Study On The Biological Effect Of Novel Antibody Against Transferrin Receptor In Vitro

[Background and Objective]Transferrin receptor（TfR）is a type II transmembrane glycoprotein on the cell membrane,which is related to the uptake of iron and affects the growth and proliferation of cells.The expression of TfR is low in normal tissues but high in the surface of tumor cells.Due to the rapid growth of tumor cells and the need to synthesize more DNA,ribonucleotide reductase requires more cofactor iron,which forces tumor cells to upregulate the expression of TfR.Previous report and our preliminary data showed that the expression level of TfR on the surface of tumor cells was 10-100 times higher than that of normal cells.Hence,the expression level of TfR is closely related to tumor stage and prognosis.TfR is an important marker for tumor targeted imaging and anti-tumor therapy because of its characteristics of extracellular segment binding,endocytosis and the role of human tumor cells pathology.However,it is a key point that how to obtain a large amount of antibody with high affinity.This research aims to obtain a new tetravalent antibody against TfR（TfR-TeAb）.On the basis of TfR scFv and bivalent antibody,TfR-TeAb was constructed and stably transfected into CHO-DHFR^-cell line（also known as DG44）.Culture conditions were optimized to maximize the production of antibody.The anti-tumor effect of TfR-TeAb was evaluated.These data could set new light for cancer therapy.[Methods]1.Construction of eukaryotic expression plasmid encoding for TfR-TeAbUsing the anti TfR bivalent antibody vector TfR-scFv-Fc as the template,the VL-Linker-VH gene fragment was obtainby overlap PCR.Then the VL-Linker-VH fragmentswere engineered into plasmid encoding for TfR-scFv-Fc and inserted between VH-Linker-VL and Fc gene fragments to obtain plasmid TfR-（scFv）₂-Fc.2.Establishment of cell line stably transfected by TfR-TeAbTfR-（scFv）₂-Fc was transfected into DHFR deficient CHO cells（DG44）by nuclear transfection.Several clone cellswere selected using MTX by limited dilution.ELISA was used to detect the concentration of antibodies in the supernatant for screening clones which highly expressed antibodies.FCM was used to detect the binding capacity of TfR-TeAb with TfR⁺cells.3.Purification and identification of TfR-TeAbClone cells were cultured in serum-free media.Supernatant was filtered and then purified using Protein A-sepharose affinity chromatography.（1）SDS-PAGE was used to analyze the purity and molecular weight of TfR-TeAb.（2）Supernatant from TfR-TeAb transiently transfected 293T cellswas incubated with hTfR⁺CHO-hTfR and the control cell line CHOvec.The activity and specificity of TfR-TeAb was detected by FCM.（3）The constant affinity of TfR-TeAb was determined by ELISA method.4.Biological effects of TfR-TeAb（1）FCM was used to detect the surface expression of TfR on HepG2 and MDA-MB-231 cells.（2）The antiproliferative effect of TfR-TeAb on HepG2 was detected by Cell Counting Kit.（3）Confocal microscopy was used to investigate whether TfR-TeAb was internalized into the cell.（4）ADCC effects of TfR-TeAb combined with NK cells on tumor cells were detected by FCM.5.Effects of GRP78 on antibody production（1）The CHO-GRP78 stable cell line was established.Then the TfR-TeAb was transfected into CHO-GRP78 and CHO cells.1)ELISA was used to detect the concentration of TfR-TeAb in the culture supernatant at different time points.2)FCM was used to detect the binding of TfR-TeAb with TfR⁺HepG2 cells,and the survival rates of CHO-GRP78 cells and CHO cells were detected by PI staining.3)The numbers of viable cells of CHO-GRP78 and CHO at each time point were determined by trypan blue staining.（2）Western Blot was used to detect the activation of caspase-3 in CHO-GRP78cells and CHO cells.[Results]1.Gene acquisition and construction of expression vectorThe VH-linker-VL-GCTAGCACCGGATCC sequence（NheI-Linker2-BamHI）was obtained by overlap PCR using our previously established VH and VL as templates.Then the target genes were cloned into Fc-pOptivec plasmid by NotI/BamHI double enzyme digestion to obtain TfR-VH-Linker-VL-Fc expre-ssion vector TfR-scFv-Fc.Eukaryotic expressed TfR-scFv-Fc can form dimers.NheI-Linker2-VL-linker（1/2）and linker（1/2）-VH-BamHI gene were obtained by PCR amplification using TfR-scFv-Fc vector as template.NheI-Linker2-VL-linker-VH-BamHI gene was obtained by overlap PCR.Confirmed by sequencing analysis,NheI-Linker2-VL-linker-VH-BamHI had been cloned and inserted into TfR-scFv-Fc plasmid by double enzyme digestion by NheI/BamHI,and the target sequenceVH-Linker-VL-Linker2-VL-linker-VH-Fc[TfR-（scFv）₂-Fc]was obtained.Dimeric TfR-（scFv）₂-Fc can form a tetravalent antibody.Sequencing showed that the TfR-TeAb eukaryotic expression vector TfR-（scFv）₂-Fc was successfully constructed.2.Transient expression and activity identification of TfR-TeAbTfR-（scFv）₂-Fc was transiently transfected into 293T cells.FCM results showed that the TfR-TeAb in the supernatant can combine with the CHO-hTfR cells with the positive rate up to 98.4%.Meanwhile the binding rate with CHOvec was only 0.2%.The results showed that TfR-（scFv）₂-Fc could express TfR-TeAb successfully.Moreover,TfR-TeAb can specifically bind to the TfR on cell membrane.3.Establishment of TfR-TeAb stable cell lineThe TfR-TeAb plasmid was transfected into DG44 cells.Six cell clones were acquiredby limited dilution.FCM results showed that the TfR-TeAb secreted by all six clones could specifically bind with CHO-hTfR cells.Three clones were finally selected and named as DG44-TfR-TeAb.4.TfR-TeAb biological character（1）SDS-PAGE showed that there was a bright stripe in the position of 80 kD in the medium-free culture supernatant.The molecule weight was as same as the theoretical value.The purity was over 90%.Non-denaturing SDS-PAGE showed that there was a bright stripe in the position of 160 kD,which suggested that TfR-（scFv）₂-Fc formed dimers automatically in DG44 cell.（2）The affinity of TfR-TeAbwith TfR was about 4.263×10⁹M^-1,while the affinity of TfR-McAb was 2.908×10⁹M^-1.This showed that TfR-TeAb had a higher affinity than that of mouse monoclonal antibody.5.Biological effects of TfR-TeAbTfR was highly expressed on the surface of HepG2 cells and MDA-MB-231 cells,and there was no significant difference between them.The results of CCK showed that the effect of antibody alone could not inhibit proliferation of HepG2.TfR-TeAb can be internalized into HepG2 cells observed by confocal micro-scopy.TfR-TeAb mediated cytotoxicity to HepG2 cells and MDA-MB-231 cells,suggesting that TfR-TeAb could mediate ADCC effects.6.Effects of GRP78 on antibody production（1）GRP78 can improve the yield of TfR-TeAb in CHO cell line.The results of ELISA showed that the concentration of TfR-TeAb in the supernatant of CHO-GRP78cells was higher than that of CHO cells on day 5,day 7 and day 9（5d,P<0.05;7d,P<0.05;9d,P<0.01）.（2）GRP78 can enhance the survival and viability of host cells.The survival rates of CHO-GRP78 cells was higher than those of CHO cells（3d,P<0.05;5d,P<0.05;7d,P<0.001;9d,P<0.001;11d,P<0.01）.The number of viable CHO-GRP78 cells was higher than that of CHO cells.At day 9,the number of CHO cells was less than 1×10⁶,while at the day 7,the number of CHO-GRP78 cells was still higher than 2×10⁶.The results showed that GRP78 could enhance the survival of host cells.（3）GRP78 may inhibit the apoptosis induced by serum deprivation viainhibiting the activation of caspase-3.The expression of cleaved-caspase-3 was detected by Western Blot in CHO-GRP78 cells and CHO cells cultured in serum-free medium.The results showed that the cleaved-caspase-3 decreased expression in CHO-GRP78 cells was lower than that in CHO（24h,P<0.05;48h,P<0.001）.[Conclusions]1.We had successfully constructed a new tetravalent antibody TfR-TeAb against TfR.We have obtained DG44-TfR-TeAb stable cell line by DHFR system.2.Purified TfR-TeAb showed correct molecular weight,good purity,high specificity and appropriate affinity.3.TfR-TeAb can bond to TfR and be inernalized into cells,which hinted that it could act as a carrier.4.TfR-TeAb mediated the ADCC effect to kill tumor cell.5.GRP78 could enhance the viability of CHO cells in serum-free culture,thus increa-sing the antibody production.Furthermore,we discovered that GRP78 can enhance the quality of produced TfR-TeAb to improve their binding ability with TfR which hinted that GRP78 has potential application value in biopharmaceutical industry.