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One-step synthesis of site-specific antibody-drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates

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Acta Pharmaceutica Sinica B 2022年第5期12卷 2417-2428页

作者： Wei Shi Wanzhen Li Jianxin Zhang Tiehai Li Yakai Song Yue Zeng Qian Dong Zeng Lin likun gong Shuquan Fan Feng Tang Wei Huang CAS Key Laboratory of Receptor Research CAS Center for Excellence in Molecular Cell ScienceCenter for Biotherapeutics Discovery ResearchShanghai Institute of Materia Medica Chinese Academy of SciencesShanghai 201203China School of Pharmaceutical Science and Technology Hangzhou Institute of Advanced StudyHangzhou 310024China University of Chinese Academy of Sciences Beijing 100049China School of Chinese Materia Medica Nanjing University of Chinese MedicineNanjing 210023China School of Life Science Liaocheng UniversityLiaocheng 252059China

Glycosite-specific antibody-drug conjugatess(gsADCs), harnessing Asn297 N-glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation ***, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an endo-N-acetylglucosaminidase(ENGase) of Endo-S2. IgG glycoengineering via ENGases usually has two steps: deglycosylation by wild-type(WT) ENGases and transglycosylation by mutated ENGases. But in the current method, we have found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the one-step glycoengineering directly from native *** studies on substrate specificity revealed that this approach has excellent tolerance on various modification of 6-Gal motif of LacNAc. Within 1 h, one-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups. These gsADCs demonstrated good homogeneity, buffer stability, in vitro and in vivo anti-tumor activity. This work presents a novel strategy using LacNAc-based substrates to reprogram the multi-step IgG glycoengineering to a one-step manner for highly efficient synthesis of gsADCs.

关键词： Site-specific ADCs ENGase LacNAc One-step assembly Potent in vivo efficacy

Development and validation of an enzyme-linked immunosobent assay for the determination of a Trulicity biosimilar in cynomolgus monkeys

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Development and validation of an enzyme-linked immunosobent ...

中国毒理学会药物毒理与安全性评价学术大会（2019年）暨粤港澳大湾区生物医药产业第一届高峰论坛

作者： Yingying Hou Jie Miao likun gong Jin Ren Qiuping Qin Department of Immunoassay and Immunochemistry Center for Drug Safety Evaluation and Research (CDSER)State Key Laboratory of New Drug ResearchShanghai Institute of Materia MedicaChinese Academy of Sciences

Background Biosimilars are highly similar to approved originator biologicals,and receive marketing authorization in more and more *** the preclinical studies of biosimilars,cynomolgus monkeys are often used due to physiological and genetic similarities to *** aimed to develop and validate an ELISA method for the determination of a Trulicity biosimilar which was a human GLP-1 protein covalently linked IgG4 Fc *** The ELISA was developed with commercially available *** assay was configured with anti-GLP-1 amide for capture and mouse anti-human IgG4 Fc labelled with horseradish peroxidise(HRP) as *** assay involved two incubation steps respectively with samples and with the labelled ***(TMB) solution reacted with hydrogen peroxide in the presence of HRP to form a blue *** development was stopped by the addition of sulfuric acid and the color absorbance was measured at 450 *** required sample dilution was 1:100 and each determination utilized 100 μl of sample *** characteristics of the ELISA were investigated with calibrators,quality controls,monkey serum samples,which included pre-study parameters,i.e.,lower and upper limits of quantification,precision,accuracy,matrix effect,dilution linearity,selectivity,specificity,sample stability,as well as in-study parameters,i.e.,parallelism and incurred sample reanalysis(ISR).Results The lower limit of quantification was 20.833 ng/ml and the upper limit of quantification was 500 ng/***-and inter-assay coefficients of variation of LLOQ,LQC,MQC,HQC and ULOQ were below 10.2%.Inter-assay accuracy of the LLOQ,LQC,MQC,HQC and ULOQ reflected by percent of bias was ranged from-10.9% to 6.3%.No matrix effect was observed with monkey *** dilution linearity was up to 1:512 and no hook effect was *** assay was able to detect the existing approved product,namely Trulicity in the same way as for the biosimilar and showed no

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Development and validation of the T-cell-dependent antibody response assay in Sprague-Dawley rats

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Development and validation of the T-cell-dependent antibody ...

中国毒理学会药物毒理与安全性评价学术大会（2019年）...

作者：欧阳璐 Xiaoli Zang likun gong Jin Ren Qiuping Qin 中科院上海药物研究所药物安全评价研究中心

Background The T-cell dependent antibody response（TDAR） is a functional assay widely used to monitor the effects of drugs on the immune *** has been an assay recommended by FDA and ICH to evaluate potential immunotoxicity of investigational new *** aimed to develop and validate two ELISAs for the measurements of anti-KLH IgM and anti-KLH IgG in Sprague-Dawley（SD） *** For anti-KLH IgG,the assay was configured with keyhole limpet hemocyanin（KLH） for capture and horseradish peroxidase（HRP）-labeled goat anti-rat IgG antibody as *** incubation steps were associated with samples and with the labelled antibody,respectively,in the ***（TMB） solution reacted with hydrogen peroxide in the presence of HRP to form a blue *** development was stopped by the addition of sulfuric *** color intensity（optical density,OD） was measured at 450 *** required dilution of samples was 1:100 and each determination utilized 100μl of sample *** anti-KLH IgM,the assay was configured with KLH for capture and HRP-labeled mouse anti-rat IgM antibody as *** assay steps were the same as those of the anti-KLH IgG *** initial dilution of samples was 1:*** characteristics of the ELISAs were investigated with calibrators,quality controls,rat serum samples,which included lower and upper limits of quantification,precision,accuracy,dilution linearity,hemolysis effect,selectivity,specificity,parallelism and sample *** For the anti-KLH IgG assay,the lower limit of quantification was 100.000 ng/ml and the upper limit of quantification was 4500.000 ng/***-and inter-assay coefficients of variation of low,medium and high quality controls were below 7.5%.Inter-assay accuracy of the low,medium and high quality controls reflected by percent of bias was ranged from-1.2% to-6.6%.No hemolysis effect was observed with rat *** dilution linearity was up to 1:87200 and no hook effect was not

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Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

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Journal of Pharmaceutical Analysis 2022年第4期12卷 645-652页

作者： Min Pei Tingting Liu Lu Ouyang Jianhua Sun Xiaojie Deng Xiaomin Sun Wei Wu Peng Huang Yi-Li Chen Xiaorong Tan Xiaoyue Liu Peng Zhu Yongzhen Liu Deheng Wang Junliang Wu Qi Wang Guifeng Wang likun gong Qiuping Qin Chunhe Wang Biotherapeutics Discovery Research Center Shanghai Institute of Materia MedicaChinese Academy of SciencesShanghai201203China School of Pharmacy University of Chinese Academy of SciencesBeijing100049China Center for Drug Safety Evaluation and Research Shanghai Institute of Materia MedicaChinese Academy of SciencesShanghai201203China Research and Development Center Dartsbio Pharmaceuticals Ltd.ZhongshanGuangdong528400China Zhongshan Institute for Drug Discovery Shanghai Institute of Materia MedicaChinese Academy of SciencesZhongshanGuangdong528400China School of Pharmacy Shanghai Engineering Research Center of ImmunotherapeuticsFudan UniversityShanghai201203China

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical *** 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 immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey *** assays were successfully 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 *** developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between 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%,*** 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 ***,these assays can be easily adopted for development of other similar immunoassays.

关键词： Monomethyl auristatin E Antibody-drug conjugates Pharmacokinetics Trophoblast cell surface antigen 2

Development and validation of a bridging electrochemiluminescent assay for the detection of antibodies against a chimeric therapeutic antibody in human serum

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Development and validation of a bridging electrochemilumines...

中国毒理学会药物毒理与安全性评价学术大会（2019年）暨粤港澳大湾区生物医药产业第一届高峰论坛

作者： Tingting Liu Wenjing Fang Xiaojie Deng likun gong Jin Ren Qiuping Qin Department of Immunoassay and Immunochemistry Center for Drug Safety Evaluation and Research(CDSER)State Key Laboratory of New Drug ResearchShanghai Institute of Materia MedicaChinese Academy of Sciences

Background All therapeutic antibodies have the potential to elicit anti-drug antibody(ADA)responses in *** can affect pharmacokinetics(PK),pharmacodynamics(PD),efficacy,safety,and bioanalytical assay *** aimed to develop and validate a bridging electrochemiluminescent assay(Bridging ECLA) coupled with affinity capture elution(ACE) for the detection of antibodies against a chimeric therapeutic antibody in human *** We raised antisera from cynomolgus monkeys by immunizing the animals with the drug through a subcutaneous *** obtained positive polyclonal antibodies served as a source of standards and quality controls applied in the assay development and *** Bridging ECLA was constructed with self-made biotinylated and ruthenium-labeled forms of the therapeutic *** was used to reduce drug interference in the *** ACE treatment,anti-drug antibodies were first captured in solution by a combination of biotinylated and ruthenium-labeled drug molecules,and then bound to solid-phased streptavidin via a Meso Scale Discovery(MSD) plate and detected by *** development,the method was further validated in the forms of a screening assay,a confirmation assay and a titration *** screening assay was used to identify samples with the ratio of ECL signal/background signal(SB) no less than the screening cut point(SCP).The screening assay positive samples were then submitted to the confirmation assay for specificity of antibodies for the therapeutic *** with percent of inhibition rate values(IR%) higher than the confirmation cut point(CCP) were subjected to the titration assay for *** method validation parameters included the determination of SCP,CCP and TCP,assay sensitivity and precision,drug tolerance,selectivity,specificity,ranges of assay quality controls,assessment of Hook effect as well as sample *** No matrix effect was observed with the use of 5-fol

关键词：

Development and validation of enzyme-linked immunosobent assays to measure the drug-unconjugated antibody,drug-conjugated antibody and total antibody of an antibody drug conjugate in cynomolgus monkeys

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Development and validation of enzyme-linked immunosobent ass...

中国毒理学会药物毒理与安全性评价学术大会（2019年）暨粤港澳大湾区生物医药产业第一届高峰论坛

作者： Jie Miao Yimin Tao Jinli Gao likun gong Jin Ren Qiuping Qin Department of Immunoassay and Immunochemistry Center for Drug Safety Evaluation and Research(CDSER)State Key Laboratory of New Drug ResearchShanghai Institute of Materia MedicaChinese Academy of Sciences

Background Antibody drug conjugates(ADCs) are an emerging class of targeted therapeutics and receiving growing attention in the pharmaceutical *** aimed to develop and validate ELISAs for the measurements of the drug-unconjugated antibody,drug-conjugated antibody and total antibody of an antibody drug conjugate in cynomolgus monkeys Methods The assay of drug-conjugated antibody was configured with biotinylated anti-eribulin5 E4 Fab for capture and mouse anti-trastuzumab antibody labelled with horseradish peroxidise(HRP)as *** assay involved three incubation steps associated with streptavidin,with samples and with the labelled antibody,*** of drug-unconjugated antibody and total antibody were configured with the mouse anti-trastuzumab antibody for capture and mouse anti-human IgG-Fc antibody labelled with HRP as *** assay involved two incubation steps associated with samples and with the labelled antibody,***(TMB) solution reacted with hydrogen peroxide in the presence of HRP to form a blue *** development was stopped by the addition of sulfuric acid and the color absorbance was measured at 450 *** required sample dilution was 1:100 and each determination utilized 100 μl of sample *** characteristics of the ELISA were investigated with calibrators,quality controls,monkey serum samples,which included pre-study parameters,i.e.,lower and upper limits of quantification,precision,accuracy,matrix effect,dilutional linearity,selectivity,specificity,sample stability,as well as in-study parameters,i.e.,parallelism and incurred sample reanalysis(ISR).Results For the assay of drug-conjugated antibody,the lower limit of quantification was312.500 ng/ml and the upper limit of quantification was 24000.000 ng/***-and inter-assay coefficients of variation of low,medium and high quality controls were no more than 5.7%.Inter-assay accuracy of the low,medium and high quality controls ref

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Development and validation of a competitive electrochemiluminescent assay for the detection of neutralizing antibodies against a therapeutic antibody in human serum

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Development and validation of a competitive electrochemilumi...

中国毒理学会药物毒理与安全性评价学术大会（2019年）暨粤港澳大湾区生物医药产业第一届高峰论坛

Background Therapeutic antibodies are large molecules able to elicit anti-drug antibody(ADA)*** responses may result in the loss of therapeutic efficacy due to the formation of neutralization antibody(NAbs).Therefore,NAb detection is important for interpretation of efficacy and safety data of therapeutic antibodies in *** aimed to develop and validate a competitive electrochemiluminescent assay by using Meso Scale Discovery(MSD) platform for the detection of neutralizing antibodies against a therapeutic antibody in human *** The positive antibody of a neutralizing property was obtained from our sponsor and served as a source of quality controls applied in the assay development and validation *** chemiluminescent method was made by utilizing self-made ruthenium-labeled therapeutic antibody and *** dissociation was conducted to reduce possible drug interference in serum *** the samples did not have Nabs,the ruthenium-labeled drug bound to the EGFR coated a MSD *** the samples contained NAb,it competed with the ruthenium-labeled antibody to bind to the *** electrochemiluminescence values were measured at 620 *** development,the method was validated extensively,including the neutralization cut point(NCP),assay sensitivity,precision,drug tolerance,analogue interference,ranges of assay quality controls as well as sample *** No matrix effect was observed with the use of 5-fold diluted human *** NCP was 9.3%(IR%).Intra-and inter-assay coefficients of variation of low,medium,and high quality controls were no more than 19.1%.Inter-assay accuracy of the low,medium,and high quality controls was good as the IR% values of the controls were all within the established target *** tolerance at 10μg/mL and 5 μg/mL of the tested positive antibody was>100 μg/mL and> 1.563 μg/mL of the drug,*** positive antibody in human serum was stable after 5 freeze/thaw cycles,at RT or at 2-8℃fo

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Validation of a potency assay for the cAMP measurement from cultured GLP-1R-GFP-293A cells activated by Liraglutide

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Validation of a potency assay for the cAMP measurement from ...

中国毒理学会药物毒理与安全性评价学术大会（2019年）暨粤港澳大湾区生物医药产业第一届高峰论坛

作者： Yimin Tao Yingying Hou Jinli Gao Wenjing zhong likun gong Jin Ren Qiuping Qin Department of Immunoassay and Immunochemistry Center for Drug Safety Evaluation and Research(CDSER)State Key Laboratory of New Drug ResearchShanghai Institute of Materia MedicaChinese Academy of Sciences

Background Bioassays used for drug potency estimation have been an integral part of the quality assessment for a biological drug *** results of a potency assay are normally used to support lot release of a *** purpose of this study was tovalidate a cell-based potency assay for cAMP measurement from cultured GLP-1 R-GFP-293 A cells activated by a glucogen like peptide-1(GLP-1)agonist such as Liraglutide and its *** Homogeneous time-resolved fluorometric assay was used for the determination of cAMP from cultured GLP-1 R-GFP-293 A cells stimulated by Liraglutide and its *** cAMP produced by cells in the biological reference product sample(BRPS),system suitability sample(SSS)and test sample(TS) or unlabeled cAMP in standards and quality control samples(QCs) competed with d2-labeled cAMP(acceptor) for binding to Eu3+-Cryptate labeled monoclonal anti-cAMP(donor).The specific fluorescent signal in the form of a fluorescence ratio(665 nm/620 nm) arisen from resonance energy transfer is inversely proportional to the concentration of cAMP in the BRPS,SSS,TS,standards and *** validation was performed to evaluate the standard curve of the cAMP assay,intra-and inter-assay accuracy and precision of cAMP quality control samples,accuracy and precision of BRPS,SSS and TS,Specificity,Dilutional linearity,and storage stability of BRPS..Results The results of standard curve and intra-/inter-assay accuracy and precision of cAMP QC samples were met the acceptance *** mean relative potency(RP) values determined from six assay batches for the SSS and TS were 101.6% and 111.1%,respectively,with CV% of 10.8% and7.0%.The vehicle was unable to stimulate the GLP-1 R-GFP-293 A cells to generate *** and GLP-1 were able to stimulate the cells to generate cAMP,but the former produced much greater RP value(621.2%),whereas the latter did not produce a dose response curve parallel to that of the *** using five mock potency test samples

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基于改进的同轴DBD等离子体装置的柴油力学性质研究

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中国石油大学学报（自然科学版） 2019年第5期43卷 163-169页

作者：刘建林于红窦晓晓宫礼坤 DAVIES G R 中国石油大学(华东)储运与建筑工程学院山东青岛266580 中国石油大学(华东)理学院山东青岛266580 新西兰梅西大学航空学院北帕莫斯顿4474

为了不断提升柴油的性能,需要采用各种技术手段对其力学参数进行改进,从而得到定量化的优化方案。提出一种简便易行的利用等离子体技术改善油品力学性能的方法。基于自行开发的同轴介质阻挡放电(DBD)装置,采用较低击穿电压即可实现放电... 详细信息

为了不断提升柴油的性能,需要采用各种技术手段对其力学参数进行改进,从而得到定量化的优化方案。提出一种简便易行的利用等离子体技术改善油品力学性能的方法。基于自行开发的同轴介质阻挡放电(DBD)装置,采用较低击穿电压即可实现放电以产生大气压等离子体,然后运用该装置对循环流动的柴油进行处理。实验中测试不同的等离子体处理时间、输入电压和柴油流量对柴油力学性质的影响,主要包括黏度、表面张力和接触角。结果表明,等离子体技术对柴油的水下接触角影响最大,对其黏度和表面张力影响相对较小。在此基础上,采用量纲分析进一步验证了部分实验结果。

关键词：同轴DBD等离子体接触角黏度表面张力量纲分析