Antibody Structure and the Generation of B-cell Diversity Chapter 4 Parham
Hans de Haard 19th of May 2010
Questions about Ab structure and generation of Ab diversity • Structure Ab
• How does an IgG molecule look like, what kind and how many chains?
• What domains/region can be discriminated with Ab and what is their
function?
• What type of epitope recognized and what does a TCR recognize?
• Generation of antibodies
• What is the basis of the great diversity of antibodies?
• Explain how antibodies are improved during B cell development.
• What is the first isotype expressed by a B cell, what about its binding
strength? What is characteristic about the mode of binding to pathogen?
• What happens during isotype switch? What isotypes are expressed and
what is their function and where do these bind the pathogen?
• What MHC is important for B cell development and which CD?
Agenda
• Structure and function of IgG and fragments
• IgG1
• Fab & Fc
• Antigen binding sites
• Generation of antibodies
• Genomic organization heavy & light chain loci
• Somatic recombination, junctional diversity & somatic hypermutations
• Heavy chain isotypes
• Therapeutic applications of antibodies
• Monoclonal antibodies
• Examples therapeutic antibodies
• EGFR as therapeutic target
Agenda
• Structure and function of IgG and fragments
• IgG1
• Fab & Fc
• Antigen binding sites
• Generation of antibodies
• Genomic organization heavy & light chain loci
• Somatic recombination, junctional diversity & somatic hypermutations
• Heavy chain isotypes
• Therapeutic applications of antibodies
• Monoclonal antibodies
• Examples therapeutic antibodies
• EGFR as therapeutic target
Structure of Immunoglobulin G
• Function antibody: • recognition pathogens by variable regions (combination
VH & VL) • recruitment cells immune system for removal tagged
pathogens by constant regions (CH)
Structure and function Immunoglobulin fragments
• Cleavage with papain generates
Fab (Fragment antigen binding)
and Fc (Fragment crystallizable)
• Differences in heavy chain C
regions define five isotypes: IgG,
IgM, IgD, IgA and IgE
• Two different light chains: kappa
(κ) and lambda (λ)
Structure of variable region
• V- and C-domains have immunoglobulin fold
• Characterized by antiparallel strands forming two β sheets (sandwich)
• Three hypervariable regions in V-domain form loops contacting antigen
Hypervariable regions make up antigen binding loops
• Variability plot reveals hypervariable
sequences of V domains
• Hypervariable (HV) or complementarity
determining regions (CDR) are flanked
by framework regions (FR)
• FR responsible for immunoglobulin fold
• CDR form loops at exposed side of
antibody
• VH and VL form two hands held
together with CDR loops
resembling the fingers
• IgG has two arms capable of
binding avidly to multivalent
targets or repetitive epitopes
within a single target
• Hinge has “linear” structure
(less stable), but gives
flexibility to binding arms
enabling avid binding
Combination heavy & light chain variable region form antigen binding unit
Antigen binding sites
• part of antigen recognized by
antibody is called antigen
determinant or epitope
• antibodies can bind avidly and
therefore strongly to repeated
epitopes
• epitopes consist of a linear or
of a discontinuous sequence
(linear vs conformational
epitope)
Shapes of epitopes
1 2 3 4
• Type (1): end of polypeptide or polysaccheride binds into pocket
formed between VH and VL
• Type (2): linear epitopes bind into shallower clefts formed by all
opposing CDRs of VH and VL
• Type (3): conformational epitopes often interact via large surface
• Type (4): pocket within antigen interacts with protruding CDR
Agenda
• Structure and function of IgG and fragments
• IgG1
• Fab & Fc
• Antigen binding sites
• Generation of antibodies
• Genomic organization heavy & light chain loci
• Somatic recombination, junctional diversity & somatic hypermutations
• Heavy chain isotypes
• Therapeutic applications of antibodies
• Monoclonal antibodies
• Examples therapeutic antibodies
• EGFR as therapeutic target
Genomic organization human heavy & light chain locus
• Light chain variable domain is product of rearranged Vλ/κ and Jλ/κ
• Heavy chain variable domain contains rearranged VH, DH and JH
• CDR3 is formed by fusion of V, (D) and J segment, therefore important in
target binding
• Lambda light chain locus contains 30 V and 4 J-C clusters, kappa 35 V, 5 J
and 1 C, and heavy chain locus 40 V, 23 D and 6J followed by 9 C isotypes
Somatic recombination
• Recombination of V1 and J by looping out a DNA segment
• Annealing of Recombination Signal Sequences (RSS) and catalyzed by RAG’s
• Diversity determined by number of possible combinations of V and J for VL
and V, D and J for VH and number of combinations of VH and VL
• Allelic exclusion prevents recombination of locus on 2nd chromosome: one B
cell only produces one antibody
Junctional diversity
• Fusion of V and J imprecise, i.e. reading
frame can be correct or incorrect
(leading to a non-functional antibody)
• More variability possible in joining of
DH-JH and VH-DH
• P-nucleotides added due to nicking, N-
nucleotides by enzyme TdT
• Contribution P- and N-nucleotides is
called junctional diversity
• Large diversity of antibodies generated
by somatic recombination, VH-VL
combinations and junctional diversity
IgM / IgD is first expressed isotype
• Rearranged heavy chain V domain in
juxtaposition of Cµ / Cδ cluster
• Alternative splicing leads to IgM (right
figure) or IgD mRNA (not shown)
• Expression on cell surface via Membrane
Coding (MC) exon or secreted (no MC)
• IgM/IgD is first format to contact target
• IgM secreted in high quantities and has
effector functions (protective immunity);
IgD low levels, no effector functions
Affinity maturation by somatic hypermutations
• IgM has low affinity binding
• During B cell development affinity improved by
somatic hypermutation
• Random mutations introduced in V gene (left), but
those giving better affinity (and therefore targeting
CDR loops ) are selected (below)
Heavy chain isotypes
• After successful rearrangement VH and VL low affinity antibody
secreted in form of pentameric IgM; high avidity (5x2 binding
units) compensates for low affinity of binding unit
• Somatic mutation increases affinity, therefore lower degree of
avidity needed: isotype switch from IgM (pentamer) to other
types (IgG, IgA or IgE; bivalent)
Isotype switching
• Isotype switching ensures
fusion of VH to other heavy
chain isotype (no change in
epitope recognition)
• Isotype light chain not changed
• Initiated by Switch (S)
sequences and catalyzed by
activation-induced cytidine
deaminase (AID)
• Looping out of DNA puts VH in
juxtaposition of new isotype
Functions of isotypes
• neutralization (e.g. blocking interaction virus with cellular receptor)
• opsonization / activation complement system leading to lysis pathogen
and ingestion by phagocytes
• activation NK cells by binding of Fc to Fc receptors
• activation mast cells by interaction with IgE receptor
(discussed in Chapter 9)
Properties of isotypes
• transport across epithelium: secretion into lumen of gut, milk, saliva,
sweat and tears to combat parasites, microbes and viruses outside the
body
• transport across placenta to supply fetus with protective antibodies
• diffusion in extravascular sites of damaged or infected tissues (related to
size of antibody)
Agenda
• Structure and function of IgG and fragments
• IgG1
• Fab & Fc
• Antigen binding sites
• Generation of antibodies
• Genomic organization heavy & light chain loci
• Somatic recombination, junctional diversity & somatic hypermutations
• Heavy chain isotypes
• Therapeutic applications of antibodies
• Monoclonal antibodies
• Examples therapeutic antibodies
• EGFR as therapeutic target
Introduction therapeutic antibodies hybridoma technology and monoclonal antibodies
• immune system generates large
collection of B cells, each
producing single type of
antibody directed against
immunogen
• B cells fused to cancer cells
yielding immortalized cells
capable of producing unlimited
quantities of a single type of
antibody (i.e. monoclonal
antibody)
Therapeutic antibodies
EGFR as therapeutic target
• Epidermal growth factor receptor plays important role in epithelial
cancers (colon, breast, head and neck, kidney, lung, pancreas and
prostate)
• Receptor dimerizes and is activated upon binding of ligand EGF
leading to proliferation (see Li et al, Cancer cell (2005))
• Erbitux prevents binding of ligand and subsequent proliferation
• Fc (IgG1) might give ADCC / CDC leading to clearance of tumor cells
by immune system
Application in current research: knowledge genomic organization as basis for Tg mice expressing human Ab’s
• Lonberg, Nature Biotechnol (2005) reviews different Tg mouse
systems for generation of human mAb’s
• For more technical details about creation of Lonberg’s Tg mouse
(Medarex / Genmab) read Tuaillon et al., PNAS (1993)
• Note how the human germline heavy and light chain loci were
introduced in mice, in which (part of) the corresponding murine loci
were knocked out
• Note how the investigators checked if rearrangements occurred and
if somatic mutations were introduced leading to improved affinities
• Also read which therapeutic antibodies were generated in such
systems and how successful these are in clinical trials
Antibody Structure and the Generation of B-cell Diversity Chapter 4 Parham
Hans de Haard 19th of May 2010
Structure of heavy chain antibodies
Discovery of VHH via phage display
pH shock
Phage display based selection on target binding
Screening for Lactococcus bacteriophage neutralizing VHH