Immunoglobulins:

structure and function

ImmunoglobulinsImmunoglobulins

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

Immunoglobulin StructureImmunoglobulin Structure

• heavy and light chains

• disulfide bonds– inter-chain– intra-chain

hinge region

carbohydrate

disulfide bond

CH1

VL

CL

VH

CH2 CH3

Immunoglobulin StructureImmunoglobulin Structure

• variable and constant regions

• hinge region

• domains– VL & CL

– VH & CH1 - CH3 (or CH4)

• oligosaccharides

hinge region

carbohydrate

disulfide bond

CH1

CL

VH

CH2 CH3

VL

Ribbon structure of IgG

mIg = BCRmIg = BCR

Immunoglobulin Fragments: Structure/Function Relationships

antigen binding

complement binding site

placental transfer

binding to Fc receptors

ANTIGEN BINDINGANTIGEN BINDING

Hypervariable regionsHypervariable regions

Immunoglobulin Fragments Structure/Function Relationships

• Fab– antigen binding– valence = 1– specificity determined

by VH and VL

papain

Fc

Fab

• Fc– effector functions

Immunoglobulin Fragments: Structure/Function Relationships

• Fab– antigen binding

• Fc– effector functions

• F(ab’)2

- Bivalent!

pepsin

Fc peptides

F(ab’)2

BINDING TO Fc RECEPTORSBINDING TO Fc RECEPTORS

(A) (A) High-affinity FcRsHigh-affinity FcRs on the surface of the cell bind monomeric Ig before it binds to antigen.

(B) Low-affinity FcRsLow-affinity FcRs bind multiple Igs that have already bound to a multivalent antigen.

complex antigen

antibodies complexed to complex antigen

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)

(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 idiotypeallotype

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)

Ig isotype Serum concentration

Characteristics, functions

12-14 mg/ml

Major isotype of secondary (memory) immune response

Complexed with antigen activates effector functions (Fc-receptor binding, complement activation

Trace

amounts

The first isotype in B-lymphocyte membrane

Function in serum is not known

Trace amounts

Major isotype in protection against parasites

Mediator of allergic reactions (binds to basophils and mast cells)

3-3,5 mg/ml

Major isotype of secretions (saliva, tear, milk)

Protection of mucosal surfaces

1-2 mg/ml

Major isotype of primary immune responses

Complexed with antigen activates complement

Agglutinates microbes The monomeric form is expressed in

B-lymphocyte membrane as antigen binding receptor

MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 1.MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 1.

MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 2.MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 2.

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

IgG

IgM

IgA

A F T E R B IR T H

breas t milkIgA

0

1 0 0 %( a d u l t )

3 3y e a r

2 546 a d u l t9 1m o n t h

maternal IgG

B E F O R E B IR T H

PRODUCTION OF IMMUNOGLOBULINS

Epithelialcell

JC C

SS

SS

C

C

SS

SS

CC

ss

Secretory IgA and transcytosis

B

JC C

SS

SS

CC

SS

SS

CCss

JC C

SS

SS

C

C

SS

SS

CC

ss

JC C

SS

SS

C

C

SS

SS

CC

ss

pIgR and IgA areinternalised

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

JC C

SS

SS

C

C

SS

SS

CC

ss

IgA and pIgR are transported to the apical surface in vesicles

B cells located in the submucosaproduce dimeric IgA

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

Antibody production during Antibody production during the primary and the secondary immune responsethe primary and the secondary immune response

Ig . C onc entra tion

na p o k

p rim er response

„A” a ntig né

IgM

IgGIgAIgE

Szekund er ’la syec ond a ry response

„A” és a ntig én

„B”

5 10 15 20 25 30

IgM

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

EFFECTOR FUNCTION OF ANTIBODIESEFFECTOR FUNCTION OF ANTIBODIES

1) Neutralization2) Opsonization followed by Phagocytosis3) ADCC4) Activation of complement (discussed later)

OPSONIZATIONOPSONIZATION

NEUTRALIZATIONNEUTRALIZATION

NK CELL DEGRANULATIONNK CELL DEGRANULATION

Antibody Dependent Cellular Cytotoxicity (ADCC)Antibody Dependent Cellular Cytotoxicity (ADCC)

MAST CELL DEGRANULATIONMAST CELL DEGRANULATION

MONOCLONAL ANTIBODIESMONOCLONAL ANTIBODIES

versus versus

POLYCLONAL ANTIBODIESPOLYCLONAL ANTIBODIES

Polyclonal antibody response Polyclonal antibody response

Ag

ImmunserumPolyclonal antibody

Ag

Ag

Set of B-cells

Activated B-cells Antibody-producing

plasma-cellsAntigen-specific

antibodies

Monoclonal antibodiesMonoclonal 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

Polyclonal antibody Monoclonal antibody(high affinity)

Number of recognized antigen determinants

several (frequent cross-reactions)

mostly one

Specificity polyspecific monospecific

Affinity Varying (diverse antibodies)

high

Concentration of non-specific immunoglobulines

high low

Cost of preparation low high

Standardisability Impossible (or uneasy) easy

Amount limited unlimited

Applicability method-dependent excellent

Features of polyclonal and monoclonal antibodiesFeatures of polyclonal and monoclonal antibodies

Possible use of monoclonal antibodiesPossible 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 antibodiesEvolution 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.

PROTECTED SUBJECT

serum antibody

PASSZÍV IMMUNIZÁLÁS

This is a case of PASSIVE IMMUNIZATION

Immune system is not activatedprompt effect

temporary protection/effect

Immunoglobulin degradation

Human immunoglobulin transgenic mouse

immunization mouse monoclonal antibodies

ENDANGERED SUBJECT

immunization

human monoclonal antibodies

humanized mouse monoclonal antibodies

- Tumor therapyTumor therapy

Monoclonals can be used for targeted chemotherapy of tumors. It is cell-type specific, but not specific to malignant cells!

- Immunsuppressive monoclonalsImmunsuppressive monoclonals

Cell-type specific immunsuppression

Monoclonals as drugsMonoclonals as drugs

Monoclonals in tumor therapyMonoclonals in tumor therapy

1. „Naked MAb”, unconjugated antibodyAnti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphomaAnti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemiaAnti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancerAnti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!)Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. humán!)

2. Conjugated antibodyAnti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin)Anti-CD20 + iodine-131 (tositumomab – Bexxar)

Immunsuppressive antibodies 1.Immunsuppressive antibodies 1.

1. Anti-TNF-α antibodiesinfliximab (Remicade): since 1998, chimericadalimumab (Humira): since 2002, recombinant human

2. Etanercept (Enbrel) – dimer fusion protein,TNF-α receptor + Ig Fc-partNot 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!)

Passive immunization Passive immunization

TypeType ApplicationApplication

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;

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 / l (normal 4.2-5.0 x 106 / 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 plasmais 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).

Myeloma multiplexHealthy individual

What happens with the B cells in myeloma multiplex?

• Electrophoresis of her serum revealed the presence of a monoclonal protein, whichon 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)

• 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 / 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 inthe bones.

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 to6000 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 outlookfor 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)

She became anemic (low red blood cell count) and neutropenic (low white bloodcell count). What was the cause of this?

The proliferation of malignant plasma cells in the bone marrow crowded outblood cell precursors. This creates a limitation on space in the bone marrow.

Questions

As her disease progressed, she became susceptible to pyogenic infection;for example, she had pneumonia twice in a short period. What is the basis ofher susceptibility to these infections?

Although her serum IgG concentration is quite elevated, almost all the IgG issecreted by the myeloma cells and is monoclonal. In fact, she has very little normal polyclonal IgG and has been effectively rendered agammaglobulinemicby 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 multiplemyeloma?

No. M-component appear in the blood as people age. About 10% of healthyindividuals in the ninth decade of live have M-component. This is called benignmonoclonal 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.

B cell tumors

Supplementary informationSupplementary information

Monoclonal antibody nomenclatureMonoclonal 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:

Prefix Target Source Suffix

-ki(n)- interleukin as target -u-human

-ci(r)- cardiovascular -o-mouse

-co(l)- colonic tumor -xi-chimeric

variable

-neu(r)- nervous system Etc.

-zu-humanized Etc.

mab

Example:Abciximabab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody used on the cardiovascular system

- Muromonab-CD3 (OKT-3) egér IgG2aAgainst 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 monoclonalInd.: allergic asthma, Churg-Strauss sy.

- Daclizumab (Zenapax):anti-IL-2 receptor humanized antibodyInd.: transplantation

- basiliximab (Simulect): as daclizumab, but chimeric!

- efalizumab (Raptiva): anti-CD11a, humanized, used in psoriasis

Immunsuppressive antibodies 2.Immunsuppressive antibodies 2.

Molecular targeted drugsName 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, chimeric

Monoclonal IgG1, chimeric

Monoclonal IgG1, humanized

Monoclonal IgG4, humanizedCalicheamicinnel konjugált

Monoclonal IgG1, murine

Monoclonal IgG2, murine

EGFR-TKI

KIT-TKI

CD52

IL-2 R

IL-2 R

CD20

HER2/neu

CD33

CD20

EpCAM

EGFR TK

TK

CLL, CML

transplantation

transplantation

Lymphoma

Breast cancer, NSC lung cancer

leukemia

lymphoma

CRC

NSCLC

GIST, CML

RadioimmunotherapyAs 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

ImmunoliposomesTargeting nucleotides or drugs in liposomes, linked to an antibody

Non-immunological targetsas abciximab (ReoPro): inhibition of thrombocyte-aggregation

Further possibilities with monoclonalsFurther possibilities with monoclonals