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Antibody Structure and the Generation of B-cell Diversity

Chapter 4 Parham

Hans de Haard 25th of April 2012

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

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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

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•  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

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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

Tonegawa: rearrangements basis of antibody diversity

•  Tonegawa and colleagues used

hybridization to demonstrate

rearrangement

•  But in the seventies of last century,

when blot based hybridization was not

yet invented

Fig. 4.24: Southernblot analysis of Ig locus - genomic DNA isolated from granulocytes (germline configuration) and peripheral lymphocytes of leukemia patient - digestion with restriction enzymes and separated on size on agarose gel - transfer from gel to membrane filter - radiolabeled piece of DNA (“probe”) used for incubation: will hybridize specifically against piece of DNA which has the same sequence

Tonegawa: rearrangements basis of antibody diversity

•  Tonegawa and colleagues cut agarose gel in small sizes and did in

solution hybridization

• Double strand of digested fragment and radiolabeled probe is

resistant to nucleases which cuts away ssDNA

•  Peaks represent fragment containing Ig locus:

“germline” detected with probe migrates

differently than rearranged DNA

from plasmocytoma cell line

•  Tonegawa received Nobel price for

his finding

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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)

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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

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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

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Antibody Structure and the Generation of B-cell Diversity

Chapter 4 Parham

Hans de Haard 25th of April 2012

4/24/12

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Structure of heavy chain antibodies

Combining benefits of antibodies and small chemicals

VH

VLCL

CH1

CH3

CH2

VHH

CH3

CH2VHH

Conventional Antibody

Heavy and light chainsBoth chains required for antigen

binding and stability

Heavy-Chain Antibody

Only heavy chainsFull antigen binding capacity

and highly stable

Ablynx’s Nanobody®®

The smallest functional fragment of a naturally occurring single-chain

antibody

Discovery of VHH via phage display

pH shock

Phage display based selection on target binding Screening for Lactococcus bacteriophage neutralizing VHH