Construction of a new anti-CD123 chimeric antigen receptor T cells and effect of anti-acute myeloid leukemia / 中华血液学杂志

Objective: To construct a new CD123- specific chimeric antigen receptor in order to provide a foundation for immunotherapy of CD123 positive leukemia. Methods: A hybridoma strain (6E11) capable of stably secreting CD123 antibody was obtained by a monoclonal screening technique, and the hybridoma cells were expanded and injected intraperitoneally to the pretreated Balb/c mice. Ascites was collected and purified to obtain the monoclonal antibody (mAb) . The affinity and specificity of 6E11 mAb were measured. The variable regions of the heavy and light chains of the 6E11 mAb were cloned by RT-PCR from the 6E11 mouse hybridoma. We generated a new CD123 specific chimeric antigen receptor with a scFv fragment derived from 6E11 antibody, designated as 6E11 CAR. T cells were transduced with lentiviral supernatant from 293T cells transfected with 6E11 CAR plasmid to generate 6E11 CAR-T cells. The specific cytotoxicity of 6E11 CAR-T against CD123(+) acute myeloid leukemia (AML) cell lines and primary AML cells in vitro were evaluated by co-culture experiments, degranulation experiments and cytokine releasing assay. Results: ① A hybridoma cell line 6E11 stably secreting anti-human CD123 antibody was developed and its variable region sequences were obtained. ② The 6E11 mAb has high affinity for CD123 protein (Kd value: 2.1 nmol/L) . The 6E11 mAb specifically recognizes CD123(+) cell line THP-1 cells and does not respond to CD123(-) cell line Jurkat cells. ③ 6E11 CAR-T cells were successfully generated with a CAR expression rate higher than 60%. ④ 6E11 CAR-T cells could specifically kill CD123(+) MV4-11 cell line but had no killing effect on the CD123(-) K562 cell line. Compared with vector-T cells, 6E11 CAR-T cells have higher killing rate to MV4-11 cells[ (98.60±1.20) %vs (20.28±6.74) %, P<0.001]. ⑤ MV4-11 cells activated 6E11 CAR-T cells significantly but not Vector-T cells[ (26.33±3.30) %vs (1.17±0.06) %, P<0.001]. ⑥ 6E11 CAR-T cells released more cytokines than vector-T cells when co-cultured with MV4-11[IL-2: (92.90±1.51) pg/ml vs (6.05±3.41) pg/ml, P<0.001; TNF-α: (1 407.20±91.95) pg/ml vs (7.86±0.85) pg/ml, P<0.001; IFN-γ: (5 614.60±170.17) pg/ml vs (8.42±2.70) pg/ml, P<0.001]. The IFN-γ, IL-2 and TNF-α in the 6E11 CAR-T group were similar to those in the Vector-T group when co-cultured with K562. ⑦ 6E11 CAR-T cells could be activated by bone marrow mononuclear cells (BMMNC) derived from CD123(+) AML patients and effectively kill these BMMNC cells from CD123(+) AML patients. Conclusion: 6E11 hybridoma cell line can stably secrete highly specific monoclonal antibodies against human CD123, which can be used to detect the expression of human CD123. It can also be used to target human CD123 protein in tumor immunotherapy. CD123 CAR-T cells with 6E11 Ig variable region sequence have specific anti-leukemic activity in vitro, which may provide a new option for further clinical research of AML.

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>

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>

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>

Mechanism of synergistic anti-tumor effect of PH II -7 with doxorubicin on multi-drug resistant HL-60/ADR cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the synergistic mechanism between PH II -7 and doxorubicin against multi-drug resistant HL-60/ADR cells and its parent HL-60 cells.</p><p><b>METHODS</b>The anti-tumor activity of doxorubicin alone and combined with PH II -7 were measured by MTT assay. RNA was extracted from the cells treated with PH II -7 for different times or doses then the expression of MRP gene was measured by RT-PCR. Confocal laser scanning microscopy and FACS were used to detect the intracellular cumulation of doxorubicin in PH II -7 treated HL-60 and HL-60/ADR cells.</p><p><b>RESULTS</b>PH II -7 has anti-tumor effect with IC50 of (0.83 +/- 0.08) micromol/L and (1.74 +/- 0.56) micromol/L for HL-60 and HL-60/ADR, respectively. It could potentiate the anti-tumor effect of doxorubicin with CDI of 0.7 and 0.43 for HL-60 and HL-60/ADR, respectively. PH II -7 and doxorubicin act synergistically in inhibiting the proliferation of HL-60 and HL-60/ADR cells and down-regulating the expression of MRP gene in a dose and time dependent manner. PH II -7 restored the intracellular cumulation of doxorubicin in HL-60/ADR cells to 55% of that in HL-60 cells.</p><p><b>CONCLUSION</b>PH II -7 can significantly hasten the cytotoxicity of doxorubicin to HL-60 and HL-60/ADR cells through down-regulating the expression of MRP. The synergistic effect was more obvious in HL-60/ADR cells.</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 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>

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>

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>

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.

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>

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.

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.

One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity / 生物工程学报

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. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.