Transcript AntibodyNoTP

Structure and Function of
Antibodies
Updated: October 02, 2014
Folder Title: Antibody
Filename: antibody(NoTP).ppt
See parts of Chapter 3, Kuby Edition 7;
B and T-Cell Receptors & Signaling
Antibody Structure: pages 80 – 94
Targets and Weapons in the Specific
Adaptive Immune Response
Responds to Antigenic Determinants (Epitopes)
Responds With:
Lymphocyte
Receptor
Primary "Weapon"
B-Cell
Membrane-bound Antibody
Extra-cellular Antibody
T-Cell
T-Cell Receptor
Extra-cellular Cytokines
Antibody Protein as the “Punter”
Hands on the End of Arms = Recognize and Grasp the Ball
Foot = Kicks the Ball
Fab = Bivalent antigen recognition (Two “hands” needed)
Fc = Effector function or business end
Need both!
Fc
Region
Fab Region
“Fc” = Fragment
Crystallizable”
“Fab” = Fragment
Antibody Binding
(not crystallizable)
Protein Structure of Antibodies
A dimeric protein
Heavy and Light Chains
2 Heavy – 2 Light (For IgG, 150K MW Antibody)
or Multiple Sets (e.g.: 4H + 4L) for IgA
or 10H + 10L for IgM (Macroglobulin; 900K MW)
Different antibody isotypes and antibody roles
IgG, IgA, IgM, IgE, IgD
Antibodies as Serum Proteins:
Which serum proteins are they?
What do they look like?
See Box 3-1, Figure 1
Kuby 7th Edition
Adding Soluble Antigen to Blood Serum
Which protein fraction decreased?
Where did it go?
Untreated Blood
Serum
Antigen Added
Tetrameric
Structure
of IgG
See Figure 4-7
Kuby, 6th Edition
p. 86
Figure 5-2, Kuby 3rd Ed.
IgG4mer
Papain Digestion
Complementarity of
Antibody – Antigen Binding
Antigen - Antibody Binding
Antibody Light Chain
Variable Region
Fig.
4-6a
Kuby
3rd Ed
AgAb
Kiss
Antibody Heavy Chain
Influenza Virus Antigen
Variable Region
Antibodies as Proteins
How Do Antibodies Bind to Virtually an Infinite
Number of Different Possible Antigens?
Proteins are not amorphous polymers.
Proteins are not promiscuous in what they bind to.
Start Here: Tuesday, September 23, 2014
Representation of Sequence Comparisons Among Light Chains
from Antibodies with Three Different Antigen Specificities
H3N-Ser-Val-Ile-Thr-Gly-Gly-Tyr-Ala... Thr-Glu-Ala-Val-Tyr-Ser-Met-COO-
H3N-Ser-Ile-Met-Thr-Arg-Leu-Tyr-Gly..Thr-Glu-Ala-Val-Tyr-Ser-Met-COOH3N-Thr-Gly-Gly-Thr-Lys-Leu-Tyr-Ile..Thr-Glu-Ala-Val-Tyr-Ser-Met-COOVariable Amino Terminal Half
Conserved Carboxyl Terminal Half
(Positions 1 to 107)
(Positions 108 - 214)
This shows the first 8
Amino acids from
the amino terminal
V-region
This shows the last 7 amino
acids from the carboxyl
terminal conserved end.
The Following Two Slides Show the Variable Amino Acid
Positions in the Amino Terminal One-half of the light Chains
(107 Residues)
And in the Amino Terminal One-fourth of the Heavy Chains
(about 110 residues)
The Heavy chain of antibodies is twice as long as the light
chains:
440 Amino acids in each of the two heavy chains
220 Amino acids in each of the two light chains
Complementarity-determining Regions (CDR’s)
from Light and Heavy Chains Come together in 3
dimensions to give the antigen-recognizing site.
Ag
Binding
Site
(Fab)
Ag Binding
Site (Fab)
Confers Biological
Activity (Kicks the
Football)
(Fc)
Following are Turning Point short answer questions.
Please put all notes on the floor.
Do not have any electronic devices other than your NXT transmitter.
No consulting with other students.
If you have a problem with your device, I can provide you with a
loaner NXT device.
If you have a problem using your NXT device, please ask Elisabeth
for help.
It is imperative that the integrity of these in-class Turning Point
quizzes be maintained at the same level as we will do with the three
written exams.
What Produces
Antibody – Antigen Specificity?
Why Do We get Specificity and
Very Tight Affinity?
Blue shows topology of pocket in MDM2 protein that binds to p53
Figure 9.13 The Biology of Cancer (©
Garland Science 2007)
How proteins recognize each other topologically
(3-dimensional surfaces)
Yellow is p53 protein showing peptide domain sequence that binds to MDM2 Control
Protein
Large surface protein epitope recognized
Smaller low molecular size epitopes recognized
Small peptide antigen binding to an Fab (Fragment-antibodybinding) fragment of a complementary antibody
Light and heavy
chain CDR
regions move to
better
complement the
antigen
Peptide antigen from an
HIV protein antigen
Conformational change in antibody upon binding antigen
(“induced fit”)
Movement of Peptide-binding Pocket
Accompanying Antigen Binding:
Fab Fragment of Antibody to Hemagglutinin Peptide
Figure 5-11
Kuby, 3rd Ed
AgAbMove
How does the
antibody protein
recognize its
complementary
antigen so precisely?
Precise topological,
spatial, and directional
arrangement of
stabilizing bonding.
Minimization of repulsive
interactions.
Immunoglobulin Isotypes
Structures and Functions
Start Here: Thursday, September 25th
See Figure 4-6,
Kuby 6th Edition
p. 85
Variable Regions
Including
hypervariable CDR’s
(Complementarity
determining regions)
κ or λ forms
See Figure 4-6,
Kuby 6th Edition
p. 85
Heavy Chain
Iso-forms
The Heavy and light chains are labeled incorrectly in the Kuby Immunology Powerpoint slides.
The figure is labeled correctly in the book.
Chain Structures of the five immunoglobulin classes in humans
(adapted from Kuby, 2nd edition)
Class
Heavy
Light
Sub-Classes
Subunit Formula
Chain
Chain
IgA
γ
α
κ or λ
γ1, γ2, γ3, γ4
γ2κ2 γ2λ2
κ or λ
α1, α2
(α2κ2)n
(α2λ2)n
n =1,2,3,4
IgM
μ
κ or λ
None
IgD
δ
κ or λ
None
δ2κ2 δ2λ2
IgE
ε
κ or λ
None
ε2κ2 ε2λ2
IgG
(μ2κ2)n
(μ2λ2)n
n = 1 or 5
Figure 5-17(a)
IgAModl
Kuby, 3rd Ed.
Structures of Four Sub-types of IgG
See Figure 4-18
Kuby, 6th Edition
p. 98
IgGSubs
Figure 5-16, Kuby, 3rd Ed.
Properties & Activities of Human Serum Immunoglobulins
(from Table 4-2, Kuby Immunology, 4th Ed. p. 96)
Property or Activity
IgG*
Mol Wt (KD)
150
H Chain
gamma
Serum Conc (mg/ml) 0.5 – 9
Serum Half-life (dys) 8 - 23
Activate Complement Yes
Cross Placenta
Yes
Membrane (mIg) Form No
Fc Binds Macrophages Yes
Mucosal Presence
No
Induces Mast Cell
No
IgA**
IgM
IgE
150 – 600
900
190
alpha
mu
epsilon
0.5 - 3
1.5
0.0003
6
5
2.5
No
Strong
No
No
No
No
No
Yes***
No
No
?
No
Strong
Yes
No
No
No
Yes
* 4 Sub-classes IgG1, IgG2, IgG3, IgG4
**2 Sub-classes IgA1, IgA2 (exists as mono-, di-, tri, tetramer)
*** mIgM is monomer. Serum IgM is pentamer
# Has no known effector function. Is a membrane-bound antigen receptor
IgD#
150
delta
0.03
3
No
No
Yes
No
No
No
Effector Functions of Antibodies
Functions of Fab Binding
Neutralization or Blocking of Target Molecule or Particle
Cross-linking and Agglutination of Target
(Bivalent Fab Binding)
Functions of Fc Region
Complement Fixation and Lysis of Target
Opsonization (Coating by Ab) and Phagocytosis of Target
Targeting by Antibody for Cell-mediated Destruction
(Antibody-dependent Cell-mediated Cytotoxicity: ADCC)
Mast Cell Attraction and Activation by Bound IgE
Immediate Type I Hypersensitivity, Type I Allergic Response, Localized
and Systemic Anaphylaxsis
Effector
Following are Turning Point short answer questions.
Please put all notes on the floor.
Do not have any electronic devices other than your NXT transmitter.
No consulting with other students.
If you have a problem with your device, I can provide you with a
loaner NXT device.
If you have a problem using your NXT device, please ask Elisabeth
for help.
It is imperative that the integrity of these in-class Turning Point
quizzes be maintained at the same level as we will do with the three
written exams.
Antibodies as Antigens
Why does this matters?
If we want to use antibodies as therapeutic agents in patients, we
have to understand and control the immunogenicity of the
antibodies,
or they will generate damaging and dangerous allergic responses,
and be cleared from the patient and would be ineffective at best.
Antibodies are not cells, so they don’t have transplantation
antigens and they don’t have to be histocompatibility matched,
but they have to go unrecognized as foreign proteins and they
cannot be allowed to generate allergic reactions in the recipient.
Start Here Tuesday September 30, 2014
Antibodies as Antigens
1. Different Heavy Chain Isotypes (gamma, alpha, mu, epsilon, delta):
Anti-isotype Antibodies (Anti-gamma, Anti-Alpha, Anti-Mu, etc)
(Also differences in constant regions of kappa and lambda light chains)
2. Different individual mouse strains (or different people):
Anti-allotype Antibodies (Antibodies from one person would raise
anti-antibodies in a non-identical twin recipient)
(1 and 2: Like any other proteins with multiple molecular forms)
3. Different antigen-recognition abilities:
Anti-idiotype Antibodies. Anti-CDR’s for different antibodies
Other proteins except for T-cell Receptors do not show these kinds of
variations and are not immunogenic in this way
Transplantation Concepts and Nomenclature: For All Proteins
From self, from identical twin, or from and to inbred animals of the
same strain:
Syngeneic, Isogeneic, Isologous
From same species but not self or identical twin:
Allogeneic
(“allo” = other)
From different species (e.g mouse to human)
Xenogeneic
(“Xeno” = foreign)
Transplantation Concepts and Nomenclature:
Specifically for Antibodies
Different Isotypes (IgG, IgM, etc.) in the Same Individual
Isotypic Determinants
The Same Isotype in Two Different Unrelated Individuals
in the Same Species (CH1, CH2, CH3, and CL constant regions)
And “Framework” Parts of VH and VL
Allotypic Determinants
(“allo” = other)
The Same Isotype in Identical Twins or in Mice of Same Inbred Strain:
Specific for Different Antigenic Determinants
6 Different CDR Regions or Idiotopes
Idiotypic Determinants
Isoforms of the same antibody in the same individual
Allotypic determinants (different individuals) are in the Constant
Regions of Heavy and Light Chains
Idiotypic determinants are in the V-regions of Heavy
and Light Chains , especially at the CDR’s)
Therapeutic Monoclonal
antibody would use
antibody matched as
isotype and matched in
the framework allotype
in V-regions (i.e
matched as close as
possible to the patient).
Then graft in the CDR’s
from mouse to get the
antigenic specificity that
is needed. Foreign
idiotopes could go
unrecognized and be
non-immunogenic.
Matched human
isotype and
human allotype.
Graft in mouse
CDR’s
Humanized monoclonal Ab
Making Monoclonal Antibodies
Now Immortal!
Following are two Turning Point short answer questions.
Please put all notes on the floor.
Do not have any electronic devices other than your NXT transmitter.
No consulting with other students.
If you have a problem with your device, I can provide you with a
loaner NXT device.
If you have a problem using your NXT device, please ask Elisabeth
for help.
It is imperative that the integrity of these in-class Turning Point
quizzes be maintained at the same level as we will do with the three
written exams.
View from OnLine at Textbook Web-site:
Molecular Animation of Immunoglobulin Structure
Molecular Animation of Cells of the Immune Response
Molecular Visualization of Immunoglobulin Structure
Molecular Visualization of Antigen-Epitope Interactions
For OnLine Access to Immunology Edition 6 Information:
http://bcs.whfreeman.com/immunology6e
Avastin for Breast Cancer:
Possible withdrawal of FDA approval. Sept. 16, 2010
http://www.cnn.com/video/#/video/health/2010/09/17/dnt.cohen.breast.cancer.cnn?iref=allsearch
View Cancer Warrior, NOVA OnLine Video, 2001
http://www.pbs.org/wgbh/nova/body/cancer-warrior.html
Judah Folkman, Endostatin, Angiostatin, and Targeting Cancer Vascularization
“Judah will cure cancer in three years” (J. D., Watson, 2001)
Anti-angiogenesis factor receptor monoclonal antibody: Avastin