Microsoft PowerPoint - 6 Immunogenicity of therapeutic proteins.pptx

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1 Pharmaceutical Biotechnology Immunogenicity of therapeutic proteins By Yuqiong Xia 2014

2 Keywords Immunogenicity, allergy, allergic, immune tolerance, neutralization Impurity, glycosylation, glycosylated Assay, tier, clinical, endogenous 2

3 Outline History of immunogenicity Mechanism, influence factors and assay Immunogenicity of monoclonal antibodies Clinical effects of induced antibodies Predicting and reducing immunogenicity 3

4 History 19 th C., animal sera treat diphtheria and tetanus ( 白喉和破伤风 ) Serious or fatal side effects Due to high doses, impurity, no quality control 4

5 History 1922, porcine and bovine insulins treat diabetes Induced antibodies, but less severe Due to animal origin 5 cb /diabetesindogs/images/6/6d/porcineinsulin.jpg

6 History , plasma-derived clotting factors and growth hormone from pituitary glands of cadavers ( 尸体的脑垂体 ) Given to children who lacked the natural immune tolerance Due to foreign proteins 6

7 7

8 History 1982, marketed human insulin produced by recombinant DNA technology Induce antibodies to patients with immune tolerance 8

9 A different kind of immune response Breaking B-cell tolerance Takes in 6 to 12 months Produce binding antibodies with no biological effect Antibodies disappear shortly after stopping treatment No memory effect 9

10 Classical immune response Vaccine-type response Occur within weeks A single injection will induce antibody response Induce high level of neutralizing antibodies Memory effect 10

11 Outline History of immunogenicity Mechanism, influence factors and assay Immunogenicity of monoclonal antibodies Clinical effects of induced antibodies Predicting and reducing immunogenicity 11

12 Mechanism of classical immune response microorganisms humoral immunity B-cell Secrete antibodies neutralization phagocytosis 12

13 Mechanism of breaking B-cell tolerance 13

14 Influence factor: sequence variation The degree of nonself and presence of aggregates Protein involved Site of the divergence from the natural sequence 14

15 Influence factor: glycosylation 15 Glycosylated β interferon is less immunological than the nonglycosylated Normally glycosylated epoetin had a lower affinity to antibodies than the nonglycosylated

16 Influence factor: impurities Cell debris Enzyme 16 Formulation Resin Container and sealing

17 Influence factor: formulation Case 1: A freeze-dried drug (human serum albumin as stabilizer) at room temperature induces immune response Case 2: Epoetin-α (Tween-80 as stabilizer) induces immune response, and causes anemia ( 贫血 ) 17

18 Influence factor: route of administration SC route induces most serious immune response IV route induces least immune response Any route can induce immune response 18

19 Influence factor: dose The effect of dose is not quite clear Highest dose could lead to lowest immune response 19

20 Influence factor: patient features Other treatment of patients Underlying disease of patients 20

21 Assay for antibodies 21 Different assays on the same sera can have 50- fold difference Lack of standardization of assay methodology

22 Assays for antibodies Many new assays need to be used together Two-tier ( 两层 ) approach Screening to Identify antibody positive sera Further analysis to determine If antibodies are neutralizing and then titer, affinity and isotype. 22

23 First tier: ELISA Sandwich Ab-Ag-Ab 23

First tier for new proteins Define an absolute sensibility is impossible Set the cut-point for the assay at a 5% falsepositive level using a group

24 First tier for new proteins Define an absolute sensibility is impossible Set the cut-point for the assay at a 5% falsepositive level using a group of normal human sera You really don t have it Test say you don t have it Test say you do have it You really do have it dialysis.wordpress.com

25 Second tier: detect neutralizing antibodies Add serum Add protein drug Detect neutralizing antibodies False negative: factors stimulating the bioassay may compensate for neutralization activity Patient serum with and without IgG should be used as control 25 Modified from

26 Outline History of immunogenicity Mechanism, influence factors and assay Immunogenicity of monoclonal antibodies Clinical effects of induced antibodies Predicting and reducing immunogenicity 26

27 Structure of IgG Y-shaped 2 H chains 2 L chains S-S bond V H and V L C H and C L 27

28 Monoclonal antibodies (mabs) Classical immune response Immune response 28

Patients are not at normal immune status Blood sampling of

29 Why human mab has immunogenicity Aggregation mab activates T-cells by itself, induce immune response by Fc remove Fc Patients are not at normal immune status Blood sampling of patients have normal circulating mabs, not just mab drug 29

30 Outline History of immunogenicity Mechanism, influence factors and assay Immunogenicity of monoclonal antibodies Clinical effects of induced antibodies Predicting and reducing immunogenicity 30

31 Clinical effects of induced antibodies The antibodies neutralize an endogenous factor with an important biological function Case 1: MDGF ( 巨核细胞生长发育因子 ) induce antibodies, and the antibodies react with endogenous thrombopoietin ( 促血小板生成素 ) Case 2: Epoetin-α induced antibodies, and the antibodies react with endogenous erythropoietin ( 红细胞生成素 ), and causes anemia ( 贫血 ) 31

32 Clinical effects of induced antibodies Antibodies influence the side effects of protein drugs Reduce the side effects Sometimes, side effects are caused by the antibody formation 32

33 Common side effects Acute anaphylaxis( 急性过敏反应 ) Hypersensitivity Skin reaction Serum sickness Still common during treatment with high dose 33

34 Outline History of immunogenicity Mechanism, influence factors and assay Immunogenicity of monoclonal antibodies Clinical effects of induced antibodies Predicting and reducing immunogenicity 34

35 Predicting immunogenicity is difficult No constant sequence variation -immunogenicity relationship A single amino acid change could make a protein immunogenic Substantial divergence from the natural sequence may have no effect 35

36 Method of predicting immunogenicity Simulation and computational models Only give limited information Binding tests T-cell proliferation assay have false positive results Many antibodies can activate T-cell or inhibit cell proliferation 36

37 Method of predicting immunogenicity For human homologues Check aggregates or impurities Animal studies are not suitable All human proteins are in principle immunogenic to animals Transgenic mice are OK 37

38 Reducing immunogenicity Change the amino acid sequence Link proteins to polymers such as PEG and low MW dextran Immunosuppressive treatments Tolerance induction 38

39 Summary Breaking B-cell tolerance Vs. classical immune response Influence factors and assay of immungenicity Special Immunogenicity of monoclonal antibodies Reducing immunogenicity 39

40 Homework 5 True or False (If false, give the reason) Since mab drug can induce immune response by its F ab part, we can remove F ab to get a less immunological mab drug. Breaking B-cell tolerance implies that a patient becomes less allergic to protein drugs. 40

University of Science and Technology of China A dissertation for master s degree Transdermal Delivery of Gut Hormone PYY3-36 for the treatment of Obes

中国科学技术大学硕士学位论文肠激素 PYY3-36 透皮给药治疗肥胖症的研究作者姓名 : 学科专业 : 导师姓名 : 完成时间 : 陈敏细胞生物学温龙平教授二一年八月三十一日 University of Science and Technology of China A dissertation