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Immunoglobulins: s tructure and f unction. Immunoglobulins. Definition : Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (antibodies) in response to an immunogen. Immunoglobulin Structure. h eavy and l ight c hains d isulfide bonds

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Immunoglobulins: s tructure and f unction


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slide2

Immunoglobulins

Definition:

Glycoprotein molecules that are

present on B cells (BCR) or produced by

plasma cells (antibodies) in response

to an immunogen

immunoglobulin structure
Immunoglobulin Structure
  • heavy andlight chains
  • disulfide bonds
    • inter-chain
    • intra-chain

disulfide bond

carbohydrate

CL

VL

CH2

CH3

CH1

hinge region

VH

immunoglobulin structure1

disulfide bond

carbohydrate

CL

CH2

CH3

CH1

hinge region

VH

Immunoglobulin Structure
  • variable andconstant regions
  • hinge region
  • domains
    • VL & CL
    • VH & CH1 - CH3 (or CH4)
  • oligosaccharides

VL

slide7

antigenbinding

complement binding site

binding to Fc receptors

placental transfer

Immunoglobulin Fragments: Structure/Function Relationships

immunoglobulin fragments structure function relationships

papain

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

Fc

Fab

immunoglobulin fragments structure function relationships1

pepsin

Fc peptides

F(ab’)2

Immunoglobulin Fragments: Structure/Function Relationships
  • Fab
    • antigen binding
  • Fc
    • effector functions
  • F(ab’)2

- Bivalent!

slide13

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.

slide14

the (Fab)2 fragment can -

  • detect antigen
  • precipitate antigen
  • block the active sites of toxins or pathogen-associated molecules
  • block interactions between host and pathogen-associated molecules

but can not activate (role of Fc region)

  • inflammatory and effector functions associated with cells
  • inflammatory and effector functions of complement
  • the trafficking of antigens into the antigen processing pathways

Why do antibodies need an Fc region?

slide15

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

Sequence variability of H and L-chain variable regions (individual, clone- specific)

Allelic variants

human immunoglobulin classes encoded by different structural gene segments isotypes
Human Immunoglobulin Classesencoded 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)
slide19

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

slide21

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

IgA and pIgR are transported to the apical surface in vesicles

s

s

s

s

s

s

s

s

s

s

C

C

C

C

C

J

J

J

J

J

C

C

C

C

C

S

S

S

S

S

S

S

S

S

S

C

C

C

C

C

C

C

C

C

C

pIgR and IgA are

internalised

S

S

S

S

S

S

S

S

S

S

Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa

C

C

C

C

C

C

C

C

C

C

B

B cells located in the submucosa

produce dimeric IgA

Secretory IgA and transcytosis

‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component

Epithelial

cell

slide22

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 and B

Antigen A

slide23

Antibody production during

the primary and the secondary immune response

slide24

EFFECTOR FUNCTION OF ANTIBODIES

Neutralization

OpsonizationfollowedbyPhagocytosis

ADCC

Activation of complement (discussedlater)

slide29

MONOCLONAL ANTIBODIES

versus

POLYCLONAL ANTIBODIES

slide30

Ag

Ag

Ag

Polyclonal antibody response

Polyclonal antibody

Immunserum

Set of B-cells

Activated B-cells

Antibody-producing plasma-cells

Antigen-specific antibodies

slide32

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
slide34

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
monoclonal antibodies as drugs
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?

evolution of monoclonal antibodies

Human

Mouse

Humanized

Evolution of monoclonal antibodies

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.

slide37

mouse monoclonal antibodies

immunization

humanized mouse monoclonal antibodies

immunization

Human immunoglobulin transgenic mouse

human monoclonal antibodies

ENDANGERED SUBJECT

PASSZÍV IMMUNIZÁLÁS

PROTECTED SUBJECT

serum antibody

This is a case of PASSIVE IMMUNIZATION

Immunesystemis notactivated

prompt effect

temporaryprotection/effect

Immunoglobulindegradation

monoclonals as drugs
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

monoclonals in tumor therapy
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)
immunsuppressive antibodies 1
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!)
slide42

Casestudy (Multiplemyeloma)

  • 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 4.2-5.0 x 106 / ml),
  • white blood cell count was 3600 / 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 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).
slide43

What happens with the B cells in myeloma multiplex?

Healthy individual

Myeloma multiplex

slide44

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.
slide45

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.
slide46

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 complainedat 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 tocure 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)/
slide47

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.

slide48

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.

slide51

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

slide52

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
slide54

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