[The influence of Ara-C on anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.].

Objective: To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.

Methods: The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C.

[Effect of calmodulin antagonist O-4-ethoxyl- butyl-berbamine on the proliferation of human breast cancer cell line MCF-7 and its relevant mechanism].

Objective: 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.

Methods: 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.

A review of ERGIC-53: its structure, functions, regulation and relations with diseases.

ERGIC-53 is a type I transmembrane protein. It includes an N-terminal signal sequence, a carbohydrate recognition domain, which is calcium-dependent and pH-sensitive, a stalk region, a transmembrane domain, and a short cytoplasmic domain; ERGIC-53 mainly acts as a receptor of a limited number of glycoprotein and transports them from ER to ERGIC and Golgi, meanwhile it has a secondary glycoprotein quality control function. Recent research has revealed that UPR, heat shock and VIPL may regulate the expression of ERGIC-53.

Haishengsu as an adjunct therapy to conventional chemotherapy in patients with non-small cell lung cancer: a pilot randomized and placebo-controlled clinical trial.

Objective: To investigate the effect of Haishengsu, an extract from Tegillarca granosa, on non-small cell lung cancer as an adjunct to conventional chemotherapy. DESIGNS/SETTINGS: Randomized, double-blind, placebo-controlled trial was conducted in 83 patients. The Haishengsu (n=42, 2.

[Three-step purification of preparative-scale antiCD20 (Fab')2].

Objective: To establish a three-step purification method of preparative-scale antiCD20 (Fab')2 using AKTA prime.

Methods: AntiCD20 (Fab')2 was extracted by hyperosmotic solution and then purified by CM sepharose FF, phenyl sepharose FF, and protein G sepharose FF.

Results: Around 8 mg anti-CD20 (Fab')2, whose purification was 96.

[Preparation and activity detection of monoclonal antibody against anti-CD3 ScFv].

Objective: To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration.

Methods: 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.

[Up-regulation of CD86 and cytokine expression in acute leukemia cells by Ara-C].

Aim: To observe the effects of Ara-c on the expression of CD86 molecule on acute leukemia cells and explore the possible mechanisms.

Methods: The expression of CD86 on U937, HL-60 and NB4 cell lines treated with or without Ara-C wa assayed by flow cytometry. The mRNA expression of CD86, NF-kappaB, IFN-gamma was examined by semiquantitative RT-PCR.

[Construction and expression of human 4-1BBL/anti-CD20 fusion protein].

Aim: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity.

Methods: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.

[The role of extracellular domain of human 4-1BBL in regulating activities of human peripheral blood lymphocytes in vitro].

Aim: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL).

Methods: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant.

[Construction and expression of diabody [CD3 x Pgp] without Etag].

Aim: To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity.

Methods: In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9.

[Design and activity determination of small molecular inhibitors of integrin alphavbeta3].

Objective: To explore the design and activity determination of small molecular inhibitors of integrin alphavbeta3 through structure-based virtual screening.

Methods: 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.

[Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7/ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine].

Objective: To investigate the reversal effect of O-(4-ethoxyl-butyl)-berbamine (EBB) on multidrug resistance (MDR) in MCF-7/ADR cell.

Methods: 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.

[An anti-P-gp/anti-CD3 bispecific antibody cytotoxic to human multidrug resistant KB cells].

Objective: To study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target.

Methods: 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.

[Cloning and expression of the extracellular domain of 4-1BBL].

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.

[Specific targeting cytotoxicity to resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody].

Objective: To study the specific targeting cytotoxicity to drug-resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody.

Methods: 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.

[Effect of calmodulin antagonist EBB on invasion of human fibrosarcoma cell HT1080].

Objective: To investigate the potential effect of EBB, a calmodulin antagonist, on invasion of human fibrosarcoma cells HT1080.

Methods: 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.

[High level expression of chimeric antibody fragment F(ab')2 directed against CD20 in Escherichia coli].

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.

[Construction and expression of single chain Fv gene against domain II of human VEGF receptor II].

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.

[Expression of chimeric anti-CD3 IgG antibody in mammalian cells and analysis of its biological activity].

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.

[Construction and expression of anti-CD3/ anti-Pgp Diabody].

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.

[One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified.

[Effect of monoclonal antibody against extracellular domain III of vascular endothelial growth factor receptor KDR on proliferation of vascular endothelial cells].

Objective: To prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity.

Methods: Extracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph.

[Prokaryotic expression and characterization of an anti-KDR single chain antibody].

Aim: To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity.

Methods: The restriction enzyme sites were added to the previously cloned V(H) and V(L) genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH.

Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor II.

Aim: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain III of vascular endothelial growth factor receptor KDR and study its biological activity.

Methods: Soluble KDR Ig domain III (KDR-III) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-III were prepared by hybridoma technique.