Basic Structure of Antibodies

1
(No Transcript)
2
? Basic Structure of Antibodies ? Obstacles to
Antibody Sequencing ? Immunoglobulin Fine
Structure ? Antibody-Mediated Effector
Functions ? Antibody Classes and Biological
Activities ? Antigenic Determinants on
Immunoglobulins ? The B-Cell Receptor ? The
Immunoglobulin Superfamily ? Monoclonal
Antibodies
3
(No Transcript)
4
Obstacles solution of Ab sequencing
? Insufficient amounts of homogenous protein
available ? The population of antibodies in the
serum r-globulin fraction consists of a
heterogeneous spectrum of antibodies. ? Pure Ig
obtained from patients with multiple Myeloma, a
cancer of Ab- producing plasma cells, made
sequencing possible In a patient, myeloma
protein can account for 95 of the serum Ig (
mostly light chains Bence-Jones proteins) ?
Light-chain sequencing from different individuals
revealed that Ig have constant variable
regions the carboxyl-terminal half of the
molecule had two basic a.a. sequences (two
types Kappa Lambda) ? Heavy-chain sequencing
revealed five basic varieties of heavy chains.
m, d, g, e, a - each of them is called a
class ? minor differences in the a.a. sequences
of the a g heavy chain led to further
classification subclass
5
Basic str. of Ab
Structural features of antibodies
? Ab molecules have a common Y-shaped str. of
four peptide chains - two identical H. L.
chains ( H2L2) - they are linked by
disulfide bridges ( H-H H-L ) - a dimer of
dimers (50KDa-25KDa)2 150KDaLow MW fraction
- intra-domain disulfide bridge forming a loop
of 60 a.a. ? The exact precise positions of
inter-chain s-s bonds differ among Ab
classes subclasses. ? The first 110 a.a. of a
light or heavy chain (VLVH) varies greatly among
antibodies of different specificity, based
on a.a. sequencing studies. ? The effector
functions are mediated by the C. regions (CH)
- one constant domain in the light chain 3 or
4 in the heavy chain
6
Antibody structure
LIGHT
HEAVY
HL
Antibody

dimer
7
Basic str. of Ab
Schematic diagram of structure of Ig
A dimer of heterodimer Based on a.a.
sequencing studies, N-terminal V region
C-terminal C region
FIGURE 4-2
8
Basic str. of Ab
Antibodies are glycoproteins with few exceptions
- possible role ? CH2/CH2 domains protrude
because of the presence of CHO in the
interior of Ab, enabling it to i) increase
the solubility of the molecules ii)
increase stability in the serum iii)
enhance interaction b/in Ab complement system
or FcR.
9
(No Transcript)
10
Ig fine structure
Immunoglobulin fine structure
? The secondary structure is formed by folding of
the extended polypeptide chain into a
antiparallel ß pleated sheet. ? The Ig fold
consists of a sandwich of two b pleated sheets
gtgtgt Ig fold domains contain 110 a.a.
residues and is stabilized by an
intra-domain disulfide bond, 60 a.a. apart ? Igs
possess multiple domains based on the Ig fold ?
comparisons of the a.a. sequences of a large of
VL VH domains. -gt the sequence variation is
concentrated in a few discrete regions
located in the loops HVR or CDR which binds Ag
-gt the remainder exhibits far less variation
Framework regions (FR) gtgtgt V domains HVR
FR
11
(No Transcript)
12
(No Transcript)
13
(No Transcript)
14
Ig fine structure
15
(No Transcript)
16
V
V
17
Ig fine structure
gtgtgt 3 complementarity-determining regions in each
Vh VL domain
FIGURE 4-9
18
HEAVY CHAIN (amino acid sequences)
QVQLVQSGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQRLEWMGE
INPGNGHTNYSQKFQGRVTITVDKSASTAYMELSSLRSEDTAVYYCARSF
TTARAFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
V
C
19
(No Transcript)
20
(No Transcript)
21
(No Transcript)
22
Ig fine structure
23
Antibody classes Biological Activites
24
Antibody classes Biological Activites
25
Antibody classes Biological Activites
Antibody classes Biological Activites

• Each class is distinguished by unique a.a.
  sequences in the H. chain constant region.
• - IgA gt IgM move across epithelial layers,
  called transcytosis
• (the presence of the J chain allows
  IgA IgM to bind to receptors
• on secretory cells)
• - IgM gt IgG3 activates classical
  complement pathway due to higher
• valency
• - IgE can induce mast-cell degranulation
• - IgG binds to FcR of phagocytes (
  opsonization ADCC ) can be
• transported across the placental tissue
• (Natural passive immunization)

26
Antibody classes Biological Activites
Passive Sero- Antibody therapy

• ? In 1890, injection of 0.2ml serum from
  tetanus-immunized rabbits into the abdominal
  cavity of mice protected from challenge of
  virulent tetanus bacteria.
• -gt protective substances appeared in serum
• -gt Immunity could be passively acquired.
• -gt In 1901, Dr. Von Behring was awarded the
  first Nobel prize in Medicine
• During the 1930s 1940s, passive immunotherapy
  based on the transfer of Ab (measles Hepatitis
  A) was used in clinical (medical) practice.

27
Serum ? (humoral immunity) (the liquid,
non-cellular component of coagulated blood)
Ab ?
Sero-therapy
Anaphylaxis ?
In 1894, Behring extracting serum from the
immunized horse using a tap. It could neutralize
diphtheria toxin in other animals or humans In
the 1930s, a fraction of serum Ig (Ab), was
responsible for these activities. gtgtgt Passive
antibody therapy
28
Sero-therapy
Tetanus toxoid
Immunized horse
Immune horse serum (tetanus antitoxin)
Patient at risk of tetanus
Patient protected
The passive transfer of immunity to tetanus by
means of antibody
29
Antibody classes Biological Activites
Antibody therapy
- Source of Immunoglobulin for passive
immunization. The pooled plasma from
thousands of donors -gt treatment with solvents
the use of detergents that was highly effective
in inactivating viruses. -gt intravenous immune
globulin (IVIG) Ig of IVIG contains 1018
Ab (mostly IgG) which may incorporate gt
107 different Ab specificities ? Better
source of IVIG ( immunodeficient patients
receive 1428 g of IVIG every 34 weeks
during the course of therapy ). ! mAb? -
Action mechanism of passively administered Ab.
i) the recruitment of the complement pathway
(IgM gt IgG3 gt IgG2) ii) Promotes
opsonization, the phagocytosis killing of the
bacteria by Mø Neutrophil IgG
iii) mediate the killing of target cells by NK
cells (ADCC) IgG iv) neutralizes toxins
viruses, or marks them for removal from the body
by phagocytes, liver kidneys.
30
Antibody classes Biological Activites
FIGURE 4-13
31
Antibody classes Biological Activites
FIGURE 4-14
32
(No Transcript)
33
Antibody classes Biological Activites
34
(No Transcript)
35
Antigenic determinants on Ig
Antigenic determinants on immunoglobulins

• Ab can themselves function as potent immunogens
  to induce Ab responses.
• powerful tools for the study of B-cell
  development
• Ab responses
• - obstacles to passive Ab therapy
• ? Three major catagories isotype, allotype,
  idiotype
• i) Isotype
• - different species inherit different
  constant-region genes
• - the isotypic determinants on an Ab from
  one species will be
• recognized as foreign in another
  species.

36
Antigenic determinants on Ig
Antigenic determinants on immunoglobulins
ii) Allotype - although all members
of a species inherit the same set of
isotypic genes, multiple alleles exist for some
of the genes. subtle (14
a.a.) differences in some, but not all, members
of a species - Ab to
allotypic determinants can arise from a blood
transfusion or be produced by a
pregnant mother in response to paternal
allotypic determinants on the fetal Ig.
iii) Idiotype - the sum of the
individual idiotope, each individual antigenic
determinant. - an idiotope can
be the actual antigen-binding site or not.
- injection of a homogeneous Ab into a
recipient who is genetically
indentical to the donor can produce anti-idiotype
Ab.
37
(No Transcript)
38
B-Cell Receptor
FIGURE 4-18
39
B-Cell Receptor
Four distinct roles of Fc binding proteins
are essential for many of the biological
functions of antibodies
? The movement of Ab across cell membranes
poly IgR for pentameric IgA to some extent,
pentameric IgM ? The transfer of lgG from mother
to fetus across the placenta FcRN ?
Trigger effector functions Opsonization or
ADCC ? Cross-linking of FcR generates
immuno-regulatory signals that affect cell
activation, differentiation, etc. which are
similar to signal transduction from BcR
40
B-Cell Receptor
41
(No Transcript)
42
(No Transcript)
43
Monoclonal antibody and Hybridoma
44
FIGURE 4-22 (a)
45
Diphtheria toxin binds to a cell-membrane
receptor(L.) a diphtheria-immunotoxin to a
tumor-associated antigen(R.)
FIGURE 4-22 (b)
46
Pre-clinical and Clinical development
programs(200 major companies in 2004)