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With the development of antibody engineering, a diverse range of structures for T cell-dependent bispecific antibodies targeting CD3 have been devised to meet the needs of various clinical therapies. T cell-dependent bispecific antibodies can be categorized into IgG-like and non-IgG-like formats based on the presence or absence of an Fc domain. The most common difficulty in manufacturing bispecific antibodies is the mispairing between light and heavy chains. To overcome this challenge, several techniques, such as knob-into-hole and CrossMab technologies, have been implemented for structural design. Given that the mechanism of action of T cell-dependent bispecific antibodies is to redirect T cells to tumor cells to induce a killing effect, abolishment of Fc domain effector function, the affinity design of the target, and the distance design of immune synapse formation are all focal points in the structural design of T cell-dependent bispecific antibodies. This review aims to provide an overview of recent advances in the structural design of T cell-dependent bispecific antibodies.

Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group ( P <0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group ( P <0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group ( P <0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group ( P <0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group ( P <0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.

Aim: To construct a recombinant eukaryotic expression vector of anti-human CD86 diabody gene and express anti-CD86 diabody through Chinese hamster ovary (CHO) cells, then analyze the capability of the diabody to recognize the tumor cells expressing CD86 and its biological effect., Methods: The antibody heavy and light chain viable region gene (V(H); and V(L);) were cloned from hybridoma cell 1D1 which secreted anti-human CD86 monoclonal antibody. Anti-human CD86 diabody gene V(H);-(GGGGS)-V(L); was constructed by SOE-PCR. Then we inserted it into eukaryotic expression vector to construct a recombinant vector pIRES2-EGFP/CD86-diabody. The recombinant vector was transfected into CHO cells with Lipofectamine(TM); 2000, and the cell clones secreting CD86 diabody were screened by G418. We used IMAC to purify CD86 diabody and quantified its concentration by BCA method. The capability of the diabody to recognize the CD86 expressed on Raji and Daudi was analyzed through flow cytometry. After Raji cells were treated with CD86 diabody for 72 h, its proliferation inhibiting effect was investigated by MTT assay., Results: We have obtained one CHO cell line that stably secreted CD86 diabody. The concentration of CD86 diabody after purification was 5.24 mg/L. The positive rates of CD86 diabody to recognize Raji and Daudi were 77.2% and 70.6%, respectively. After CD86 diabody treatment for 72 h, the inhibition rate of Raji cells was 37%., Conclusion: The anti-human CD86 diabody which we obtained successfully could recognize CD86 expressed on tumor cells specifically, and inhibit the proliferation of these tumor cells effectively.

Objective: To analyze the drift of T cell receptor (TCR) Vα and Vβ gene family CDR3 spectratype in response to ovarian carcinoma cells mediated by an anti-human ovarian carcinoma/CD3 bispecific single-chain antibody (BHL-1), and explore the mechanism of the bispecific single-chain antibody-mediated T cell immune response., Methods: Immunoscopic spectratyping technique was used to analyze the TCR repertoire diversity (CDR3 spectratype distribution) of the T cells from 6 healthy donors before and after stimulation of the cells with human ovarian carcinoma in the presence of BHL-1. The predominant usage of TCR α and Vβ chain CDR3 was analyzed after the stimulation, and sequence analysis was performed for the CDR3 region of the monoclonal T cells., Results: The spectratypes of Vα and Vβ gene family TCR CDR3 region showed a Gaussian distribution before stimulation of the T cells from the 6 donors. After stimulation of the T cells, CDR3 spectratype drift occurred in the T cells, and some TCR Vα and Vβ families showed an anomalous and oligoclonal expansion. Different CDR3 sequences of the Vα and Vβ gene family TCR were found in the monoclonal T cells stimulated with BHL-1., Conclusion: CDR3 spectratype drift occurs in TCR α and Vβ chains of T cells after stimulation with human ovarian carcinoma cells and BHL-1, indicating that the predominant usage of TCR Vα and Vβ families is associated with the specific T cell immune response mediated by BHL-1.

Aim: To construct and express an anti-VEGFR2/anti-CD3 bispecific single-chain antibody (bscVEGFR2×CD3)and to identify its binding specificities to CD3 and VEGFR2., Methods: The gene encoding anti-VEGFR2/anti-CD3 bispecific single-chain antibody was designed and synthesized. Bispecific single-chain antibody (bsc-Ab) DNA was subcloned into a eukaryotic expression vector pcDNA3.1(+), then transfected into Chinese hamster ovary (CHO) cells and stable expression cell lines were selected. Expressed Bsc-Ab was purified by His-tag affinity chromatography and confirmed by 120 g/L SDS-PAGE and Western blotting. Antigen binding activity of the bsc-Ab was analyzed by FACS., Results: The plasmid DNA containing bispecific single-chain fragments were confirmed. BscVEGFR2×CD3 was secreted by CHO into the supernatant. Six stable expression cell lines were established. The molecular weight of bsc-Ab was correct indicated by SDS-PAGE and Western blotting. The bsc-Ab could specifically bind to CD3(+); jurkat cells and VEGFR2(+); A375 cells., Conclusion: An anti-VEGFR2/anti-CD3 bispecific single-chain antibody is successfully constructed and expressed, and the antibody has specific binding capacity to CD3 and VEGFR2.

We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.

Several studies have demonstrated the role of 4-1BBL in T cell activation. Furthermore, enhanced 4-1BB/4-1BBL interaction has been shown to amplify T-cell-mediated antitumor immunity in several mouse models. However, when applied in humans, it was difficult to generate sufficient T cells ex vivo and whole cell vaccines to transfer back into patients. To overcome this difficulty, we have focused on producing the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein. In this report, PCR and overlap PCR were used to construct the human 4-1BBL extracellular domain/anti-CD20 Fab' expression vector. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC; its antigen binding activity was examined by rosetting assay. The data of DNA sequence showed that the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 microg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells and Raji cells as shown by cellular rosetting. In conclusion, the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was induced to express in E. coli 16C9. The results of some biological activity experiments indicated that the fusion protein could bind to stimulated Jurkat cells and Raji cells. Furthermore, 4-1BBL-negative tumors can be converted into 4-1BBL-positive tumors by the fusion protein without the need for 4-1BBL gene transfer to the malignant cells.

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. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity., Results: The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively., Conclusion: The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.

Objective: To study and compare the biologic activity of two anti-PSA/anti-CD3 bispecific single-chain antibodies., Methods: Flow cytometry (FCM) was used to detect the binding activity of two antibodies to CD3-positive cell line Jurkat and prostate carcinoma cell line LNCaP. The effect of the two antibodies in mediating tumor cell lysis in vitro was determined by using the 51Cr-release test. For in vivo evaluation of the two antibodies activity, a nude mouse model was used. The mice were inoculated with LNCaP prostate cancer cells., Results: FCM showed that both the antibodies could bind Jurkat and LNCaP cells with high specificity. The percentages of the cells bond by the bispecific single-chain antibodies were 56.3% and 55.4%, and those by the multivalent antibodies were 74.0% and 83.0% respectively. Both the antibodies mediated a specific lysis of LNCaP cells in vitro, with activated CTLs as effector cells, and significantly reduced tumor growth of nude mice in vivo as compared with the untreated controls and the group treated with CTLs only (P <0.05). The experiment also showed that the multivalent antibody had a better activity than the bispecific antibody in binding antigens, mediating lysis of LNCaP cells and reducing tumor growth (P < 0.05)., Conclusion: Both the anti-PSA/anti-CD3 bispecific single-chain antibody and multivalent antibody have good biologic activity, and the formation of the tetramerization of single-chain antibody can improve its biologic activity.

Aim: To construct a recombinant vector which expresses bispecific single chain antibody (BsscFv) against human gamma-seminoprotein and CD3 molecule and evaluate its biologic activity., Methods: The BsscFv gene was constructed by the splicing overlap extensive (SOE) PCR and then a flexible peptide linker was inserted between anti-human gamma-seminoprotein single chain Fv gene and anti-CD3 single chain Fv gene. The fusion gene was subcloned into the pSectag2-B plasmid and was expressed in HeLa cell lines. After being analyzed by SDS-PAGE and Western blot, the expressed product was purified through a Ni(2+)-NTA superflow affinity chromatography column. Flow cytometry (FCM) was used to detect the binding activity of BsscFv to CD3(+) cell line Jurkat cells and prostate carcinoma cells LNCaP. In vitro killing effect on target cells (LNCaP) mediated by BsscFv was determined by chromium(51)-release test. The effect of CTLs mediated by BsscFv on inhibiting tumor growth was observed by utilizing nude mice bearing prostate cancer cells., Results: DNA sequencing indicated that BsscFv gene consisted of 1,500 bp, encoding 500 amino acids. SDS-PAGE and Western blot analysis showed that the expressed product with relative molecular mass of 61,000 existed in culture supernatant of Hela cells. Flow cytometry analysis demonstrated that the binding rate of BsscFv to LNCaP cells and Jurkat cells was 54.1% and 53.7%, respectively. In vitro, BsscFv mediated cytotoxicity of CTLs to LNCaP cells as confirmed by chromium(51)-release assay. In prostate cancer nude mouse model, BsscFv inhibited tumor's growth as compared with control group., Conclusion: The BsscFv against human gamma-seminoprotein and CD3 molecule possesses certain biologic activity, and in vitro and in vivo it can mediate cytotoxicity of CTLs to prostate cancer cells.

After having successfully constructed and expressed the gene of the anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3 x CD20), here we analyzed its in vitro bioactivity of mediating the lysis of Ramous human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Obvious opoptosis characters were observed by Annexin V/PI(AV/PI) stained and scanning electron microscope. As evaluated by non-radioactive cytotoxity assay, the bscCD3 x CD20 showed potent bioactivity of mediating human B-lymphoma cells lysis in the presence of T-enriched human PBL. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose and time-dependent effect on mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5microg/mL and E:T used at 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBL. Among the upregulated apoptogenes, ATM and P53 showed an increase of 187 times and 15 times respectively, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the bscCD3 x CD20.

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., Results: 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., Conclusion: 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.

Aim: To prepare monoclonal antibodies (mAbs) against membrane antigens on human erythrocytes and characterize their properties., Methods: BALB/c mice were immunized with the membrane antigens of human type O erythrocytes. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. The antibodies to common antigen on human erythrocytes were screened by hexadimethrine bromide (polybrene) test tube method and then the agglutinating antibodies (complete antibodies) were weeded out by slide hemagglutination assay. The hybridoma cells secreting non-agglutinating antibodies (incomplete antibodies) were cloned by limiting dilution method. The stability of the obtained hybridoma cells and the properties of the mAbs were identified., Results: One hybridoma cell 2E8 was obtained, which secreted non-agglutinating antibody. The mAb 2E8 belonged to IgG1, could agglutinate H antigen, and had no species cross-agglutination reaction. The titers of culture supernatant and ascites of 2E8 were 1:1,024 and 1:64x10(6), respectively. When the affinity of mAb 2E8 was evaluated by agglutination reaction, erythrocytes began to agglutinate after 7 seconds and the clots exceeded 1 mm(2) in 3 minutes., Conclusion: The non-agglutinating mAb against H antigen was prepared successfully. The mAb 2E8 has good titer, affinity and specificity, which lays the foundation for preparation of bispecific antibodies (BsAb).

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., Results: 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., Conclusion: 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.

The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into Flp-In expression vector pcDNA5/FRT and transfeced into Flp-In CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300 microg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellular rosetting showed that it can react specifically on Jurkat (CD3+) and Ramous (CD20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can observed by microscope. All these results would lighten the further study of its biological functions in vitro and in vivo.

Objective: To fuse the genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and exploit a new way to improve the functional affinity and biological activity of antibody., Methods: Genes of p53 tetramerization domain and anti-CD3/anti-prostate-cancer BsAb was fused by technique of DNA sub-cloning. The fusion gene confirmed by sequencing was subcloned into the pSectag2-B plasmid. Then the recombinant plasmid was transfected into HeLa cells. The expression products, which were analyzed by both SDS-PAGE and western blotting, were purified with Ni(2+)-NTA superflow affinity chromatography. mBsAb-pSectag2-B plasmid was added into the suspensions of human peripheral blood mononuclear cells (PBMCs) and PC-3 cells respectively. Flow cytometry was used to examine the binding rate of multivalent anti-prostate-cander/anti-CD3 bispecific scFv with PBMCs and PC-3 cells. T cells were isolated from the PBMCs. PC-3 cells were labeled with Na(2)[(51)Cr]O(4) used as target cells. Labeled PC-3 cells, T cells, and different concentrations of mBsAb were mixed. Natural release control well with labeled target cells only and maximum release control well with labeled target cells and 10% SDS were prepared. The supernatants were extracted. gamma calculator was used to calculate the counts per minute (cpm) values to calculate the specific release rate of (51)Cr., Results: Sequencing showed a fragment from mBsAb-pSectag2-B with the size of 1.7 kb corresponding to the predicted value. SDS-PAGE and Western blotting showed expression of 67 000 D protein in the supernatant of culture fluid of HeLa cells transfected with MBsAb-pSectag2-B plasmid. The binding rates of multivalent anti-prostate-cancer/anti-CD3 bispecific scFv with PBMC and PC-3 cells were 70.4% and 81% respectively, significantly higher than those of anti-prostate-cancer/anti-CD3 bispecific scFv. In the presence of mBsAb the activated T cells lysed PC-3 cells in positive correlation with the antibody concentration and effective cell/target cell ratio and with a lysis rate significantly higher than those of the control groups., Conclusion: Multivalent anti-CD3 x anti-prostate-cancer BsAb exhibits much higher functional affinity and biological activity than anti-CD3 x anti-prostate-cancer BsAb, which may break a new path to the improvement of functional affinity and biological activity of antibody.

Aim: To prepare bispecific monoclonal antibody (bsmAb) against HIV p24 and human group A erythrocytes, and set up an indirect hemagglutination test for detecting HIV p24., Methods: Hybridoma cells 2-E(4) secreting anti-HIV p24 mAb and hybridoma cells S(2) secreting anti-human group A erythrocyte mAb were naturalized with 8-Ag and 5-BrdU respectively, making them sensitive to HAT. Then the two hybridoma cells sensitive to HAT were fused by routine method and hybrid-hybridoma cells secreting the bsmAb against HIV p24 and human group A erythrocytes were screened. The bsmAb was used to establish an indirect hemagglutination test for detecting HIV p24., Results: 6 hybrid-hybridoma cell lines were obtained. An indirect hemagglutination test for detecting HIV p24 was set up by using the bsmAb, and its sensitivity reached 400 ng/L., Conclusion: The bsmAb against HIV p24 and human group A erythrocytes is prepared and a rapid indirect hemagglutination test for detecting HIV p24 is developed by using the purified bsmAb.

Objective: To study the specific targeting cytotoxicity mediated by an anti-CD(3)/anti-CD(20) diabody., Methods: The diabody was purified by affinity chromatography and identified by Western blot assay, size exclusion chromatography, FACS and rosetting assay; the effect of the anti-CD(3)/anti-CD(20) diabody mediated lysis of CD(20)-expressing tumor cells was assayed by (51)Cr release assay in vitro and by human B cell tumor nude mice xenograft model in vivo., Results: The anti-CD(3)/anti-CD(20) diabody could bind both Jurkat cells (CD(3)(+)) and Daudi cells (CD(20)(+)) and appeared to be potent in targeting activated peripheral blood mononuclear cell (PBMC) to lyse Daudi cells in vitro. Furthermore, it inhibited tumor growth and prolonged the survival of mice bearing xenografted Raji cells., Conclusion: The diabody proved to be a potent agent for targeting peripheral blood lymphocytes to lyse CD(20) antigen expressing tumor cells in vitro and in vivo.

Objective: To study anti-tumor affects of bispecific monoclonal antibody targeted human monocytes-macrophages on hepatoma cells., Methods: The fragment of monoclonal antibody HAb18F(ab')2 which has high affinity to hepatoma cells and the fragment of monoclonal antibody antimonocytes/macrophages MAb7F(ab')2 were cross-linked with N-succinimidyl 3-(2-pyridl-dithil) propionate., Results: Heteroaggregated monoclonal antibodies HAb18F(ab')2-MAb7F(ab')2 could bind to hepatoma cells as well as monocytes-macrophages. They were purified with FPLC Sephacryl S-200. The bispecific character was identified with Indirect Immunofluorescent Staining., Conclusion: In vitro cytotoxicity assay showed that HAb18F(ab')2-MAb7F(ab')2 could significantly enhance the lysis of human hepatoma cells by human monocytes-macrophages.