PART 4: (1/2) Central Processing Unit (CPU) Basics CHAPTER 14: P ROCESSOR S TRUCTURE AND F UNCTION.
Immunoglobulins: s tructure and f unction
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Transcript of Immunoglobulins: s tructure and f unction
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
Immunoglobulin Fragments: Structure/Function Relationships
antigen binding
complement binding site
placental transfer
binding to Fc receptors
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
(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.
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)
NK CELL DEGRANULATIONNK CELL DEGRANULATION
Antibody Dependent Cellular Cytotoxicity (ADCC)Antibody Dependent Cellular Cytotoxicity (ADCC)
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).
• 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.
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