A Study About The Preparation Of Anti-TfR-Diabody And Its Anti-tumor Effect In Vitro

ObjectiveTransferrin receptor(TfR)is the main receptor for cells to take up iron.There are two types of TfR:TfR1 and TfR2.Of these,TfR1 is called CD71 which is lowly expressed on normal tissue cells,but is highly expressed on tumor cells.The affinity between TfR and transferrin on tumor cells is 10 to 100 times higher than normal cells,and its expression level is closely related to tumor stage and prognosis.How to obtain anoptimal antibody in the targeted tumor therapy is a key issue to be solved.The purpose of this project was to establish a novel anti-TfR-Diabody based on mouse-derived anti-TfR-VH,anti-TfR-VL and pOptiVEC-Fc expression vectors in our laboratory,and explore its biological effect.This research would lay a foundation for the application of anti-TfR antibody in targeted tumor imaging and therapy.Methods1.Construction of anti-TfR bivalent antibody expression vector TfR-scFv-Fc-pOptiVECThemouse-derived anti-TfRmAb,stored in our lab,was used as templates.The primers were designed to obtain scFv by the Overlap-PCR.After enzyme digestion,the product was subcloned into the pOptiVEC-Fc expression vector which contained the full length of human-derived IgG1 Fc fragment to obtained the eukaryotic expression vector TfR-scFv-Fc-pOptiVEC.2.Establishment of anti-TfR bivalent antibody transformed cell line(1)The TfR-scFv-Fc-pOptiVEC expression vector was transiently transfected into293T cells,three days later,the culture supernatant was collected to detectthe antibody concentration by ELISA.The binding activity and specificity of the supernatant were detected by FCM using CT11(CHO cell line stably expressing human TfR)and C14(no-load control CHO cell line);(2)The linearized TfR-scFv-Fc-pOptiVEC plasmid was transfected into DG44 cells(DHFR-deficient CHO cells).Then cells were cultured in serum-free Opti mediasupplemented with methotrexate(MTX).Single clones were picked by limiting dilution method.Their supernatants were collected and screened by FCM for their binding with TfR+Molt4 cells.3.Purification and identification of anti-TfR bivalent antibodyThe culture supernatant of transfected cells was collected,and filtered with a 0.22μm filter.Then the product was purified by preloading Protein A column.The molecular weight and purity of antibody were detected by SDS-PAGE.The antibody concentration was detected by ELISA and affinity constant with TfR was calculated accordingly.4.Biological Effects of anti-TfR bivalent antibody(1)Differential expression of TfR on various tumor cell lines(including HepG2,HepG2.215,MDA-MB-231,A549,Molt4,U266 and KG-la)was determined by FCM;(2)3×104target cells were incubated with 6×105 PBMCs(E:T ratio=20:1)in the media supplemented with anti-TfR bivalent antibody(the final concentration:6.25pmol/ml)for 5h.Then the cell death was evaluated using 7-AAD staining by FCM.The ADCC effect mediated by anti-TfR bivalent antibody was calculated;(3)3×104 target cells were incubated with complement(dosage:20μl/100μl)in the media supplemented with anti-TfR bivalent antibody(the final concentration:6.25pmol/ml)for 3h.Then the cell death was evaluated using 7-AAD staining by FCM.The CDC effect mediated by anti-TfR bivalent antibody was calculated.Results1.TfR-scFv-Fc-pOptiVEC expression vector was successfully preparedUsing the anti-TfR antibody signal peptide-VH vector and VL vector as templates,the VH-Linkerl-VL-Linker2(NheI/BamHI)sequence was obtained using overlap-PCR with a short linker peptide(G4S)3.The agarose gel electrophoresis showed that the size of the sequence was consistent with the predicted 829bp.Subsequently,VH-Linker 1-VL-Linker2(NheI/BamHI)sequence and the plasmid pOptiVEC-hFc were double-digested with NotI/BamHI.Then the two were ligated to form TfR-scFv-Fc expression plasmid TfR-scFv-Fc-pOptiVEC.Restriction,endonuclease digestion and sequencing confirmed the successful construction of Anti-TfR-scFv-Fc eukaryotic expression vector.After TfR-scFv-Fc is expressed in mammalian cells,it can form a double-chain antibody(bivalent antibody)through disulfide interactions in the hinge region of the Fc segment,named TfR-Diabody.The TfR-scFv-Fc-pOptiVEC was then transiently transferred into 293T cells,ELISA assay showed that the concentration of TfR-Diabody was 13.88mg/L.97.9%CT11,but only 3.42%C14 could bind with TfR-Diabody,which suggested that the antibody in the culture supernatant could bind to the TfR-expressing cell with good specificity.For TfR-scFv-Fc-pOptiVEC stably transfected DG44 cell clones,the antibody yield was about 1.03μg/ml.Nearly 100%TfR+Molt4 cells could bind with the antibody.2.Identification of TfR-DiabodySDS-PAGE showed that there was a band with molecular weight of about 60kDa emerged in the gel.Native PAGE identified that the TfR-Diabody was a dimer with molecular weight about 120kDa,which suggested that TfR-scFv-Fc can successfully form a dimer after expression.The anti-TfR bivalent antibody was name as TfR-Diabody.The affinity constant of TfR-Diabodywas 2.974×109 M-1,comparable to its parental TfR mAb(2.908×109M-1).3.Biological Effect of TfR-DiabodyThe FCM method was used to detect the ADCC effect mediated by TfR-Diabody combined with PBMCsto the above 7 tumor cell lines.The results showed that TfR-Diabody could successfully mediate ADCC effect to kill HepG2,MDA-MB-231,Molt4,U266 cells,but not to HepG2.215,A549,and KG-la cells although they also expressed moderate TfR.These differential ADCC effects might result from the different sensitivities of tumor cell lines to the biologically active molecules released by ADCC effectors.And the cytotoxic functions of PBMCs might vary in different healthy blood donors.Complement-dependent cytotoxicity(CDC)is another mechanism by which antibodies can mediate specific target cell lysis.After binding with tumor cells,TfR-Diabody effectively lysed the HepG2,MDA-MB-231,A549,Molt4,U266,KG-la cells in the presence of complements,suggesting that TfR-Diabody effectively mediated CDC to kill TfR+tumor cell lines.However,HepG2.215 cells showed resistance to TfR-Diabody-mediated CDC when compared with HepG2 cells.The mechanism is worthy of further study.ConclusionsIn summary,these results suggested that:1.a new eukaryotic expression vector of anti-TfR bivalent antibody was successfully constructed and stably expressed by DG44 cells;2.The TfR-Diabody possessed excellent binding activity and specificity with TfR+tumor cells as its parental antibody.3.The human-type Fc portion could effectively mediate ADCC and CDC to kill various TfR+human tumor cells lines.The novel TfR-Diabody retains the high affinity of mouse-derived variable regions to TfR antigen,and the effector functions of human-type Fc segment.This novel human/mouse chimeric anti-TfR antibody would reduce the anaphylactic complications to subsequent administration of murine mAb.The above results indicate that the TfR-Diabody constructed in this subject provides a new strategy for its application in human targeted anti-tumor research.