Enzyme-linked immunosorbent assays for quantification of MMMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera / 药物分析学报

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosor-bent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were suc-cessfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation be-tween serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias％ranged from-12.2％to-5.2％,precision ranged from-12.4％to-1.4％,and the relative standard deviation(RSD)was less than 6.6％and 8.7％,respectively.The total error was less than 20.4％.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Biocompatibility of KLD-12 Peptide Hydrogel as a Scaffold in Tissue Engineering of Intervertebral Discs in Rabbits / 华中科技大学学报（医学）（英德文版）

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals.Rabbit MSCs were cultured in the hydrogel for 2 weeks.Live cells were counted by using Caicein-AM/PI fluorescence staining.MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%,0.03%,and 0.05%.Hemolysis test,skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues.Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape.Cell survival rate was 92.15%(mean: 92.15%±1.17%)at the 7th day and there was no difference in survival rate between day 7 and day 14.Cell proliferation test showed that the A value of the KLD-12solutions was not significantly different from that of control groups(complete culture media)(P>0.05)at the 24th and 48th h.The hemolysis rate of KLD-12 solution was 0.112%.Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0.The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes.It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs,and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.

Experimental Study on Self-assembly of KLD-12 Peptide Hydrogel and 3-D Culture of MSC Encapsulated within Hydrogel In Vitro / 华中科技大学学报（医学）（英德文版）

o-fiber hydrogel in vitro. MSCs in KLD-12 peptide hydrogel grew well and proliferated with the culture time. KLD-12 peptide hydrogel can serve as an excellent injectable material of biological scaffolds in tissue engineering of IVD.