Preparation Of Chimeric Antibody Against Human B7-2 Molecule And Analysis Of Its Biological Effects

Objective: preparing anti-human B7-2 chimeric antibody and studying its anti-tumor effect.Method and Result:1 Preparation, purification and titer assay of ch1D1Eukarya expression cell line CHO-ch1D1 has been cultured and selection with compression by G418, then was sub-cloned and selected cell line which high expresses and stably secrete ch1D1, it has been cultured, culture supernatant was collected and purified through Protein G affinity chromatography, on the peak of specific elution collect chimeric antibody, quantitated by method of ultraviolet spectrophotometer, the rate of production is 30mg/L or so. ch1D1 combined with gene transfected cell L929-B7-2 on dosage 4μg,2μg,1μg and 0.5μg, positive rate was 98.4%,97.6%,97.2%and 59.1% respectively by indirect immunofluorescence and flow cytometry (FCM). The titer of ch1D1 was set as 1μg/5×105cell.2 ch1D1 recoginized membrane B7-2 moleculeThrough indirect immunofluorescence and FCM, we examined the recognition of ch1D1 to surface molecule B7-2 on Raji,Daudi(B lymphoma cell line),Jurkat(T lymphoma cell line ) and L929-B7-2, positive rate was97.5%,96.5%,2.1% and 97.2%. The result showed that ch1D1 could combine membrane B7-2 with high affinity, corresponded to parent antibody 1D1.3 ch1D1 suppressed proliferation and induced apoptosis of RajiThe suppression effect of ch1D1 on Raji was investigated by MTT assay, under the condition of co-culture of Raji and ch1D1 with a final concentration 10μg/mL, meanwhile, apoptosis rate of Raji under induction of ch1D1 using FCM. ch1D1 alone can suppress the reproduction of Raji cell, with the inhibition rate of 27.1%, when ch1D1 react in combination with B7-1 human-mouse chimeric antibody ch4E5, the inhibition rate raised to about 56.3%, has statistics deviation in contrast to ch1D1 alone (P<0.05). In addition, the apoptosis rate of Raji was 12.21% when co-culture with ch1D1, 22.75% when in combination with ch4E5, 12.44% when co-culture with parent antibody 1D1.4 ch1D1's Fc fragment mediated ADCC and CDC effect on RajiPeripheral blood monocytes (PBMCs) were used as effect cells, Raji as target cells together with ch1D1 for co-culture to analyze the ADCC effect of PBMCs on Raji and CDC effect of complement on Raji via MTT assay at 4h, 24h, 48h and 72h, respectively. The killing rate of ch1D1-mediated PBMCs on Raji cell was 25.0%,52.0%,71.3%and72.7%, respectively. The complement comes from human serum with dilution 4 times, the killing rate of ch1D1 mediated complement on Raji cell was 39.4%,54.6%,60.3% and 66.1%. There is significant deviation compared to the human IgG1 and parent antibody group 1D1(P<0.05).5 ch1D1 inhibited tumorigenesis of Raji in vivoBALB/c nude mouse which were 6w old and female were selected in experiment. There are three group such as: A Raji cell alone without antibody, Raji 5×10~6/200μL; B Raji with ch1D1; C Raji with 1D1; inoculating subcutaneous on left axillary fossa. The antibody group ch1D1 and 1D1 acted on Raji with concentration 50μg/mL respectively in vitro, then inoculated after 30min and room tempreture. It is observed that the rate and the time of tumor generation and the size of tumor and so on. The result of experiment shows that no tumor generate with ch1D1 or 1D1 during 70d, nude mouse were survival and state well; in contrast, tumor generated at inoculation 30d without antibody, the rate of tumor formation was 70%, average diameter 2.8mm, at 70d average diameter 13.3mm.Conclusion: Anti-human B7-2 chimeric antibody ch1D1 has been prepared successively, ch1D1 has good effect to anti-tumor in vivo and vitro.