A Study About The Preparation Of Anti-TfR/CD3 Bispecific Antibody And Its Anti-tumor Effect

ObjectiveTransferrin receptor?TfR?is a relatively specific marker of tumor cells which is low expressed in normal tissue cells,but increase expression on the surface of tumor cells.The literature reports and previous research by our group have shown that the affinity between TfR and transferrin on the surface of tumor cells is 10 to 100 times higher than normal cells.Besides,TfR is highly expressed on the surface of rapidly proliferating tumor cells,and the expression level of TfR is related to tumor stage and prognosis.Transferrin receptor has attracted attention as an important specific marker for tumor-targeted imaging diagnosis and targeted therapy with its extracellular segment binding properties,endocytosis and tumor cell action characteristics.However,in the targeted biotherapy of tumors,how to obtain antibodies that specifically target effector cells is a key issue to be solved.Based on the preliminary construction of anti-TfR scFv and bivalent expression vector,this project further designed and synthesized a TfR bispecific T-cell engager?TfR-BiTE?against human TfR and CD3 by DNA recombination technology and explore its effects and mechanism in anti-tumor,which may lays the foundation for its application in targeted anti-tumor research.Methods1.Gene acquisition and construction of bispecific antibody expression vectorThe pET-28a-CD3-scFv vector was used as a template to design primers,and the target fragment NheI-signal-CD3-scFv-BamHI was obtained from the template by molecular biological methods.The target gene fragment was inserted into the pOptiVEC-TfR-scFv-His vector betweenTfR-scFv sequence and the His tag sequence by restriction enzyme digestion and ligation reaction,to construct a Single-TfR-CD3-His bispecific antibody eukaryotic expression vector.The NotI-CD3-signal-V_H-Linker and Linker-CD3-V_L-NheI genes were obtained from the pGM-T-CD3-V_H and pGM-T-CD3-V_L vectors by PCR.NotI-signal-CD3-V_H-Linker-CD3-V_L-NheI was synthesized by overlap extension PCR?overlap?technique.The NotI-signal-CD3-V_H-Linker-CD3-V_L-NheI and TfR-?scFv?₂-His vectors were cut with NotI/NheI,respectively,and then ligase-ligated to obtain an expression vector,and named as Single-CD3-TfR-His.NotI-signal-CD3-V_H-G4S-TfR-V_L-MluI was obtained from Single-CD3-TfR-His vector by overlap extension PCR?overlap?technique,and then placed into pBudCE4.1-IRES-DHFR vector downstream of the pEF-1?promoter,it was formed and named pEF-1?-CD3-V_H-TfR-V_L vector.Using the same method,the HindIII-signal-TfR-V_H-G4S-CD3-V_L-myc-SalI-XbaI gene fragment was obtained from the Single-TfR-CD3-His vector and put into pEF-1?-CD3-V_H downstream of the pCMV promoter of the-TfR-V_L vector,a CD3-TfR-Diabody vector is formed.The IRES-DHFR sequence was further placed on the CD3-TfR-Diabody vector to form a complete Double-Diabody expression vector.2.TfR-BiTE vector transfection,antibody expression and identification?1?TfR-BiTE vector transfection:The linearized plasmid was stably transfected into DHFR^-/-DG44 cells by nuclear transfection.The serum-free medium was used for selective culture.After the cells were normally grown,methotrexate?MTX?was used for pressure screening to obtain a positive antibody stable cells.Flow cytometry?FCM?was used to detect the binding ability of cell supernatants to TfR⁺tumor cells and CD3⁺lymphocytes,and a polyclonal cell line capable of secreting bispecific antibodies was screened.Finally,positive monoclonal cell lines were selected by limiting dilution and flow cytometry.?2?Expression of TfR-BiTE antibody:The cell culture supernatant stably expressing TfR-BiTE was collected,and the TfR-BiTE antibody was purified by prepacking the column with a nickel metal gel.?3?Identification of TfR-BiTE antibody:Flow cytometry was used to detect the binding of TfR-BiTE to TfR⁺tumor cells?HepG2?and CD3⁺T cells?PBMCs?,respectively.Western blot was used to determine the relative molecular weight of TfR-BiTE.TfR-BiTE was ligated to effector cells and tumor cells by confocal microscopy.3.Stimulation of PfMCs by TfR-BiTEUnstimulated PBMCs were stained with CFSE and co-cultured with TfR⁺tumor cells?HepG2 or HT1080?and TfR-BiTE for 24 h?or 48 h?.Flow cytometry was used to detect activation?CD69⁺GrB⁺?and proliferation?average fluorescence intensity of CFSE?of PBMCs?effector T cells?after TfR-BiTE stimulation.Flow cytokine detection was used to detect the secretion level of Th1/Th2 cytokines in the co-culture supernatant.4.TfR-BiTE anti-tumor effect in vitroCFSE-labeled unstimulated PBMCs were co-cultured with TfR⁺tumor cells?HepG2/HT1080/HepG2.215/Molt4?and TfR-BiTE for 24 h?or 48 h?.Flow cytometry was used to detect the killing effect of tumor cells?CFSE^-7-AAD⁺?.5.TfR-BiTE anti-tumor effect in vivoNCG mice were subcutaneously inoculated with Luc-HepG2 cells,freshly isolated PBMCs were injected on day 7,and TfR-BiTE was injected at a dose of 20?g/mouse for7 consecutive days,followed by monitoring tumor volume and body weight changes in mice.The bioluminescence invivoimaging was performed after the experiment was terminated when the tumor volume was close to 2000 mm³.Separate tumors to take pictures.IHC was used to analyze the infiltration of T cells in tumor tissues for evaluation of the antitumor effect of TfR-BiTE.The damage of liver and kidney tissue tissue was analyzed by H&E staining.Results1.Acquisition of genes and construction of expression vectorsThe TfR-scF and CD3-scFv were obtained by genetic engineering methods using the anti-TfR-scFv and CD3-scFv related vectors constructed in the previous study group,and the two were connected in different ways to construct three different structures.It was confirmed by cloning/enzyme digestion and sequencing that the construction of Single-TfR-CD3-His and Single-CD3-TfR-His eukaryotic expression vector was successful.2.Expression and activity identification of TfR-BiT?1?The supernatant was obtained by transient transfection of 293T cells,and incubated with HepG2 cells and PBMCs cells to identify the bispecific antibody binding activities of the three structures,among which Single-TfR-CD3-His was better,followed by Double-Diabody,and finally Single-CD3-TfR-His.Moreover,in mediating effector cell PBMCs killing tumor cells HepG2,the two single-chain Single-TfR-CD3-His and Single-CD3-TfR-His bispecific antibodies are better than the double-stranded structure Double-Diabody.?2?Stable expression and identification:Two single-chain structural bispecific antibodies,Single-CD3-TfR-His and Single-TfR-CD3-His expression vectors were linearized and transfected into eukaryotic cells DG44.Stable expression cell lines were screened by using serum-free medium with methotrexate?MTX?.Stably transfected cell culture supernatants bind well to TfR-expressing HepG2 cells and CD3-expressing T cells,similar to monoclonal antibodies,and Single-TfR-CD3-His binds better than Single-CD3-TfR-His.?3?Purification and molecular weight identification:The stably expressed Single-TfR-CD3-His?TfR-BiTE?cell culture supernatant was purified by pre-packing a nickel metal gel to obtain a TfR-BiTE antibody.Western blot showed that the molecular weight of TfR-BiTE was 56 kDa.?4?TfR-BiTE mediates the connection between target cells and effector cells:Confocal images showed that in the presence of TfR-BiTE,the effector cells surround the tumor cells in a rose-like garland shape,which was not observed in the control group.3.TfR-BiTE stimulates the activation,proliferation and secretion of cytokines of PBMCs?1?TfR-BiTE-activated T cells are dependent on TfR⁺tumor cells:When no target cells are present,TfR-BiTE can activate T cells and express early activation marker CD69molecule,but the expression of killing activity marker GrB is only about 25%.In contrast,when there is a target cell,the expression of GrB increases as the concentration of TfR-BiTE increases,and when the concentration of TfR-BiTE increases to 100 ng/ml,GrB The expression reached the plateau,about 70%.In addition,in CD4⁺T cells,CD69⁺GrB⁺cells were only 30%,while in CD8⁺T cells,the proportion of CD69⁺GrB⁺cells was as high as 70%,which was more than twice that of CD4⁺T cells.?2?TfR-BiTE promotes T cell proliferation through TfR⁺tumor cells:When TfR⁺tumor cells were present,the average fluorescence intensity of CFSE in T cell subsets was significantly reduced,and in the concentration range of 0.1¹000 ng/ml,the higher the concentration,the more obvious the decrease in mean fluorescence intensity.In the CD8⁺group,when the TfR-BiTE was 1000 ng/ml,the average fluorescence intensity of CFSE in the TfR⁺tumor cell group was significantly reduced,3.7 times that of the tumor cells absent,4.2 times that of the mAb Mix and CD3 mAb groups.The results suggest that TfR-BiTE can induce T cell proliferation through TfR⁺tumor cells,and in the concentration range of 0.1¹000 ng/ml,the higher the concentration,the more obvious the proliferation.Furthermore,the proliferation of CD8⁺T cells was more pronounced than that of CD4⁺T cells.?3?TfR-BiTE stimulated the secretion of cytokines by PBMCs:In the presence of TfR⁺tumor cells,when the concentration of TfR-BiTE was 1000 ng/ml,the secretion of cytokines was significantly increased compared with the mAb Mix group,and IFN-?increased by 98%,TNF increased by 448%,IL-10 increased by 257%,IL-6 increased by1.42%,IL-4 increased by 329%,and IL-2 increased by 4418%[%average cytokine increase=?TfR-BiTE 1000 ng/ml group-mAb Mix group?/mAb Mix group×100].The results indicate that TfR-BiTE can rely on TfR⁺tumor cells to stimulate PBMCs to secrete a large number of cytokines,of which IL-2 and TNF are most obvious,which may be the mechanism of TfR-BiTE mediates effector cells to kill tumor cells.4.TfR-BiTE mediates the killing properties of PBMCs on TfR⁺tumor cells?1?TfR molecular expression dependence:In the co-culture system,as the concentration of TfR-BiTE increases,the death of Luc-HepG2 cells of TfR⁺tumor cells increases.However,there was essentially no death in MX-1 cells without TfR expression.The results suggest that TfR-BiTE has a dependence on TfR expression when mediating PBMCs killing tumor cells.?2?TfR molecular specificity:In the co-culture system,TfR-BiTE significantly inhibited the killing of tumor cells after adding a high concentration of TfR mAb or soluble recombinant protein TfR.The results suggest that high concentrations of TfR mAb can compete with TfR-BiTE for binding to TfR on tumor cells,thereby inhibiting TfR-BiTE-mediated killing of tumor cells.Similarly,a high concentration of soluble recombinant TfR protein can neutralize a portion of TfR-BiTE,reducing its effective concentration,thereby reducing the killing of tumor cells.?3?TfR molecular abundance correlation:Molt4,HepG2,HT1080 and HepG2.215,the TfR expression abundance of the four tumor cells gradually decreased,and the TfR-BiTE-mediated killing effect also gradually decreased.This result suggests that TfR-BiTE mediates the positive correlation between PBMCs killing tumor cells and TfR molecular abundance.In addition,thePBMCs from different donorsdo not have the same killing effect on the same tumor cell.5.Inhibition of tumor by TfR-BiTE in vivoNCG mice were subcutaneously inoculated with Luc-HepG2 cells,PBMCs and TfR-BiTE were injected on day 7,TfR-BiTE was injected for 7 consecutive days,and the experiment was terminated when the tumor volume of the mice was close to 2000 mm³.The tumor volume of the mice showed that the tumor volume of the NCG mice injected with the TfR-BiTE group was significantly smaller than that of the control group.Further tumor immunohistochemistry?IHC?analysis showed that there was significant CD3⁺lymphocyte infiltration in the tumor tissues of the TfR-BiTE group,while no infiltrating lymphocytes were observed in the control.At the same time,there was no difference in body weight between the mice in each experimental group,and no significant damage was observed in the H&E staining results of the mouse liver and kidney tissue.These results suggest that TfR-BiTE can recruit T cells to the tumor site,exert anti-tumor effects,and have no systemic toxicity.ConclusionThe experimental results of this study indicated that:1.The novel anti-TfR×CD3bispecific antibody TfR-BiTE eukaryotic expression vector was successfully constructed and stably expressed in eukaryotic cells;2.Respectively,TfR-BiTE not only can be combined with TfR⁺tumor cells and CD3⁺lymphocytes,but also mediates tumor cell and effector cell junction,providing a theoretical basis for tumor cell killing;3.In the presence of TfR⁺cells,TfR-BiTE can promote the activation and proliferation of CD3⁺T cells,and the activation and proliferation of CD8⁺T cells are more significant than CD4⁺T cells;4.TfR-BiTE-activated T cells showed higher expression of Granzyme B and cytokine expression,and IL-2 and TNF expression increased significantly;5.TfR-BiTE is capable of mediating CD3⁺T cell killing of TfR⁺tumor cells and is characterized by dependence,specificity and abundance of TfR expression.At the same time,it has a concentration dependence of TfR-BiTE in a range of 0.1¹000 ng/ml;6.In Luc-HepG2tumor cell xenograft animal model,TfR-BiTE can recruit CD3⁺T cells to tumor tissue to inhibit tumor Growth with no significant damage to the liver and kidney of the metabolic organs.The results show that the TfR-BiTE constructed by this subject lays a good foundation for its application in targeted anti-tumor research.