1. Antibody (immunoglobulin)
Serum protein electrophoresis (SEP)
Basic structure and function of immunoglobulin.
Monoclonal antibody, its modification and application

2. Serum protein electrophoresis

4. Buffer pH 8.6

10. Serum electrophoresis

11. Immunoglobulin (antibody, BCR): universal adaptor

12. 典型抗體分子的構造(1)
BCR and Ab
N 端 構造 功能 異
C端序列
作用機制 同
免疫球蛋白
辨識抗原
C 端

13. Proteolytic digestion of IgG
Variable region
Fab: papain digest
F(ab')2: pepsin digest
Constant region (Fc)

14. Different domains have different functions

15. 抗體分子與專一性抗原之交互作用(1) 局部區域的高度變異序列形成抗原結合位 Hypervariable region
Framework region
CDR (complementarity-determining region)

19. General structures of five classes of antibody

22. Structure and formation of secretory IgA

23. Antigenic determinants of immunoglobulin
Isotypes: all variants present in serum of normal individual
types: heavy and light chain
classes: IgG, IgA, IgM, IgD, IgE subclasses: IgG1, IgG2, IgG3, IgG4
subgroups: VHI, VHII, VHIII
Allotypes: genetically controlled alternative forms
Idiotypes: individually specific to each immunoglobulin molecule

24. Immunoglobulin superfamily

25. Generation of diversity (G.O.D.)
VDJ rearrangement of heavy chain
VJ rearrangement of light chain
random combination of heavy and light chain
Imprecision of rearrangement

26. V-region genes are constructed from gene segments

27. Overview of B cell development

28. Model of allelic exclusion

29. Immunoglobulin concentration changes along the age

30. Serum immunoglobulin vary among different healthy persons
IgG: mg/dl
IgA: mg/dl
IgM: mg/dl
IgD: 0-40 mg/dl

31. Antibody response Clonal selection
Primary and secondary antibody response
Isotype switch: IgM----->IgG
Affinity maturation: Somatic mutation

32. Monoclonal antibody Myeloma (HGPRT-) Spleen from immunized mice
HAT medium
Selection
Limiting dilution and cloning

33. Method Immunization with antigen Spleen and myeloma cell fusion
Polyethylene glycol (PEG): polymer of ethylene oxide
HAT medium: selection of hybridoma
Screening
Cloning by limiting dilution: Poisson distribution
Large scale production and storage: can be purified by protein A affinity column
cell line (10-50 mg/ml)
ascites (1-10 mg/ml)

34. Main pathway (de novo) Secondary Pathway ~~~~~~~~~~~~~~~~~~~~~~ DNA
Aminopterin Block
DNA
NUCLEOTIDES
RNA
HGPRT TK
Hypoxanthine
Thymidine
Secondary Pathway
Enzyme-deficient
(HGPRT-) myeloma cell
Hybrid cell
Normal splenocyle
spontaneous death
HAT-dependent death
ACTIVE GROWTH

35. Application of monoclonal antibodies: universal adaptor
Antigen purification: affinity column
Diagnosis: detect Ag or Ab
pathogen
cell surface marker
soluble antigens e.g. AFP
cancer diagnosis: immunoimaging
Immunotherapy
Cancer therapy
Autoimmune disease treatment
Allergy prevention
Tissue transplantation

36. Application of monoclonal antibody

37. Application of mAb as immunotherapeutic drugs

38. Monoclonal antibodies that recognize tumor-specific antigens might be used in a variety of ways to help eliminate tumors

39. MAT: the magic bullet
Therapeutic results were disappointing until recently.
Two major hurdles were encountered.
Biologic activity in humans severely limited due to the host’s immune response to foreign mAbs (production of human anti-mouse Abs). Causes neutralization of activity and toxicity.
Large-scale production

40. Mouse mAb may cause serum sickness in vivo

41. Approaches to reduce or eliminate human anti-mouse antibodies.
Chimeric Antibodies
Cloned antibody genes are generated in which the variable regions from the original mouse mAb are combined with human antibody-constant regions.
Highly effective in reducing human anti-mouse antibodies (HAMA). However, the variable (Ag-recognition) domains are still foreign; HAMA often still occurs neutralizing efficacy.

42. Generation of chimeric mouse-human mAb

43. Antibodies can be engineered to reduce their immunogenicity in humans
humanized
chimaetic
Red: mouse
White: human

44. Chimeric Antibodies

45. Humanized mAbs
All portions of the mAb not required for antigen binding, including framework residues in the variable region, are replaced with human sequences.
Gene engineering, expression, evaluation
< 10% of optimized mAb retains mouse sequence
Highly-effective (but time-consuming) approach

46. 抗體分子與專一性抗原之交互作用 局部區域的高度變異序列形成抗原結合位 Hypervariable region
Framework region
CDR (complementarity-determining region)

47. Humanized mAb
CDR: complementary determination region

48. Antibodies can be engineered to reduce their immunogenicity in humans
humanized
chimaetic
Red: mouse
White: human

49. Modification of mouse mAb

50. Immunotoxin: magic bullet

51. Antibodies in action

52. MAT in Organ Transplantation
Rejection of allografts is an immunological response exerted primarily by T-cells. mAb approaches against T-cell antigens has proven effective.
CD25 (IL2-Receptor a chain). Highly effective approach for the prevention of graft rejection episodes and graft loss (e.g., renal).
basiliximab (chimeric)
dacliximab (humanized)

53. MAT in Coronary Artery Disease
Atherosclerotic lesions (plaques) involve platelet aggregation followed by coagulation and thrombus formation. Platelet receptors involved in early steps of plaque formation.
basis of “one-aspirin-a-day” concept
limited efficacy in preventing platelet aggregation
Abciximab (chimeric mAb) has been developed/approved as an effective treatment for the prevention of thrombosis and plaque formation in high risk patients.
Targets the platelet receptors (integrins)
Prevents platelet aggregation

54. MAT in Autoimmune Diseases
TNFa is a proinflammatory cytokine that has been implicated as a causal factor in a variety of inflammatory diseases including Rheumatoid Arthritis.
Infliximab is a chimeric mAb targeted to TNFa. has displayed remarkable activity against Rheumatoid Arthritis and other inflammatory diseases (Crohn’s Disease).
Approved in First for Crohn’s Disease; now R.A.

55. Antibodies can be used to alleviate and suppress autoimmune disease
Anti-inflammatory effects of anti-TNF-a therapy in rheumatoid arthritis

56. MAT in Cancer (I)
Most intensively studied area (25 years of research).
Cancer poses the highest hurdles.
Also being evaluated in modified versions involving the conjugation to radio-ionizing particles, immunotoxins, immunoliposomes.

57. MAT in Cancer (II) Rituximab (chimeric mAb)
Targets CD20; a cell surface protein expressed exclusively on B cells.
Approved for the treatment of B cell malignancies (NHL, CLL, ALL).
Trastuzumab (humanized mAb)
Her2 is a form of the EGF-Receptor that undergoes amplification in approximately 25% of breast cancers.
1st identified as a rat oncogene called neu (HER2/neu)
Patients with amplified HER2 expression suffer a very poor prognosis.
Trastuzumab (Herceptin) has displayed impressive activity against HER2+ breast cancer both alone and in combination.

58. Summary
Antibodies are universal adaptors which can bind to any specific target both in vitro and in vivo.
Humanized monoclonal antibody reduce its immunogenecity without change its specificity.
MAT provides another way to develop new therapy against cancer, autoimmune disease…etc. (serum therapy)

59. New approaches under development
Phage-display libraries expressing human VH and VL chains are assembled into libraries and expressed as combinatorial matrices.
Assayed for immunoreactivity against antigen of interest.
Transgenic Mouse Approach
A procedure in which the endogenous mouse Ig gene loci is replaced by homologous recombination with their human homologs.
Immunization of transgenic mouse followed by standard hybridoma procedures produces a fully humanized mAb.

61. Advantages of phage display
More efficiently than through conventional 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 108 to 109 members) of functional antibodies to generate a larger repertoire of antibodies than those available through conventional hybridoma technology.
Easy isolation and expression of the cloned gene in a bacterial host.
Excellent potential to further improve binding properties of the selected antibody by protein engineering techniques.

62. PNAS 101:2536, 2004 Feb.

63. Selection of phage library and screening of phage antibodies
Two human nonimmune single-chain variable region fragment (scFv) libraries (a total of 2.7x1010 members) constructed from B cells of 57 unimmunized donors were used for selection of scFvs against the purified S1-C9

64. Generation human mAb by transgenic mice