1. Immunoglobulins: structure and function

2. Immunoglobulins 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
disulfide bond
carbohydrate CL VL
CH2
CH3
CH1 VH
hinge region

4. Immunoglobulin Structure
variable and constant regions
hinge region
hinge region
carbohydrate
disulfide bond
CH1 CL VH
CH2
CH3
domains
VL & CL
VH & CH1 - CH (or CH4)
oligosaccharides VL

5. Ribbon structure of IgG

6. mIg = BCR

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

8. ANTIGEN BINDING

9. Hypervariable regions

10. Complementary Determining Region (CDR)

11. X-Ray Crystallography of Fab Lysozyme Complex.
X-Ray Crystallography of Fab Lysozyme Complex. Top panel: Anti-lysozyme Fab (light chain in yellow, heavy chain in blue) binds to lysozyme (green). A critical residue of lysozyme, Gln 121, is shown in red. Center panel: Computer-generated image of separated components. Bottom panel: Rotation of separated components to show the contact residues in red (16 in each case) involved in Fab/lysozyme binding. Gln 121 (white) projects slightly from the lysozyme contact area, but is accommodated by a small cleft formed by the CDRs on the Fab binding surface. Reproduced with permission from Amit A. G. et al. (1986) Three-dimensional structure of an antigen–antibody complex at 2.8 angstroms resolution. Science 233, 747–753.
Rotation of separated components
to show the contact residues in red
(16 in each case) involved in Fab/lysozyme binding.

12. BINDING TO Fc RECEPTORS

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

14. Immunoglobulin Fragments Structure/Function Relationships
Fab
antigen binding
valence = 1
specificity determined by VH and VL
papain Fc
effector functions Fc
Fab

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

16. 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)

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

18. 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)

19. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 1.

20. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 2.

21. 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

22. PRODUCTION OF IMMUNOGLOBULINS

23. Pathological consequences of placental transport of IgG
(hemolytic disease of the newborn)
Passive anti-D IgG
anti-Rh
IgM

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

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

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

27. Isotype Switching during B Cell Development
Isotype Switching during B Cell Development During the initial stages of a B cell’s primary response to antigen, it produces and secretes IgM. Later in the primary response or during subsequent responses, different heavy chain isotypes may be expressed by the progeny of the original IgM-producing clone. Such “switching” occurs at the DNA level, resulting in the production of an Ig protein with the same V region but a different C region. Thus, over the lifetime of a B cell clone, it may produce antibodies of the same specificity but different isotypes.

28. EFFECTOR FUNCTION OF ANTIBODIES

29. OPSONIZATION

30. NEUTRALIZATION

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

32. MAST CELL DEGRANULATION

33. MONOCLONAL ANTIBODIES POLYCLONAL ANTIBODIES
versus
POLYCLONAL ANTIBODIES

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

36. 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

37. Features of polyclonal and monoclonal antibodies
Polyclonal antibody
Monoclonal antibody
(low affinity)
(high affinity)
Number of recognized antigen determinants
several (frequent cross-reactions)
one (but frequent cross-reactions)
mostly one
Specificity
polyspecific
often polyspecific
monospecific
Affinity
Varying (diverse antibodies)
low
high
Concentration of non-specific immunoglobulines
Cost of preparation
Standardisability
Impossible (or uneasy)
easy
Amount
limited
unlimited
Applicability
method-dependent
excellent

38. 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

39. 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?

40. Evolution of monoclonal antibodies
Mouse
Human
Humanized
Chimeric
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.The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anti-cancer drugs). Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice). The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients
Humanized antibodies are distinct from chimeric antibodies. The latter also have their protein sequences made more similar to human antibodies, but the fraction of foreign protein is larger than in humanized ones.
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.

41. humanized mouse monoclonal antibodies human monoclonal antibodies
PASSZÍV IMMUNIZÁLÁS
immunization
mouse monoclonal antibodies
Human immunoglobulin transgenic mouse
humanized mouse monoclonal antibodies
immunization
human monoclonal antibodies
PROTECTED SUBJECT
serum antibody
ENDANGERED SUBJECT
immunsystem is not activated
prompt effect
temporary protection/effect
Immunoglobulin degradation

42. Monoclonals as drugs - 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

43. Monoclonals in tumor therapy
„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)

44. Immunsuppressive antibodies 1.
Anti-TNF-α antibodies
infliximab (Remicade): since 1998, chimeric
adalimumab (Humira): since 2002, recombinant human
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!)

45. Passive immunization Type Application 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;

46. 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 106 / ml (normal x 106 / ml),
white blood cell count was / ml (normal 5000 / ml).
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 mg / dl),
that of IgA 14 mg / dl (normal 150 – 250 mg / dl) and that of IgM 53 mg / dl
(normal mg / dl).

47. Case study (Multiple myeloma)
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.

48. Case study (Multiple myeloma)
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 106 / ml. At the same time her white blood count had
fallen to 2600 / ml.
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.

49. Case study (Multiple myeloma)
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)/

50. Questions
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.

51. 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 / ml) 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.

52. What happens with the B cells in myeloma multiplex?
Healthy individual
Myeloma multiplex

53. B cell tumors

54. Supplementary information

55. 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 system

56. Immunsuppressive antibodies 2.
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

57. Molecular targeted drugs
Name
Type
Target
Indications
Alemtuzumab
(Mabcampath)
Daclizumab
(Zenapax)
Basiliximab
(Simulect)
Rituximab
(Rituxan/Mabthera)
Trastuzumab
(Herceptin)
Gemtuzumab
Ibritumomab (Y90)
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
EpCAM
EGFR TK TK
CLL, CML
transplantation
Lymphoma
Breast cancer, NSC lung cancer
leukemia
lymphoma
CRC
NSCLC
GIST, CML

58. Further possibilities with monoclonals
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