vivo.</p><p><b>RESULTS</b>The diabody was produced in E.coli in a soluble functional form and could bind both Jurkat cells (CD3(+)) and K562/A02 cells (Pgp(+)). The binding rates were 86.25% and 86.26%, respectively. It could inhibit tumor growth by 98.57% and prolonged the survival of mice bearing xenografted K562/A02 cells.</p><p><b>CONCLUSION</b>The diabody was proved to be a potent agent for mediating T lymphocyte cytotoxicity to lyse Pgp expressing tumor cells in vitro and in vivo.</p>

Establishment of an imatinib resistant cell line K562/G01 and its characterization / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a BCR/ABL+ cell line with resistance to imatinib, and investigate the possible mechanisms of the acquired resistance.</p><p><b>METHODS</b>K562 cells were cultured in gradually increased concentrations of imatinib over a period of several months to generate their resistance line. MTT assay, RT-PCR, Western blotting, and FISH were used to study the possible molecular mechanisms of the resistance.</p><p><b>RESULTS</b>A resistant cell line, K562/G01, was established with 15.2 +/- 3.0-fold resistant to imatinib as compared with that of the parental sensitive cell line. The resistant cell line also had the cross-resistance to a broad spectrum of other anticancer agents excepting for DOX. There was no difference between the two cell lines in terms of the cell morphology, proliferation doubling time, and fraction distribution of cell cycle. K562/G01 cells showed increased levels of BCR/ABL, mdr1 mRNA and their coding proteins and the increased tyrosine kinase activity. No point mutation in the BCR/ABL ATP-binding site was detected while the copies of BCR/ABL fusion gene were increased in K562/G01 cells.</p><p><b>CONCLUSION</b>An imatinib-resistant human leukemia cell line, K562/G01, was established. The mechanisms of resistance of K562/G01 cells to imatinib involved increased expression of BCR/ABL and mdr1/P-gp, amplification of BCR/ABL fusion gene, and increased activity of BCR/ABL.</p>

Molecular design and biological activity of BCR-ABL tyrosine kinase inhibitors / 中国医学科学院学报

<p><b>OBJECTIVE</b>To discover BCR-ABL tyrosine kinase inhibitors through structure based virtual screening.</p><p><b>METHODS</b>Docking screening against the distinctive inactive conformation of the catalytic domain of BCR-ABL tyrosine kinase was performed on 3D database. The MTT assay was performed to assess the viability of the tumor cells treated with selected compounds. The amount and kinase activity of BCR-ABL protein were detected in the presence of compounds by Western blot analysis and immunoprecipitation.</p><p><b>RESULTS</b>From the top 1,000 compounds with the best DOCK energy score, 15 compounds were selected for biological assay. Eight out of 15 compounds showed notable inhibitory activity against Ph+ human K562 cells with IC50 values ranging from 10 to 200 micromol/L. In cell-based assays of ABL tyrosine phosphorylation, the ability of two kinds of novel, structurally diverse, lead compounds to inhibit ABL kinase activity was observed. However, no significant differences in the amount of BCR-ABL protein were noted on the ABL immunoblot in the presence of these lead compounds.</p><p><b>CONCLUSIONS</b>Two promising lead compounds were discovered to inhibit BCR-ABL tyrosine kinase activity. Virtual screening technique has been proven to narrow down the size of screening compound libraries to the most prospective drug candidates with high success rates.</p>

High level expression of chimeric antibody fragment F(ab')2 directed against CD20 in Escherichia coli / 生物工程学报

The use of tumor antigen specific antibody for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. In previous work, the human-mouse chimeric antibody fragment Fab' directed against CD20 was constructed from the new anti-CD20 antibody HI47 (a mouse IgG3, K). The chimeric antibody fragment Fab' could reduce its antigenicity, but the yield, quality and affinity of chimeric antibody fragment Fab' restrict its use. To improve affinity of chimeric antibody fragment Fab', a new phasmid pYZcpp3, which expresses chimeric antibody fragment F(ab')2, was constructed by adding a sequence encoding a small peptide, (CPP)3, to C-terminus of heavy chain constant region of chimeric antibody fragment Fab'. Using the pYZcpp3 to transform E. coli. 16c9, the genetically engineered bacteria 10916# was obtained. 10916# can secret the soluble chimeric antibody fragment Fab' and F(ab')2 into periplasmic. The yield was up to 360 mg/L with the percent of F(ab')2 up to 45% in 19L fermentor by the high density fermentation technology. Without denaturation and renaturation, the F(ab')2 has possessed the native three-dimensional structure. The purity of F(ab')2 was more than 90% after the purification of protein G affinity chromatography and S200 size exclusion chromatography. The F(ab')2 could distinguish and bind to Raji cells (CD20+) by FACS. F(ab')2 could inhibit the proliferation of Raji cells in vitro by MTT, IC50 was 22.8 microg/mL. HI47 and its chimeric fragments F(ab')2 induced a significant level of apoptosis (23.5%, 20.8%, respectively), independent of any cross-linking agents, in Raji cells after 24 h incubation. The chimeric antibody fragment F(ab')2 directed against CD20 is possible to apply to tumor therapy in clinic in the future.

Construction and expression of single chain Fv gene against domain II of human VEGF receptor II / 生物工程学报

The genes encoding for the light and heavy chain variable regions (V(H) and V(L)) has been cloned by RT-PCR from a murine hybridoma that produced monoclonal antibody (mAb) Ycom1D3, which was against domain III of human vascular endothelial growth factor receptor II (KDRIII) and were then connected to each other by a short peptide linker containing 15 amino acids (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant Ycom1D3-ScFv gene was cloned into the expression vector pAYZ and induced to express in E. coli 16C9. SDS-PAGE and Western blot analysis showed that the recombinant Ycom1D3-ScFv gene was expressed in E. coli 16C9 and the relative molecular weight of the fusion protein is 30kD which was consistent with the theoretically predicted value. ScFv expression was in the form of an inclusion body and the purified fusion protein was obtained after a series of purification steps including cell breakage, inclusion body solubilization, TALON metal affinity chromatography and protein refolding. Flow cytometric analysis showed that the ScFv fragment can react with human umbilical vein endothelial cells (HUVECs) which express KDR on the cell surface. In Conclusion, Recombinant Ycom1D3-ScFv gene has been successfully constructed and expressed in E. coli 16C9, which could be useful in both diagnostic and therapeutic applications.

Identification of differentially expressed genes involved in multidrug resistance in K562/A02 by cDNA microarray / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the molecular mechanism underlying multidrug resistance (MDR) and identify unknown genes that might be involved in drug resistance development in K562/A02 cells.</p><p><b>METHODS</b>K562/A02 was induced by gradually increasing the ADM concentration in culture medium of K562 cells, the differential expression of associated genes between K562 and K562/A02 was determined with cDNA microarray. Overexpression of neurofilament protein NF-H gene in K562/A02 cells was confirmed with RT-PCR and immunocytochemistry. Anti-sense oligodeoxynucleotides were transfected into K562/A02 cells by lipofectamine in order to further analyze the role of NF-H in drug resistance.</p><p><b>RESULTS</b>Comparing with the expression profiles, we found upregulation of 5 transcripts and downregulation of 7 transcripts in response to MDR of K562/A02 cells. The overexpression of NF-H, one of the 5 upregulated genes, was confirmed. After being treated with antisense oligodeoxynucleotides of NF-H and mdr1, the cellular adriamycin concentration increased significantly, but antisense NF-H alone did not have significant effect on drug resistance phenotype.</p><p><b>CONCLUSION</b>The development of MDR in K562/A02 cells is multifactorial. NF-H may be involved in the drug resistance of K562/A02, which may provide a new marker of diagnosis and a new target of therapy.</p>

Effects of 3-substituted aryl oxindole(PH II-7) on cell cycle of tumor cells / 药学学报

<p><b>AIM</b>To study the antitumor mechanism of 3-substituted aryl oxindole (PH II-7) and determine its effects on cell cycle distribution of tumor cells.</p><p><b>METHODS</b>The cell cycle distributions were determined with FACS. The cell cycle regulation-related proteins of K562 lysates were analyzed with Western Blot. The inhibition of PH II-7 on DNA synthesis of tumor cells were estimated though 3H-thymidine incorporation and the tyrosine kinase activity of EGFR of A431 lysates was measured with ELISA.</p><p><b>RESULTS</b>PH II-7 effected cell cycle distribution of several tumor cells, including multidrug resistant tumor cell lines, and accumulation of cells in the G0-G1 stages was observed. The cell cycle regulation-related proteins CDK2, Rb and c-myc were inhibited by PH II-7 in a dose dependent manner, whereas the expression of CyclinE was increased after exposure to PH II-7. Furthermore, PH II-7 2.0 mg.L-1 was shown to inhibit the incorporation of 3H-thymidine into DNA, and 21.89%-41.29% of the PTK activity of EGFR in A431 lysates was inhibited by PH II-7 2-8 mg.L-1 in a dose-dependant manner.</p><p><b>CONCLUSION</b>PH II-7, a new anti-tumor agent, blocks the transition of cell cycle of tumor cells from G1 to S phase by inhibition CDK2.</p>

Structure-based design, synthesis and evaluation of bioactivity of anti-P-gp peptide mimetic / 药学学报

<p><b>AIM</b>To design and evaluate the small peptide mimetic of anti-P-glycoprotein (P-gp) antibody (PHMA02).</p><p><b>METHODS</b>From the three dementional structure analysis of computer modeling of PHMA02 CDR loops, a small peptide mimetic was designed and determined by flow cytometry.</p><p><b>RESULTS</b>Anti-P-gp peptide mimetic functionally similar to PHMA02 was developed. The peptide mimetic competitively inhibits PHMA02 binding to P-gp and partially block the P-gp function as a drug efflux pump in K562/A02 cells.</p><p><b>CONCLUSION</b>Some special conformational properties of CDR loops of antibody might serve as lead structures for develop new biological peptide mimetics. Antibody-structure-based design would develop new drug in the future.</p>

Construction and expression of anti-CD3/ anti-Pgp Diabody / 生物工程学报

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.

Expression of chimeric anti-CD3 IgG antibody in mammalian cells and analysis of its biological activity / 生物工程学报

The anti-CD3 antibody can improve success rate of organs transplant. HIT3a, a mouse anti-CD3 antibody, was chimerized by using gene engineering methods to decrease its immunogenity. The anti-CD3 genes, heavy chain and light chain, were cloned using PCR from the vector pCANTAB 5E containing anti-CD3 scFv gene fragment, and two PCR fragments were recombined into the expression vector pKN100 with human antibody light constant domain and pG1D105 with human antibody heavy constant domain, respectively. The two vectors were co-transfected into CHO cells using liposome. The anti-CD3 antibody was detected by ELISA and Western blot assay in supernatant of transfected CHO cells culture. The primary results of competitive assays by FACS showed that anti-CD3 antibody could partially block the sites through which parent antibody (HIT3a) bind to CD3+ Jurkat cells. The result of 3H-TdR incorporation showed that the chimeric anti-CD3 antibody could stimulated proliferation of peripheral blood mononuclear cells (PBMC) as the parent antibody. In this thesis, the results of some experiments indicated that the chimeric anti-CD3 antibody expressed in CHO cells was an antibody with native biological activity, and it is possible to apply to in clinic in the future.