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IMMUNGLOBULINS
STRUCTURE and FUNCTION
Immunoglobulin structure
Valency, affinity, avidity
Direct and indirect functions
Immunoglobulin types
Fc receptors
Immunoglobulin transport
Monoclonal and polyclonal antibodies
Passive immunization
Therapeutic antibodies
Chimera and humanized monoclonal Ab
Tumortherapy, Immunsuppression
Case study (mieloma multiplex)
B cell malignancies
More and more therapeutic monoclonal antibodies
CARs and TRUCKs
IMMUNOGLOBULINS
Definition:
Glycoprotein molecules that are
present on B cells (BCR) or produced by
plasma cells (antibodies) in response
to an immunogen
immunoglobulin ~ antibody
STRUCTURE
• heavy and light
chains
• disulfide bonds
– inter-chain
– intra-chain
disulfide
bond
carbohydrate
CL
VL
CH1
VH
CH2
hinge region
CH3
STRUCTURE
• variable and
constant regions
• hinge region
immunoglobulin domen
disulfide
bond
carbohydrate
• domains
– VL & CL
– VH & CH1 - CH3
(or CH4)
• oligosaccharides
CL
VL
CH1
VH
CH2
hinge region
CH3
Ribbon structure of IgG
mIg = BCR
Hypervariable region - Complementary Determining Region (CDR)
3 CDR regions in a V domain
VH & VL domains 3+3 CDR
(the circled part from the previous slide)
IMMUNOGLOBULIN FRAGMENTS
STRUCTURE/FUNCTION RELATIONSHIPS
antigen
binding
complement binding site
placental
transfer
binding to Fc
receptors
IMMUNOGLOBULIN FRAGMENTS
STRUCTURE/FUNCTION RELATIONSHIPS
papain
• Fab
– antigen binding (valence=1,
monovalent)
– specificity determined by
VH and VL domains
– some effector function is
impaired
VH
VL
Fc
• Fc
(effector functions)
Fab
IMMUNOGLOBULIN FRAGMENTS
STRUCTURE/FUNCTION RELATIONSHIPS
pepsin
• F(ab’)2
+
– bivalent antigen
binding (valency=2)
F(ab’)2
Some effektor functin is impaired.
It can mediate agglutination/precipitation (other seminar)
Fc
peptides
MODEL OF ANTIGEN BINDING
EFFECTOR FUNCTION:
NEUTRALIZATION
neutralized pathogen / toxin
toxin effect or pathogen adhesion is blocked by steric hindrance
Why do antibodies need an Fc region?
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 associated with
complement system
•
the trafficking of antigens into the antigen processing
pathways (opsonized phagocytosis)
NEUTRALIZATION
•
EFFECTOR FUNCTIONS OF
ANTIBODIES
1. Neutralization
(variable domain associated)
2. Marking the antigens for effector functions
(variáble domain and Fc dependent)
•
•
•
phagocytosis (oppsonization)
ADCC and IgE mediated mast cell activation
activation of the complement system (next seminar)
Fc part is recognized by Fc receptors
Different antibodies have different Fc part
VARIABILITY IN DIFFERENT REGIONS OF THE IG
DETERMINES IG CLASSES OR SPECIFICITY
isotype
allotype
idiotype
(Classes/subclasses)
Sequence variability of H/Lchain constant regions
Allelic variants
Sequence variability of H
and L-chain variable
regions (individual, clonespecific)
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)
Az Fc receptors
FcγR :
FcεR :
FcαR :
γ  IgG binding
ε  IgE binding
α  IgA binding
OPSONIZATION
Phagocytosis is ineffective/slow without opsonization
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.
ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC)
low affinity FcR mediated NK cell / Basophyl granulocyte degranulation
MAST CELL DEGRANULATION
Mast cell pre-armed with
antigen specific IgE
Cross-linking of IgE via bound
pathogen/allergen
degranulation
pathogen expulsion
(allergy symptoms)
FEATURES OF ANTIBODY-ANTIGEN
INTERACTION - valency and avidity
Valency: numbers of bonds
between antigen and 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 given antibody
MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 1.
Ig isotype
Serum
valence concentration
Characteristics, functions
Trace
amounts
 Major isotype of secondary
(memory) immune response
 Complexed with antigen activates
effector functions (Fc-receptor
binding, complement activation
 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
12-14 mg/ml
2
free IgM pentamer (star shape)
2
2
4
1-2 mg/ml
10
 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
Antigen bound IgM (crab shape)
MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 2.
THE ROLE OF THE HINGE REGION
ANTIBODY PRODUCTION DURING THE PRIMARY
AND THE SECONDARY IMMUNE RESPONSE
antigénfüggő
alacsony
100-1000x erősebb
az első válasznál
magas
ANTIBODY PRODUCTION DURING THE PRIMARY
AND THE SECONDARY IMMUNE RESPONSE
Ig. Concentration
level
of antibodies
secondary response against
Szekunder
’lasyecondary resp
antigen
A
primary response
primer response
against antigen A
IgG
IgA
IgE
IgM
IgM
primary
response
against
antigen B
5
„A”
antig é nA
Antigen
10
15
20
25
„A” és „B”
30
Antigen
A and B
antigén
napok
napok
ANTIBODY ISOTYPE AND FUNCTION
IgG
Isotype distribution in
the body
PRODUCTION OF IMMUNOGLOBULINS
BEFORE BIRTH
AFTER BIRTH
breast milk
IgA
100%
(adult)
maternal IgG
IgM
IgG
IgA
0
3
month
6
9
1 2 3 4 5 adult
year
Fc receptors
and transporting Fc receptors
FcRn
„neonatal” Fc receptor
Placenta, endothelial and
epithelial cells, etc.
IgG transfer, IgG salvage
pIgR
poli Ig receptor
Epithelial cells
IgA, IgM transfer
Az IgG placental transport:
neonatal Fcg receptor (FcRn)
human FcgRn
humán MHC
Class I
(the structure of FcRn and MHC I is related)
FcRn, FcγRn
FcRn can transport IgG and albumin
Located in different cell types:
- endothelial cells
- epithelial cells (digestive and respiratory system)
- different immune cells eg. professional APC (cross presentation)
FcRn (FcγRn)
function
pinocytosis
IgG is bound with high
affinity in acidic
conditions
IgG can be released at the opposite side
of the epithelia: transcytosis
IgG can be transported to various sites of the body
circulation  tissues
tissues  epithelial surfaces (IgG is more abundant in the rectum and in some upper respiratory tract than IgA)
maternal circulation  foetal circulation
Az FcRn salvages IgG from lysosomal degradation
SECRETORY IgA AND TRANSCYTOSIS
SS
‘Stalk’ of the pIgR is
degraded to release IgA
containing part of the pIgR
(the secretory component)
SS
ss
J
SS
S
S
ss
S
S
SS
SS
SS
SS
ss
ss
SS
S
S
J
J
S
S
ss
J
S
S
S
S
S
S
S
S
S
S
S
S
SS
B
J
ss
SS
Epithelial
cell
pIgR and IgA are
internalised
ss
SS
S
S
J
S
S
J
S
S
SS
S
S
SS
IgA and pIgR are
transported to
the apical
surface in
vesicles
M
U
C
U
S
SS
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
The cargo of pIgR (poli immunoglobulin receptor)
Immunoglobulins with ”J-chain”: IgA, IgM
(Do not confuse this J chain with the VJ/VDJ rearrangement’s J region!)
multimeric IgM can exist
without J chain, but not
transported
The destination
epithelial surfaces, sercretums (saliva, breast milk)
SUMMARY OF THE
EFFECTOR FUNCTIONS OF
ANTIBODIES
(next seminar)
MONOCLONAL ANTIBODIES
and
POLYCLONAL ANTIBODIES
POLYCLONAL ANTIBODY RESPONSE
Ag
Immunserum
Polyclonal
antibody
Set of B-cells
Ag
Activated B-cells
Antibodyproducing
plasma-cells
Antigen-specific
antibodies
PRUDUCTION OF MONOCLONAL ANTIBODIES
MONOCLONAL ANTIBODIES
• products of one B lymphocyte clone
• homogeneous in antigen specificity, 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 monoclonal ABs:
– anti-inflammatory antibodies (autoimmune diseases)
– tumor therapy
FEATURES OF POLYCLONAL AND MONOCLONAL ANTIBODIES
Polyclonal antibody
Monoclonal antibody
(high affinity)
Number of recognized
antigen determinants
several (frequent crossreactions)
mostly one
Specificity
polyspecific
monospecific
Affinity
Varying (diverse
antibodies)
high
Concentration of nonspecific immunoglobulines
high
low
Cost of preparation
low
high
Standardisability
Impossible (or uneasy)
easy
Amount
limited
unlimited
Applicability
method-dependent
excellent
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?
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
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.
PASSZÍV IMMUNIZÁLÁS
mouse monoclonal
antibodies
immunization
humanized mouse
monoclonal antibodies
immunization
PROTECTED
SUBJECT
serum antibody
ENDANGERED
SUBJECT
Human immunoglobulin
transgenic mouse
human monoclonal
antibodies
This is a case of PASSIVE
IMMUNIZATION
Immune system is not activated
prompt effect
temporary protection/effect
Immunoglobulin degradation
Passive immunization II.
Type
Usage
Intramuscular
HBV-Ig; Varicella-Zoster-Ig;
Anti-D profilaxis
Intravenal (IVIG)
Humoral immunodeficiencies
(Bruton agammaglobulinaemia; different
deffects resulting
hypogammaglobulinaemia)
Anti-toxin, anti-viral antibodies
(snake venom, Ebola)
Natural passive immunization:
maternal IgG placental transfer
maternal IgA in breast milk
Passive immunization III.
”Antisera” / Policlonal antibodies
Antivenom
snakes, scorpions, spiders, dangerous
toxic sea creatures
Slow, gradial hyperimmunization of large
animals (horses)
Purification of the gamma globulin fraction from
the sera of the hyperimmunized animals
(Purified animal proteins (Ig) are milder
immunogens than raw serum in the patients body.
Repeated therapy could be possible)
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
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)
IMMUNSUPPRESSIVE ANTIBODIES
1. Anti-TNF-α antibodies
infliximab (Remicade): since 1998, chimeric
adalimumab (Humira): since 2002, recombinant human
2. Etanercept (Enbrel) – dimer fusion protein,
soluble TNF-α receptor + IgG Fc-part
Not a real monoclonal antibody.
IgG Fc part  t1/2, transport
Indications of anti-TNF-α therapy:
• Rheumatoid arthritis
• Spondylitis ankylopoetica (Bekhterev's disease)
• Psoriasis vulgaris, arthritis psoriatica
• Crohn’s disease, colitis ulcerosa
(usually – still – not in the first line!)
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
white blood cell count was 3600 / ml
(normal 4.2-5.0 x 106 / 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).
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.
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.
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 immunoglobulin molecule)/
Q&A
The serum IgG from her was assumed to be monoclonal because it migrated as
a tight band on electrophoresis in an agarose gel, and because it reacted with
antibodies to lambda but not to kappa chains. What other evidence could be
brought to bear to prove the monoclonality of this IgG?
The IgG could also be shown to belong a single subclass of IgG, that is IgG1,
IgG2, IgG3, or IgG4. Further more, it would be possible to show that a single
variable-region gene was rearranged to form this IgG.
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.
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.
What happens with the B cells in myeloma multiplex?
Healthy individual
Myeloma multiplex
SUPPLEMENTARY
INFORMATION
B CELL TUMORS
FcγRII mediated B cell feedback regulation
FcγRIIb
FcγRIIb
B cell
B cell
Phosphorilated ITIM motifs on FcγRIIb
recruits phosphatases to interfere signal
transduction
Inhibition of the signal transduction of B
cell receptor
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:
Prefix Target
Source
Suffix
-ki(n)- interleukin as target -u-human
-ci(r)- cardiovascular
-o-mouse
variable
mab
-co(l)- colonic tumor
-xi-chimeric
-neu(r)- nervous system
Etc.
-zu-humanized
Etc.
Example:Abciximabab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody
used on the cardiovascular system
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
MOLECULAR TARGETED DRUGS
Name
Type
Target
Indications
Alemtuzumab
Monoclonal Ab, humanized
CD52
CLL, CML
Monoclonal IgG1, chimeric
IL-2 R
transplantation
Monoclonal IgG1, chimeric
IL-2 R
transplantation
Monoclonal IgG1, chimeric
CD20
Lymphoma
Monoclonal IgG1, humanized
HER2/neu
Monoclonal IgG4, humanized
CD33
Breast cancer, NSC
lung cancer
leukemia
Monoclonal IgG1, murine
CD20
lymphoma
Monoclonal IgG2, murine
EGFR-TKI
KIT-TKI
EpCAM
EGFR TK
TK
CRC
NSCLC
GIST, CML
(Mabcampath)
Daclizumab
(Zenapax)
Basiliximab
(Simulect)
Rituximab
(Rituxan/Mabthera)
Trastuzumab
(Herceptin)
Gemtuzumab
Calicheamicinnel konjugált
Ibritumomab
(Y90)
Edrecolomab
Gefitinib
Imatinib
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
Chimeric Antigen Receptors (CAR)
Effector cytotoxic or cytokin producing helper T cells transfected with
engineered antigen receptor construct.
first generation second generation third generation
chimeric antigen receptors (CARs)
linker
variable domains of
”tumour antigen”
specific antibody
antibody
hinge region
signal transduction
domains directly
linked to the receptor
VH
VL
CAR T cells could also be co-transfected with inducible pro-inflammatory,
cytotoxic, immunstimulatory cytokine gene constructs:
TRUCK (T cells redirected for universal cytokine killing)