Pharmaceutical Biotechnology Lesson one

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1 Pharmaceutical Biotechnology Antibodies, vaccines and adjuvants By Yuqiong Xia 6/10/2017

2 生物技术制药 2 技术基础生物技术药物生物技术制药概念药物的开发药物的生产细胞因子生长因子激素类药物血液制品和治疗性酶抗体疫苗和佐剂核酸药物药物的发现专利的申请临床前试验临床试验生物药物的来源生物药物的生产和纯化生物药物的分析干扰素白介素造血生长因子

3 多克隆抗体概念制备医疗应用抗体和疫苗单克隆抗体疫苗概念制备 : 杂交瘤技术医疗应用概念佐剂 3

4 Outline Polyclonal antibody preparations Concept Preparation Medical application Monoclonal antibody Vaccine 4

5 What is polyclonal antibody A collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope Antibodies that are secreted by different B cell lineages within the body 5

6 Application of polyclonal antibody For passive immunization against infectious diseases and other harmful agents IV injection Transitory effect: 2-3 weeks 6

7 Types of polyclonal antibody Predominant antibody: IgG Two types Antisera, from animal Immunoglobulin preparation, from human 7

8 Outline Polyclonal antibody preparations Concept Preparation Medical application Monoclonal antibody Vaccine 8

9 Preparation of antisera 9

10 Preparation of antisera Immunize healthy animals with antigen repeat injection of antigen Draw blood from animal 1-2 L every days for horse Collect into sterile containers Purify antibody from blood Analyze desired antibody quantitatively Bioassay or immunoassay Formulation NaCl, glycine, preservative 10

11 Purify antibody from blood Method 1 Form blood clot ( 血液凝块 ) Recover antibody-contained antisera by centrifugation Precipitation or HPLC to purify antibody Method 2 Add heparin or other anticoagulant Remove cellular elements Precipitation or HPLC to purify antibody 11

12 Preparation of immunoglobulin Similar to that of antisera Immunize people Draw blood Analyze desired antibody Formulation Donor People who have recently been immunized against the antigen People who recently recovered from an infection caused by the antigen 12

13 Outline Polyclonal antibody preparations Concept Preparation Medical application Monoclonal antibody Vaccine 13

14 Medical application of polyclonal antibodies Anti-D immunoglobulin To prevent haemolytic disease of the newborn (HDN, 新生儿溶血性贫血 ) Normal immunoglobulin (immune globulins) Against many diseases Hepatitis B immunoglobulin To prevent Hepatitis B Tetanus immunoglobulin ( 破伤风免疫球蛋白 ) To manage tetanus-prone wound Antiserum against toxins of snakes and spiders 14

Haemolytic disease of the newborn (HDN) 1 2 3 1. RhD- mother carrying RhD+ baby 2. Mother produces RhD+ antibody 3.

15 Haemolytic disease of the newborn (HDN) RhD- mother carrying RhD+ baby 2. Mother produces RhD+ antibody 3. Haemolysis of red cells of second baby 15

16 Anti-D immunoglobulin To HDN of the newborn Administration Administer anti-d immunoglobulin immediately postdelivery, suppress an immune response Source Serum or plasma of Rh- people who have been immunized against Rh-D antigen 16

17 Normal immunoglobulin (immune globulins) Source Plasma, serum or placentas ( 胎盘 ) of normal, healthy donors Components Antibodies against diphtheria, measles, poliomyelitis, hepatitis A, rubella and varicella ( 白喉, 麻疹, 脊髓灰质炎, 甲肝, 风疹, 水痘 ) More than 90% IgG Application Provide passive immunization against these diseases 17

Hepatitis B immunoglobulin Source Serum or plasma of donors who exhibit high titers of anti-hepatitis B surface antigen (HBsAg) antibodies Medical application Protect persons

18 Hepatitis B immunoglobulin Source Serum or plasma of donors who exhibit high titers of anti-hepatitis B surface antigen (HBsAg) antibodies Medical application Protect persons who contact hepatitis B, such as newborns whose mothers have been infected with the virus hepatitis B 18

19 Tetanus immunoglobulin Application To manage tetanus-prone wounds Source Serum or plasma of human donors who have been immunized with tetanus toxin 19

20 Antiserum against toxins of snakes Application Treat people who have suffered bites from snakes Source The serum/plasma of horses that have been injected with (usually inactivated) venom ( 毒液 )or venoms 20

21 Outline Polyclonal antibody preparations Monoclonal antibody Concept Structure Preparation: hydridoma technology Medical application Vaccine 21

is possible to produce mabs for detection or purification -mab: mab drugs 22

22 What is monoclonal antibody Antibodies made by identical immune cells Monovalent affinity, bind to the same epitope Given almost any substance, it is possible to produce mabs for detection or purification -mab: mab drugs 22

23 History of mab 1900s, Paul Enrlich, magic bullet 1970s, B-cell multiple myeloma ( 多发性骨髓瘤 ) can produce single types of proteins 1975, Kohler and Milstein, hybridoma technology 1988, Greg Winten, humanize mab 23

24 The application of mabs Oncology 9 mabs, 2 immunoconjugates Transplantation 3 mabs Inflammatory diseases 6 mabs, 3 Fc-fusion proteins Cardiovascular area 1 mabs 24

25 The advantage of mabs Safe Avoid unnecessary exposure to non-target organs Safer than chemotherapeutic treatment Molecular targeted medicine 25

26 Outline Polyclonal antibody preparations Monoclonal antibody Concept Structure Preparation: hydridoma technology Medical application Vaccine 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 Structure of IgG 2 heavy chains (2 H-chains) Variable regions: V H Constant regions: C H, effector recognition and binding 2 light chains (2 L-chains) Variable regions: V L Constant regions: C L, effector recognition and binding 28

29 Different humanized mabs 29 0% 60%-70% 90%-95% 100% Decreased immunogenic risks of generating anti-therapeutic antibodies (ATA)

30 Murine mabs Form neutralizing human anti-mouse antibodies (HAMA) Muromomab-CD3 (OKT3) First generation mab of murine origin Treat acute transplant rejection 50% patients produced HAMA HAMA interferes OKT3 s binding to T-cells 30

31 Chimeric mabs Human constant regions and mouse variable regions Antigen specificity is the same as mouse antibodies Human Fc region make the in vivo half-life longer Induce human anti-chimeric antibodies (HACA) For infliximab (one chimeric mab), 61% patients had HACA response 31

32 Humanized mabs Only CDR(complementary determing region, also named epitope) is from murine; other parts are from human Produce human anti-human antibody (HAHA) For trastuzumab (a humanized mab), 0.1% patients have HAHA response 32

33 Human mabs Fully humanized mabs Production Phage display library Using transgenic XenoMouse with human heavy and light chain gene fragments Adalimumab, 5% patients produced HAHA 33

34 34

Key structural components of mabs Fv, Fragment variable V H V L scfv 35 http://www.mdpi.

35 Key structural components of mabs Fv, Fragment variable V H V L scfv 35

Key structural components of MAbs Fab, Fragment antigen binding F(ab ) 2, two Fab s F(ab ) 2 36

36 Key structural components of MAbs Fab, Fragment antigen binding F(ab ) 2, two Fab s F(ab )

Key structural components of MAbs Fc mabs elicit cytotoxicity after Fc interact with its receptors (FcγR) and complement fixation CH2 domain or hinge

37 Key structural components of MAbs Fc mabs elicit cytotoxicity after Fc interact with its receptors (FcγR) and complement fixation CH2 domain or hinge region joining CH1 and CH2 are crucial regions for binding to FcγR Modify Fc regions can enhance or decrease the cytotoxity of mabs VH CH1 37 Fc CH2 CH3

Antibody derivatives and fusion proteins Fc have similar half life as fullsized antibody 38 Bind to a drug and reduce side effects of the drug F(ab

38 Antibody derivatives and fusion proteins Fc have similar half life as fullsized antibody 38 Bind to a drug and reduce side effects of the drug F(ab ) 2, Fab, scfv have short half life Fc is fused to cytokines, growth factor enzymes, etc., and used to increase half life and stability of cytokines

39 Outline Polyclonal antibody preparations Monoclonal antibody Concept Structure Preparation: hydridoma technology Medical application Vaccine 39

40 Hybridoma technology Milstein and Kohler, hybridoma technology 1975 present the technology 1984 Noble Prize 1986 market authorization 40

1 2 Hybridoma technology to produce mabs 3 5 6 4 7 8 1. Immunization of a mouse 2. Isolation of B cells from the spleen 3. Cultivation of myeloma cells 4.

41 1 2 Hybridoma technology to produce mabs Immunization of a mouse 2. Isolation of B cells from the spleen 3. Cultivation of myeloma cells 4. Fusion of myeloma and B cells 5. Separation of cell lines 6. Screening of suitable cell lines 7. in vitro (a) or in vivo (b) multiplication 8. Harvesting 41

42 Phase display technique to screen mab 42

43 Outline Polyclonal antibody preparations Monoclonal antibody Concept Structure Preparation: hydridoma technology Medical application Vaccine 43

44 Medical application of mabs mabs in Cancer Therapy Treat cancer mabs in Solid Organ Transplantation Reduce immune response mabs in Anti-inflammatory Therapy Treat inflammation 44

45 Cancer cells img jpg

46 The target of therapeutic mabs CD cell Alemtuzumab Epidermal growth factor receptor (EGFR): HER-2 Trastuzumab Vascular endothelial growth factor (VEGF) Bevacizumab 46

47 CD (cluster of differentiation) 分化抗原 Cell surface molecules providing targets 47

48 Alemtuzumab Target CD52 cell surface glycoprotein kda not shed, a good therapeutic target CD52+ lymphocytes, monocytes, NK cells, macrophages and eosinophils CD52+ disease chronic lymphocytic leukemia( 慢性淋巴细胞白血病 ), non- Hodgkin lymphoma( 非霍奇金淋巴瘤 ), acute lymphoblastic leukemia ( 成淋巴细胞性白血病 ) 48

49 HER-2 Same as EGFR 2, belong to EGFR transmembrane glycoproteins constitutively expressed in many normal epithelial tissues skin and hair follicle ( 毛囊 ) overexpressed in many cancers colon( 结肠 ), rectum( 直肠 ) and breast cancer 49

50 HER-2 50

51 Trastuzumab (Herceptin ) A recombinant DNA derived humanized mab (IgG1 kappa) Target the extracellular domain of HER-2 receptor Induce auto-phosphorylation of the tyrosine kinase( 酪氨酸激酶 ) internal domain, resulting in decreased tumorigenic ( 致瘤的 ) potential and possibly reversal of chemo-resistance 51

52 52 Trastuzumab (Herceptin ) slows cancer growing

53 VEGF in cancer Tumor reaches mm, it can no longer supply itself with nutrients and oxygen from nearby small blood vessels Tumor secretes proteins called vascular endothelial growth factor (VEGF) VEGF attach to nearby blood vessels and stimulate growth 53

54 VEGF in cancer VEGF can induce neovascularization ( 新血管形成 ) for malignant cells in an autocrine fashion High levels of VEGF are correlated with poor prognosis( 预后 ), disease recurrence, and metastases( 转移 ) in many tumors. 54

55 Bevacizumab (Avastin ) VEGF inhibitor (the only FDA approved ) a normal human IgG1 (93%) + the VEGF binding residues from a murine neutralizing antibody (7%) Produced in CHO cells 55

56 Bevacizumab can inhibit VEGF in cancer 56 Bevacizumab blocks the signals from the tumor that prompt blood vessel growth. Without nutrients and oxygen, the tumor stops growing.

57 Outline Polyclonal antibody preparations Monoclonal antibody Vaccine Concept Traditional vaccine Vaccines by recombinant DNA technology Adjuvant 57

58 Vaccine Vaccine contains antigens derived from or related to a pathogen Exploit the natural defense mechanisms of our immune systems 58

59 Vaccine Upon vaccine administration, both humoral and cell-mediated immune system are activated Long-term immunological protection Subsequent administration of one or more repeat doses are needed 59

60 Traditional vaccine Live attenuated bacteria BCG 卡介苗 Dead or inactivated bacteria pertussis vaccine 百日咳 Live attenuated virus Measles 麻疹 Inactivated virus Hepatitis A 甲肝 Toxoids Tetanus 破伤风疫苗 Pathogen-derived antigens Hepatitis B 乙肝 60

61 Vaccines by recombinant DNA technology Produced in E. coli or Saccharomyces cerevisiae ( 酿酒酵母 ) Advantages Safe Abundant No side effects 61

62 Adjuvants Administration of many vaccines on their own stimulates a poor host immune response Adjuvants can elicit an earlier, more potent and longer-lasting immune response 62

63 Why adjuvants help? Depot formation of antigen Lead to slow release of antigen from the site of injection, and therefore long-term effect Enhanced antigen presentation to the cells of the immune system Direct induction of immuno-stimulatory substances, such as ILs and other cytokines 63

Mineral-based adjuvants Most commonly employed are aluminium hydroxide and aluminium phosphate Formulation Mix the antigen in solution with a pre-formed aluminium phosphate (or hydroxide) precipitate

64 Mineral-based adjuvants Most commonly employed are aluminium hydroxide and aluminium phosphate Formulation Mix the antigen in solution with a pre-formed aluminium phosphate (or hydroxide) precipitate Adsorption of the antigen to the aluminium-based gel ensues, named aluminium-adsorbed vaccines. Action mode Depot formation at the site of injection Activate complement, lead to a local inflammatory response Advantage Safe and effective 64

65 Summary Polyclonal antibody poly Anti-serum, immunoglobulin preparation Anti-D immunoglobulin Monoclonal antibody mono mab Fab, Fc, VH, VL, CH, CL Hybridoma technology Anti-CD52, anti-her2, anti-vegf Vaccine Adjuvants: aluminum hydroxide/phosphate 65

Microsoft PowerPoint - 14 Monoclonal Antibodies.pptx

Microsoft PowerPoint - 14 Monoclonal Antibodies.pptx Pharmaceutical Biotechnology Monoclonal Antibodies By Yuqiong Xia 2014 Keywords Monoclonal antibody (mab), epitope, myeloma Chimeric, complement, complementary Phage display, hinge, cytotoxity Apoptosis,