1. Monoclonal Antibodies Dr. Aws Alshamsan Department of Pharmaceutics Office: AA87 Tel: 4677363 aalshamsan@ksu.edu.sa

2. Objectives of this lecture By the end of this lecture you will be able to: 1.Define terms such as monoclonal, polyclonal, isotype, idiotype, allotype, CDR, and hybridoma 2.Compare monoclonal-antibody production methods 3.Identify different mAb types 4.List some applications of mAb in medicine

3. Antibody Response

5. Antibody Structure

6. Complementarity Determining Regions

7. Antigen Antibody Interaction

9. Ab Isotypes

10. Ab Fragments

13. Ab types differences

14. Polyclonal v.s. Monoclonal

15. Affinity and Avidity Affinity: the strength of binding between a single binding site and a single ligand. Avidity: the strength of binding between a molecule and a complex ligand, e.g. if there are multiple binding sites then the avidity may be increased by increasing the number of binding sites or by increasing the affinity of those binding sites.

17. Affinity and Avidity, continued IgM is produced early in an immune response when the affinity for antigen often is low; as an immune response continues, antibody affinity is improved, this is combined by “class switching” to the use of smaller molecules (IgG, IgE and IgA). The increased affinity compensates for the decrease in number of binding sites in maintaining the overall avidity for antigen.

18. Polyclonal Response

19. Polyclonal antibody –Antigens possess multiple epitopes –Serum antibodies are heterogeneous, To increase immune protection in vivo To reduces the efficacy of antiserum for various in vitro uses –To response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen –To have clear advantages for the organism in vivo Monoclonal antibody –Derived from a single clone, specific for a single epitope –For most research, diagnostic, and therapeutic purposes

20. mAb Types

23. mAb nomenclature Source stem Suffix omAb ximAb zumAb u

25. mAb Production Hybridoma Technology Phage Display Method

26. Hybridoma Technology 1975, by Georges Köhler and Cesar Milstein - Be awarded a Nobel Prize in1984

29. (1) Immunisation of a mouse (2) Isolation of B cells from the spleen (3) Cultivation of myeloma cells

30. (4) Fusion of myeloma and B cells (using PEG) (5) Separation of cell lines

31. (6) Screening of suitable cell lines

32. (7) in vitro (a) or in vivo (b) multiplication (8) Harvesting

34. Selected by using HAT medium (Hypoxanthine- Aminopterin-Thymidine) Myeloma cells are unable to grow B cells are able to survive, but can not live for extended periods HAT Selection

36. Two different pathways to synthesis nucleotide in mammalian cells (Folic acid analog)

37. Phage Display - Introduction The display of functional foreign peptides or small proteins on the surface of bacteriophage particles. An important tool in protein engineering A powerful way to screen and select for peptides on the basis of binding or molecular recognition

38. Phage Display Principle

39. More efficient than hybridoma system. Cheaper to produce recombinant antibodies using bacteria, rather than mammalian cell line. Easier to maintain and grow bacterial cultures for recombinant antibody production. Bypass immunization in antibody selection. Bypass the use of animal cells for production of antibodies. Producing the combinatorial library (ideally with 10 8 to 10 9 members) of functional antibodies to generate a larger repertoire of antibodies than those available through conventional hybridoma technology. Phage Display Advantages

41. Plantibodies "plantibodies" are antibodies produced by genetically-engineered corps e.g. corn, potatoes and tobacco plant "plantibodies" are cheaper and arguably safer than mammalian mAbs

42. Clinical Applications of Monoclonal Antibodies A.Diagnosis 1.Pregnancy test 2.ELISA 3.Western Blot 4.Flow Cytometry 5.Radioimmunoimaging B.Therapy A.Passive Immunotherapy B.Active Immunotherapy C.Drug Targeting

43. Pregnancy test

44. ELISA

45. Western Blot

46. Flow cytometry

47. Radioimmunoimaging

48. Passive immunotherapy

50. Active immunotherapy

51. Active immunotherapy using Bispecific mAb

52. Bispecific mAb

53. Drug targeting

54. You are now able to: Define terms such as monoclonal, polyclonal, isotype, idiotype, allotype, CDR, and hybridoma Compare monoclonal-antibody production methods Identify different mAb types List some applications of mAb in medicine