ADCC讲座.pdf

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1、 2010,Promega Corporation.Confidential and Proprietary.Not for Further Disclosure.Promega ADCC检测法检测法 Confidential and Proprietary.Not for Further Disclosure.Biological Product 2 Source:FDA webinar,Feb 15,2012 Confidential and Proprietary.Not for Further Disclosure.什么是什么是Biologics:生物制药生物制药 US Biologi

2、cs by 2010 Sales($billions)nature biotechnology,volume 29,number 12,DECEMBER 2011 3 9.7%growth 单克隆抗体药(mAb)占生物制药市场的 1/3 mAb=$48 billion in 2010,expected$86 billion by 2015(CAGR)of 12.4%Confidential and Proprietary.Not for Further Disclosure.Biologics Workflow 4 Target Discovery POTENCY Therapeutic Pr

3、otein Screen/Optimization Pre-clinical Trials Therapeutic Protein Manufacturing Post Launch Patient Response SCREENING STABILITY COMPARABILITY LOT RELEASE POTENCY&STABILITY IMMUNOGENICITY Bioassay Use Is Critical Throughout Biopharmaceutical Drug Development&Monitoring Clinical Trials Precision and

4、accuracy are critical New solutions can solve shortcomings of current bioassays Confidential and Proprietary.Not for Further Disclosure.生物制药面临的挑战生物制药面临的挑战 A company acquired a late-stage mAb drug and needed to switch the manufacturing cell line and process to fit into their standard process FDA requ

5、ested that an assay examining the mAb mechanism of action(MOA)be used to demonstrate similarity between process change Current MOA based assays measuring antibody dependent cellular cytotoxicity(ADCC)had poor reproducibility,high variability and was therefore not suitable for use The company develop

6、ed a novel reporter assay with low variability,high reproducibility,and used it to successfully demonstrate similarity and make the manufacturing change 5 Confidential and Proprietary.Not for Further Disclosure.什么是什么是ADCC:抗体依赖性的细胞介导抗体依赖性的细胞介导 的细胞毒的细胞毒 抗体依赖的细胞介导的细胞毒（antibody-dependent cell-mediatedcy

7、totoxicity）作用是指表达IgGFc受体的NK细胞、巨噬细胞和中性粒细胞等，通过与已结合在病毒感染细胞和肿瘤细胞等靶细胞表面的IgG抗体的Fc段结合，而杀伤这些靶细胞的作用。IgG抗体可介导这些细胞发挥ADCC作用，其中NK细胞是能发挥ADCC作用的主要细胞。在抗体介导的ADCC作用的发生过程中，抗体只能与靶细胞上的相应抗原表位特异性结合，而NK细胞等效应细胞可杀伤任何已与抗体结合的靶细胞，故抗体与靶细胞上的抗原结合是特异性的，NK细胞等对靶细胞的杀伤作用是非特异性的。6 Confidential and Proprietary.Not for Further Disclosure.T

8、arget cell Y=NFAT-RE-luc Reduce variability by replacing NK cells with genetically engineered stable cell line Increase signal with robust reagents FcgRIIIa Antibody 传统传统ADCC 检测检测 vs 报告基因检测报告基因检测 Effector engineered Jurkat cell Reporter-based ADCC bioassay Glo Target cell Y Primary NK(Effector)cell

9、Antibody FcgRIIIa Variability of assay results largely due to source of NK cells Spontaneous lysis of target&effector cells results in high background Classic ADCC bioassay Specific signal is from target cell Signal is from effector cell 7 Confidential and Proprietary.Not for Further Disclosure.靶标肿瘤

10、细胞 Target Cell Y IgGs Fcg gRIIIA CD3 z z dimer ZAP70 Syk Y Effector Cell 效应细胞 LAT PLC-g g 1and 2 Intracellular Ca2+release Calcineurin Calmodulin Activated calcineurin NFAT Luc NFAT-RE IP3 R Lck Scientific Basis for ADCC Reporter Assay Source:Leibson,PJ Immunity 1997 Confidential and Proprietary.Not

11、 for Further Disclosure.Design Requirements for ADCC Reporter Bioassay 波动性低 人工构建的效应细胞替代 NK cells (Jurkat FcgRIIIA/NFAT-RE-luc2)冻存细胞溶解即用的模式，细胞即为检测试剂 检测过程和检测试剂简单易用 可应用于悬浮或贴壁的各种靶细胞 满足生物检测法的精确要求 可以用于质控治疗性抗体药物的效力和稳定性 可以区分是由于抗体糖基化的小改变还是ADCC效应中Fc功能的生物相关活性引起的小改变 9 Confidential and Proprietary.Not for Furthe

12、r Disclosure.ADCC报告基因反应是一定需要结合抗体才能实现报告基因反应是一定需要结合抗体才能实现 Target cells,effector cells and specific antibody No Target cells No Effector cells or no FcgRIIIa No antibody or non-specific antibody Assay signal is dependent on:Presence of Target cells +Presence of FcgRIIIa receptor +Appropriate specific a

13、ntibody 10 Confidential and Proprietary.Not for Further Disclosure.Cells as Reagents Frozen,Thaw-and-Use*Accomplished with control of cell propagation conditions and defined freezing protocol Test performance versus freshly cultured cells Check consistency from batch to batch Produce ample cell bank

14、s to provide long-term supply Minimizes cell-culture variability and assay preparation time 11*can still used freshly cultured cells if frozen format fails Confidential and Proprietary.Not for Further Disclosure.Equivalent Results with Frozen,Thaw-and-Use Cells A.Fresh cells from continuous cultureB

15、.Frozen,thaw-and-use cellsShown are representative data from ADCC reporter assay using fresh(A),or frozen thaw-and-use(B)Jurkat effector cells and Wil2-S cells.Target cells are CD20+WIL2-S Similar EC50s and fold-induction of luciferase activity 12 Confidential and Proprietary.Not for Further Disclos

16、ure.ADCC Reporter Assay Protocol mAb Response induction(6-20 hr)Bio-Glo Assay Reagent Receptor antigen-expressing Target cells,thaw-and-use or continuously cultured cells 1.阳性对照抗体或待测抗体与靶细胞共孵育.2.加入Promega的带有FcgRIIIa受体和 NFAT-RE luc2 luciferase人工效应细胞系，孵育(6-20h).3.加入检测试剂Bio-Glo Assay Reagent，检测发光 Glo En

17、gineered Jurkat Effector cells in thaw-and-use format 13 Confidential and Proprietary.Not for Further Disclosure.ADCC Reporter Bioassay Kit Configurations Engineered Jurkat cells Glo Detection Reagent Control Antibody Target WIL2-S or Raji Cells Core Kit Complete Kit Core Kit Engineered Jurkat Effector cells(NFAT-RE-luc2/FcgRIIIa);frozen,thaw-and-use Bio-Glo Luciferase Assay System RPMI Medium Low IgG Serum Complete Kits(2 types)Above PLUS:Target WIL2-S cells;frozen,thaw-and-use or Target Raji cells;frozen thaw-and-use Control Ab(CD20)+Medium Serum