1. Immunoglobulins: structure and function

2. Immunoglobulins Definition Definition : Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (antibodies) in response to an immunogen

3. Immunoglobulin Structure heavy and light chains disulfide bonds –inter-chain –intra-chain hinge region carbohydrate disulfide bond C H1 VLVL CLCL VHVH C H2 C H3

4. Immunoglobulin Structure variable and constant regions hinge region domains –V L & C L –V H & C H1 - C H3 (or C H4 ) oligosaccharides hinge region carbohydrate disulfide bond C H1 CLCL VHVH C H2 C H3 VLVL

5. Ribbon structure of IgG

6. mIg = BCR

7. Immunoglobulin Fragments: Structure/Function Relationships antigen binding complement binding site placental transfer binding to Fc receptors

8. ANTIGEN BINDING

9. Hypervariable regions

10. Immunoglobulin Fragments Structure/Function Relationships Fab –antigen binding –valence = 1 –specificity determined by V H and V L papain Fc Fab Fc –effector functions

11. Immunoglobulin Fragments: Structure/Function Relationships Fab –antigen binding Fc –effector functions F(ab’) 2 - Bivalent! pepsin Fc peptides F(ab’) 2

12. BINDING TO Fc RECEPTORS

13. (A) High-affinity FcRs (A) High-affinity FcRs on the surface of the cell bind monomeric Ig before it binds to antigen. Low-affinity FcRs (B) Low-affinity FcRs bind multiple Igs that have already bound to a multivalent antigen. complex antigen antibodies complexed to complex antigen

14. Why do antibodies need an Fc region? detect antigen precipitate antigen block the active sites of toxins or pathogen-associated molecules block interactions between host and pathogen-associated molecules the (Fab) 2 fragment can - inflammatory and effector functions associated with cells inflammatory and effector functions of complement the trafficking of antigens into the antigen processing pathways but can not activate (role of Fc region)

15. (Classes/subclasses) Sequence variability of H/L- chain constant regions Sequence variability of H and L-chain variable regions (individual, clone- specific) Allelic variants Variability in different regions of the Ig determines Ig classes or specificity isotype idiotype allotype

16. Human Immunoglobulin Classes encoded by different structural gene segments (isotypes) IgG - gamma (γ) heavy chains IgM - mu ( μ ) heavy chains IgA - alpha ( α ) heavy chains IgD - delta ( δ ) heavy chains IgE - epsilon (ε) heavy chains light chain types kappa (κ) lambda (λ)

17. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 1.

18. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 2.

19. Features of antibody-antigen interaction Valency: numbers of antigens / antibody Affinity: the strength of interaction between a specific antigen and one binding site of the antibody Avidity: sum of affinities of the binding sites of a a given antibody

20. PRODUCTION OF IMMUNOGLOBULINS

21. Epithelial cell J C C S S S S C C S S S S C C ss Secretory IgA and transcytosis B J C C S S S S C C S S S S C C ss J C C S S S S C C S S S S C C ss J C C S S S S C C S S S S C C ss pIgR and IgA are internalised ‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component J C C S S S S C C S S S S C C ss IgA and pIgR are transported to the apical surface in vesicles B cells located in the submucosa produce dimeric IgA Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa

22. secondary response against antigen A primary response against antigen A level of antibodies napok primary response against antigen B Antigen A days Antigen A and B Antibody production during the primary and the secondary immune response

23. Antibody production during the primary and the secondary immune response

24. EFFECTOR FUNCTION OF ANTIBODIES 1)Neutralization 2)Opsonization followed by Phagocytosis 3)ADCC 4)Activation of complement (discussed later)

25. OPSONIZATION

26. NEUTRALIZATION

27. NK CELL DEGRANULATION Antibody Dependent Cellular Cytotoxicity (ADCC)

28. MAST CELL DEGRANULATION

29. MONOCLONAL ANTIBODIES versus POLYCLONAL ANTIBODIES

30. Polyclonal antibody response Ag Immunserum Polyclonal antibody Ag Set of B-cells Activated B-cells Antibody- producing plasma-cells Antigen-specific antibodies

32. Monoclonal antibodies - product of one B-lymphocyte clone - homogeneous in antigenspecificity, affinity, and isotype - can be found in pathologic condition in humans (the product of a malignant cell clone) - advantages against polyclonal antibodies: antibodies of a given specificity and isotype can be produced in high quantity and assured quality. -therapeutic usage of monoclonals: anti-TNF-α therapy in rheumatology, tumor therapy

33. Polyclonal antibodyMonoclonal antibody Number of recognized antigen determinants several (frequent cross- reactions) mostly one Specificitypolyspecificmonospecific AffinityVarying (diverse antibodies) high Concentration of non- specific immunoglobulines highlow Cost of preparationlowhigh StandardizationImpossible (or uneasy)easy Amountlimitedunlimited Applicabilitymethod-dependentexcellent Features of polyclonal and monoclonal antibodies

34. Possible use of monoclonal antibodies - Identifying cell types Immunohistochemistry Characterization of lymphomas with CD (cluster of differentiation) markers - Isolation of cells Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral blood!) - Blood group determination (with anti-A, anti-B, and anti-D monoclonals) - Identification of cell surface and intracellular antigens Cell activation state - Targeted chemotherapy CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma Prevention of organ rejection after transplantation

35. Monoclonal antibodies as drugs? Mouse monoclonal antibodies may elicit an immune response upon administration in human subjects. (see immunogenicity-determining factors!) How can we solve this problem?

36. Evolution of monoclonal antibodies Mouse Chimeric Human Humanized Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

37. PROTECTED SUBJECT serum antibody PASSZÍV IMMUNIZÁLÁS This is a case of PASSIVE IMMUNIZATION Immune system is not activated prompt effect temporary protection/effect Immunoglobulin degradation Human immunoglobulin transgenic mouse immunization mouse monoclonal antibodies ENDANGERED SUBJECT immunization human monoclonal antibodies humanized mouse monoclonal antibodies

38. Tumor therapy - Tumor therapy Monoclonals can be used for targeted chemotherapy of tumors. It is cell-type specific, but not specific to malignant cells! -Immunsuppressive monoclonals Cell-type specific immunsuppression Monoclonals as drugs

39. Monoclonals in tumor therapy 1.„Naked MAb”, unconjugated antibody Anti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphoma Anti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemia Anti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancer Anti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!) Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. humán!) 2.Conjugated antibody Anti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin) Anti-CD20 + iodine-131 (tositumomab – Bexxar)

40. Immunsuppressive antibodies 1. 1.Anti-TNF-α antibodies infliximab (Remicade): since 1998, chimeric adalimumab (Humira): since 2002, recombinant human 2.Etanercept (Enbrel) – dimer fusion protein, TNF-α receptor + Ig Fc-part Not a real monoclonal antibody, no Fab end, the specificity is given by TNF-receptor! Indications of anti-TNF-α therapy: Rheumatoid arthritis Spondylitis ankylopoetica (SPA - M. Bechterew) Psoriasis vulgaris, arthritis psoriatica Crohn-disease, colitis ulcerosa (usually - still – not in the first line!)

41. Passive immunization TypeApplication Intramuscular (less effective due to lower dose) HBV-Ig; Varicella-zoster-Ig; Intravenous (IVIG)Bruton-agammaglobulinaemia; variable and mixed immunodeficiencies with hypogammaglobulinaemia; Anti-venom antibody treatment;

42. Case study (Multiple myeloma) In 1989, a 55-year-old housewife, who had been in good health her entire life, began to experience excessive fatigue. Her physician did not find abnormalities on physical examination. The blood sample revealed mild anemia; red blood cell count was 3.5 x 10 6 /  l(normal 4.2-5.0 x 10 6 /  l), white blood cell count was 3600 /  l (normal 5000 /  l). The sedimentation rate of her red blood cells was 32 mm / h (normal <20 mm / h). (Sedimentation is accelerated when fibrinogen or IgG content of the blood plasma is elevated.) The concentration of IgG was found to be 3790 mg / dl (normal 600 - 1500 mg / dl), that of IgA 14 mg / dl (normal 150 – 250 mg / dl) and that of IgM 53 mg / dl (normal 75 -150 mg / dl).

43. Myeloma multiplexHealthy individual What happens with the B cells in myeloma multiplex?

44. Electrophoresis of her serum revealed the presence of a monoclonal protein, which on further analysis was found to be IgG with lambda light chains. normal serum serum from the patient Radiographs of all of her bones did not show any abnormality. No treatment was advised. Case study (Multiple myeloma)

45. In April 1991 her serum IgG was 4520 mg / dl, and in January 1992 it was 5100 mg / dl. By November 1992, her anemia had worsened and her red blood cell count had fallen to 3.0 x 10 6 /  l. At the same time her white blood count had fallen to 2600 /  l. In December 1992, she experienced the sudden onset of upper arm pain and headache. Radiographs of the skull and the left upper arm showed ‘punched out’ lesions in the bones. Case study (Multiple myeloma)

46. She was treated with melphalan (methylphenylalanine mustard), corticosteroids, and irradiation. Her symptoms improved. In April 1993, further chemotherapy was given because of the persisting elevation of her serum IgG. The treatment reduced her serum IgG level from 8200 mg / dl to 6000 mg / dl. In February and in May 1995, she was found to have pneumonia. She was treated successfully with antibiotics. She recovered from this episode in the hospital and remained fully active. She required blood transfusion for her anemia and complained at times of bone pain. Her serum IgG was stable at 6200 mg / dl. Although she was in relative good health as our case history ended, her outlook for survival was very poor. Recently, bone marrow transplants have been used to cure patients with multiple myeloma. /Myeloma proteins have played an important part in the history of immunology. (Bence-Jones protein, subclasses of IgG, amino acid sequence of immuno- globulin molecule)/ Case study (Multiple myeloma)

47. She became anemic (low red blood cell count) and neutropenic (low white blood cell count). What was the cause of this? The proliferation of malignant plasma cells in the bone marrow crowded out blood cell precursors. This creates a limitation on space in the bone marrow. Questions

48. As her disease progressed, she became susceptible to pyogenic infection; for example, she had pneumonia twice in a short period. What is the basis of her susceptibility to these infections? Although her serum IgG concentration is quite elevated, almost all the IgG is secreted by the myeloma cells and is monoclonal. In fact, she has very little normal polyclonal IgG and has been effectively rendered agammaglobulinemic by her disease. In addition, her white blood cell count is decreased and she has too few neutrophils (<1000 /  l) to ingest bacteria in the bloodstream and lungs effectively. A monoclonal immunoglobulin in the serum is called an M-component (‘M’ for myeloma). Is the presence of an M-component in serum diagnostic of multiple myeloma? No. M-component appear in the blood as people age. About 10% of healthy individuals in the ninth decade of live have M-component. This is called benign monoclonal gammopathy. Without bone lesions and presence of many malignant cells in the bone marrow, the diagnosis of multiple myeloma cannot be made. Some people have IgM M-components in their blood. This is due to another malignancy of plasma cells called Waldenström’s macroglobulinemia, which differs in many ways from multiple myeloma and is a more benign disease.

49. B cell tumors

50. Supplementary information

51. Monoclonal antibody nomenclature The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary names to a group of medicines called monoclonal antibodies. This scheme is used for the World Health Organization’s International Nonproprietary Names. Components of nomenclature: Example:Abciximab  ab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody used on the cardiovascular systemAbciximab

52. -Muromonab-CD3 (OKT-3) egér IgG2a Against CD3 pan-T-cell antigen, after transplantation; It is rarely (or not) used nowadays (mouse protein!); ongoing trials in diabetes mellitus, with the humanized version -Omalizumab (Xolair): Anti-IgE humanized IgG1k monoclonal Ind.: allergic asthma, Churg-Strauss sy. -Daclizumab (Zenapax): anti-IL-2 receptor humanized antibody Ind.: transplantation -basiliximab (Simulect): as daclizumab, but chimeric! -efalizumab (Raptiva): anti-CD11a, humanized, used in psoriasis Immunsuppressive antibodies 2.

53. Molecular targeted drugs NameTypeTargetIndications Alemtuzumab (Mabcampath) Daclizumab (Zenapax) Basiliximab (Simulect) Rituximab (Rituxan/Mabthera) Trastuzumab (Herceptin) Gemtuzumab Ibritumomab (Y 90 ) Edrecolomab Gefitinib Imatinib Monoclonal Ab, humanized Monoclonal IgG1, chimeric Monoclonal IgG1, humanized Monoclonal IgG4, humanized Calicheamicinnel konjugált Monoclonal IgG1, murine Monoclonal IgG2, murine EGFR-TKI KIT-TKI CD52 IL-2 R CD20 HER2/neu CD33 CD20 EpCAM EGFR TK TK CLL, CML transplantation Lymphoma Breast cancer, NSC lung cancer leukemia lymphoma CRC NSCLC GIST, CML

54. Radioimmunotherapy As Zevalin, Bexxar – monoclonal + isotope Antibody-directed enzyme prodrug therapy (ADEPT) An enzyme is linked to the antibody, and the enzyme will make citotoxic drug from the later administered prodrug Immunoliposomes Targeting nucleotides or drugs in liposomes, linked to an antibody Non-immunological targets as abciximab (ReoPro): inhibition of thrombocyte-aggregation Further possibilities with monoclonals