A Comparative Research On Pharmacokinetics And Pharmacodynamics Of A Humanized Anti-CD20 MAb Basing On Comprehensive Bioanalytical Methods

AIM:To study characteristic changes of its pharmacokinetics(PK),pharmacodynamics(PD)and immunogenicity in animals after anti-CD20 monoclonal antibody was humanized,and develop different assays based on anti-CD20 humanized monoclonal antibody(anti-CD20zumab)and systematically compare with each other.METHODS:The sandwich Enzyme linked immunosorbent assay(ELISA)was employed to study the PK property of anti-CD20zumab in cynomolgus monkeys,while the PD profile of anti-CD20zumab was evaluated by a flow cytometry assay(FCA)at the same time.The bridging-ELISA was utilized to observe immunogenicity of anti-CD20zumab in cynomolgus monkeys and rats.Three sandwich ELISAs based on different capture antibodies that binding to different sites of anti-CD20zumab and a FCA based on Wil2-S cells were developed to quantitatively determine anti-CD20zumab.in cynomolgus.monkeys' plasma and systematically compared with each other.RESULTS AND CONCLUSIONS:1.After cynomolgus monkeys were given a single vd doses of 10,30,100 mg·kg-1(1:3:10)of anti-CD20zumab,the ratio of the maximum concentration(Cmax)(1:2.7:10.7)and the ratio of the area under concentration-time curve(AUC(0-t))(1:2.9:13.7)exhibited that pharmacokinetics profile of anti-CD20zumab showed linearity pharmacokinetics characteristic dramatically in the dose range of 10?30 mg·kg-1 and nonlinearity pharmacokinetics characteristic certainly following the increase of dose to 100mg·kg-1.When the dose increased to 100mg·kg-1,drug system exposure showed nonlinearity increase,half-life of terminal(t1/2)extended significantly,and clearance rate(CL)dropped dramatically.2.After cynomolgus monkeys were given 4 weekly doses of 30 mg·kg-1 of anti-CD20zumab and Rituximab,they exhibited the similar pharmacokinetics characteristic,and their average plasma concentrations and the main pharmacokinetic parameters were not different statistically.The vast majority of individuals emerged different degrees of drug accumulation after last administration of anti-CD20zumab and Rituximab,but there was a small part of individuals showing that drug exporsure after last administration was lower significantly than that after first administration.After last administration,the coefficient of variation(CV)of the pharmacokinetic parameters between different individuals of two groups was significantly increased compared with first administration.3.At 30 minutes after administration,the binding rate of antibody and CD20 on B cell surface of cynomolgus monkeys was higher than 99%for all groups,and B cells began to be cleared.At 1 hour after administration,B cells were cleared to extreme and the measured values of them were less than 0.2%for all group.4.B cells have started to recover at 7th or 14th day after administration for the low-dose and middle-dose groups,while B cells still didn't recover at 28th day after administration for high-dose group,showing a certain dose-response relationship.5.After cynomolgus monkeys were given 4 weekly doses of 30 mg·kg-1 of anti-CD20zumab and Rituximab,the function of anti-CD20zumab to clear B cells was significantly stronger than Rituximab,the individual difference of scavenging reaction in rituximab's group was more obvious and some individuals appeared to resistance with the increasing of the frequency of administration.6.After cynomolgus monkeys were given 4 weekly doses of 30 mg·kg-1 of anti-CD20zumab and Rituximab,some individuals produced anti-drug antibodies(ADA)in the body and the time of initial appearance of ADA was about 14 days.The positive rates of ADA were 15.6%and 37.5%,suggesting that the immunogenicity of rituximab in cynomolgus monkeys was stronger than anti-CD20zumab and the results were in line with the purpose of humanized transformation.7.After rats were given 4 weekly doses of 20 mg·kg-1 of anti-CD20zumab and Rituximab,there was no ADA detected for two groups,suggesting that the immunogenicities in rats were low for them.Failed to see the improvement of immunogenicity from the results in rats directly after humanized transformation,which may be due to that immune receptivity of the rats in vivo to this molecule was higher and small number of animals was observed.8.The correlation ananlysis of PK and PD showed that the recovery of B cell in cynomolgus monkeys will directly affect the change of plasma concentrations of anti-CD20zumab.9.The correlation ananlysis of PK and immunogencity showed that the appearance of ADA in cynomolgus monkeys can also directly affect the change of plasma concentrations of anti-CD20zumab.10.All validation parameters of four assays can meet the reqirement of PK of biologics,and each of them can be used to quantitatively determine anti-CD20zumab in cynomolgus monkeys,plasma.Thus,this analysis provided different quantitative assays for the class of drugs to PK study.