| Background:Human immunoglobulin for intravenous injection(IVIG)is isolated and purified from 3000-10000 healthy plasma and is widely used clinically.IVIG plays an immunomodulatory role of Fc fragment of IgG,but the indication of biological activity of Fc fragment needs to be supplemented.In recent years,antibody-dependent cellmediated cytotoxicity(ADCC)has been widely used in the toxicity evaluation of antibody-mediated drugs.However,as an important antibody drug,ADCC biological activity of IVIG in clinical studies is still in the preliminary stage and is waiting to be explored.Objective:In this study,we systematically studied ADCC biological activity of IVIG.Firstly,the method for determinating the ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established.Secondly,the effect of IgG dimer content on the biological activity of ADCC of IVIG was studied.Finally,when IVIG exerted the biological effect of ADCC,the effect on expression of FcyRIIIA on the surface of effector cells,the release of killer substances,and the regulation of cytokine secretion were detected.This study will provide methodological basis for supplementing and improving the evaluation index of biological activity of Fc fragment of IVIG and provide technical support for further improving the quality standard.Method:1.(1)Jurkat-NFAT-Luc-CD16 reporter cell line was co-cultured with PLC/PRF/5 cells to detect the luciferase released after IVIG activated FcyRIIIA on the surface of Jurkat-NFAT-Luc-CD16 reporter cell line and it reflected ADCC biological activity IVIG.The method to detect ADCC biological activity of IVIG was established by optimizing a series of experimental conditions.(2)The above established methods were validated from the aspects of accuracy,precision,repeatability,etc.(3)The established methods were used to analyze the biological activity of ADCC in different manufacturers of IVIG.2.(1)The buffer solution of IVIG was replaced by protein ultrafiltration technology and its pH was changed,and it was storing at 4℃ for 6 months to increase the content of IVIG IgG dimer.Then different contents IgG dimer of IVIG were prepared with pH7.2 PBS and were analyzed by HPLC.(2)The biological activity of ADCC of IVIG with different IgG dimer contents were studied by established method,exploring the influence of biological activity of ADCC of IVIG with different IgG dimer contents.3.(1)The regulation of IVIG on the expression of FcγRⅢA of effector cells were investigated by real-time qPCR and flow cytometry.(2)The biological effects of IVIG on ADCC mediated by FcγRⅢA were studied by time-resolved immunoassay,enzyme linked immunosorbent assay,enzyme linked immunospot assay and flow cytometry.It includes the killing effect of IVIG on tumor cells,the expression of active marker of NK cells CD107A,the killing substance perforin and granzyme B released by effector cells when IVIG mediates ADCC and the regulation effect on cytokines released by effector cells.Results:1.(1)There was a good dose-effect relationship between IVIG and Jurkat-NFATLuc-CD16 cell surface FcγRⅢA.and the fitted four-parameter equation R2=0.99.(2)Within-run and between-run analysis including three independent tests,initial working concentration relative light unit(RLU)and the relative standard deviation(RSD)of the concentration for 50%of maximal effect(EC50)were less than 11%,The RSD of recoveries were all less than 10%.RSD of within-tun starting working concentration were 10.86%,10.80%for EC50,5.13%for between-run starting working concentration,7.25%for EC50.(3)The RLU of biological activity dose effect curves of ADCC from manufacturers A to E IVIG were 0.96,0.93,0.96,0.97 and 0.99,respectively.The RLU of initial working concentration of manufacturer E reached 10550,while that of manufacturer A was only 4646.The biological activity difference of ADCC between the two products was up two times(P<0.005),and there was a difference in biological activity between manufacturer A and manufacturer D(P<0.01).2.(1)Four groups of different IgG dimer contents of IVIG were prepared by protein ultrafiltration technique.Groups 1 to 4 were determined by HPLC to be 3.27%,4.38%,5.00%and 15.04%,respectively.(2)The ADCC biological activity of four groups were 1237,1110,710 and 570.When the IgG dimer content of IVIG was 3.27%,the RLU was 1237.When the IgG dimer content reached 5.00%,the RLU decreased to 710(P<0.05).When the IgG dimer content of IVIG reached 15.04%,comparing with 3.27%IgG dimer,the biological activity of ADCC was the lowest,decreased by 2.2 times(P<0.01).3.(1)IVIG can up-regulate the expression of FCG3A encoding FcγRⅢA in effector cells and the expression of CD 16 on effector surface.Compared with the control group,the experimental group doubled the expression of FCR3G and increased the expression of CD16 on effector cells to 1.8 times(P<0.05).(2)IVIG had the biological activity of ADCC and had different biological activities of ADCC.The maximum ADCC activity reached 40%at the concentration of 1.25mg/ml which killed more than twice of K562 cells than the control group(P<0.05).(3)IVIG downregulate the expression level of CD107A on the surface of NK cells on 3.4 times(P<0.05).(4)Compared with the control group,the secretion of perforin and granzyme B secreted by effector cells decreased after IVIG treatment.IVIG inhibited the release of perforin and granzyme B at 4h,12h and 24h,with no statistical difference(P>0.05).(5)In the experimental group,after IVIG was co-incubated with effector cells,compared with the control group,the secretion of RANTES was increased by3.4 times(P<0.0001),and the secretion of PDGF-BB was increased by 15.5 times(P<0.005).Conclusion:1.In this study,the method for determinating the ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established.This method has the advantages of strong specificity,good repeatability and high accuracy,and is suitable for the evaluation of the biological activity of ADCC for IVIG.The ADCC activity of IVIG from different manufacturers was analyzed.The biological activity of IVIG ADCC from different manufacturers was different.2.IgG dimer content affects the biological activity of ADCC of IVIG.The number of binding sites of Fab fragment in dimer IgG decreased and the binding to target cells decreased,resulting in reduced binding to effector cells and decreased biological activity of ADCC.3.IVIG may mediate ADCC to kill tumor cells K562 by up-regulating the expression of FcγRⅢA on effector cells.However,IVIG has certain inhibitory effect on NK cells,inhibiting the activation of NK cells and the release of NK cell-killing substances.At the same time,IVIG can regulate effector cells to release RANTES and other cytokines to activate effector cells and promote immune regulation.This study is of great significance for clarifying the role and mechanism of IVIG mediated ADCC and providing evidence for IVIG ADCC research. |
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