Antibodies

Antibody-Antigen Interactions

&

Monoclonal AntibodiesCellular and Molecular Immunology

Pathology 5210 & N-5211February 23, 2009

Dr. Coffman

Antibodies are bifunctional molecules

• Key components of host defense system• One end interacts with foreign antigen (can

interact with at least two epitopes)• Other end interacts with components of host

defense - Fc receptors on specific cells, complement proteins

• Links antigen recognition with specific host responses

Where antibodies are found

• Serum and tissue fluids• Bound (noncovalently) to Fc receptors

on the surface of many cell types• Integral membrane protein on the

surface of unstimulated B cells (the only place that antibodies exist as integral membrane proteins)

MostlyIgG

Electrophoresis of serum proteins - densitometric scan of gel

Ig-mediated Effector Functions

• Opsonization• Complement activation• Antibody-dependent cell-mediated

cytotoxicity (ADCC)• IgG crossing placenta to provide

passive immune protection• Activation of immune and inflammatory

cells

Basic Immunoglobulin Structure

• 2 identical light chains (kappa or lambda)• 2 identical heavy chains (alpha (IgA), delta

(IgD), epsilon (IgE), gamma (IgG), or mu (IgM))

• Constant and variable immunoglobulin domains

• Carbohydrates for solubility and protease protection

• Disulfide bonds hold domains together, link heavy chains together, and link light chains to heavy chains

Can you spot the error in this slide?

Immunoglobulins are constructed from multiple genetic modules

Immunoglobulin hinge regions impart flexibility

Membrane-bound Immunoglobulins

• Secreted immunoglobulins bind to Fc receptors on the surfaces of many cell types

• Immunoglobulins that are integral membrane proteins are present only on unstimulated B cells

• Stimulation of these B cells causes them to produce the secreted form of the antibody that they expressed on their cell membranes

Immunoglobulins areintegral membraneproteins and contain atransmembrane helix

Secreted immunoglobulins:splicing event removestransmembrane helix andreplaces it with a hydrophilicsequence

Immunoglobulins in B cell activation

On the surface of anunstimulated B cell

On the surface of anunstimulated B cell

MC = membrane-codingSC = secretion-codingpA5 = first polyadenylation

site (used in secretedform)

pAm = second polyadenylationsite (membrane form)

Splicing of primary transcriptdetermines whether pAm siteis used (membrane form) orpA5 site is used (secreted form)

Immunoglobulin Superfamily Members that Bind Immunoglobulins

Immunoglobulin Superfamily Members that Recognize Antigens

Other Immunoglobulin Superfamily Members

Light Chain Constant and Variable Domains

All immunoglobulin superfamily members contain domains similar to these

IgG• Major human serum immunoglobulin

(~70% of total circulating Ig)• Principal antibody mediator of the secondary immune

response• Four IgG subclasses - IgG1, IgG2, IgG3, IgG4• Only antibody class that can cross the placenta and

provide passive protection to the fetus• Opsonization, complement activation, antibody-

dependent cell-mediated cytotoxicity (ADCC)

4 subclasses of human IgG

- size of the hinge region- no. & position of the interchain

-S-S- bond- IgG1>IgG2>IgG3>IgG4 in serum conc.- 90% - 95% homologous in DNA sequences

Long hingeregion moresusceptibleto proteases, CH2 moreeasily bound

by complement

IgM• 10% of the total antibody pool, almost all in the

circulation• Principal antibody mediator of the primary immune

response• Pentamer structure - heavy chains have C terminal

tail pieces that make S-S bonds with J chain• Agglutinates antigen - increases antigen visibility• Proficient at complement fixation• Antigen receptor on naïve B cells

IgM Pentamer-monomer on the B cell membrane, secreted form is a pentamer

- 5% - 10% serum Ig- 1st Ab in neonates- 1st Ab in primary

response- more efficient in

agglutination & complement fixation

- J (joining) chain allowsIgM to transport acrossthe epithelium.

- also present in secretions

IgA• Two major forms - monomer (IgA1) in circulation,

dimer (IgA2) is the predominant antibody in mucosal secretions (occasionally IgA2 is trimeric)

• Secretory component protects IgA2 dimer from proteases secreted by mucosal bacteria; the secretory component is a fragment of the polyIg receptor involved in transporting IgA2 from the basolateral surface of mucosal epithelial cells through the cells and secreted upon reaching the lumen

Secretory IgA

Secreted IgA dimer with a secretory component

Formation of Secretory IgA

Transcytosis

IgD

• Relatively rare• Surface marker for B lymphocyte

maturation• Functions as a co-stimulator in antigen

presentation• Functions in B cell development

IgD on surface

IgE• Present only in trace amounts as a free monomer in the

circulation• Binds to very high affinity Fc receptors on the surface of tissue

mast cells and circulating basophils• Binds to moderate affinity Fc receptors on the surface of

monocytes and lymphocytes• Primary antibody mediator of hypersensitivity, allergies, and

asthma• Primary antiparasite antibody; sensitizes worms and other

parasites for destruction by eosinophils

Allergen cross-linkage of receptor-bound IgE on mast cells induces degranulation

Classification ofImmunoglobulins

by antigenicdeterminants

Isotypes

Allotypes

Idiotypes

Determined by gene

Determined by allele

Determined byvariable region

Antibody-Antigen Interactions

The interaction of two molecular surfaces: the paratope on the

antibody and the epitope on the antigen

CDR = complementarity determining region

Antigens bind to amino acids from both light and heavy chains

Antibody-antigen binding is mediated by noncovalent forces

One antibody-antigen interaction is mediated by many of these individual interactions

Antibody-antigeninteractions aremediated bymultiplenoncovalentinteractions

Light ChainCDR1

(green)

HeavyChainCDR2(green)

Specific antigen binding is due to multiple noncovalentinteractions and a good fit (lack of steric hindrancesor large pockets)

Antigen (red,white,blue)

Antigen

Light ChainHeavy Chain

Good 3D fit = Tight binding

What is an epitope?

A molecular surface on an antigen that is bound by an antibody.

Properties of epitopes

• Average epitope size ~400 Å2

• For an average protein antigen, 14-18 antigen amino acids interact with 14-18 antibody amino acids

• All potential interactive groups are used: for protein antigens, peptide bond NH and O as well as all R groups (NH3

+, COO-, OH, SH, aromatic rings, aliphatic carbon chains) are used

Immunogenicity requires more than Antigenicity

• Immunogenicity: the ability to evoke an immune response

• Antigenicity: the ability to bind an antibody• Many small molecules can bind tightly to

antibodies but cannot activate B cells • Antigenic small molecules can be made

immunogenic by attaching them to a large molecule (hapten-carrier)

Properties of epitopes that lead to immune responses

• Foreignness (non-self structure)• Molecular size (larger = more immunogenic)• Chemical complexity - homopolymers usually poor

immunogens (exception: polyvinylpyrrolidine), co-polymers of 2-3 different units may be immunogenic

• Degradability - molecules that cannot be degraded and processed by APCs are poor immunogens (ex: peptides containing D-amino acids are not immunogenic)

Other factors in immunogenicity

• Genetic constitution of immunized animal -individuals differ in their ability to respond to particular antigens due to genetic constraints

• Method of antigen administration - ip (intraperitoneal), sc (subcutaneous), iv (intravenous), im (intramuscular), etc.

• Amount of antigen administered• Use of an adjuvent to promote response

Size considerations of antigens

• Under 1,000 Daltons, nonimmunogenic (usually need to couple molecules of this size to proteins in order to raise antibodies)

• 1,000-6,000 Daltons, may or may not be immunogenic

• Over 6,000 Daltons, usually immunogenic

Classes of molecules as antigens

• Carbohydrates - non-self carbohydrates (bacterial capsules, ABO blood group antigens) are often antigenic

• Lipids - usually not immunogenic• Nucleic acids - poor immunogens themselves but

good as haptens• Proteins - excellent antigens• Metal ions - only immunogenic as haptens• Small organic compounds - usually only

immunogenic as haptens

Components of Ab-Ag binding

• Three dimensional fit: tight binding if there is not much room for water molecules and no steric hindrances

• The sum of multiple noncovalent interactions: tight binding if there are many favorable hydrogen bonds and ionic interactions and no unfavorable interactions (ex: two close COO-groups)

AffinityAnd

Avidity

Affinity: describes the interaction of twomolecular surfaces - one surface onthe antigen (epitope) and onesurface on the antibody (paratope)

Keq =[AB]

[Af][Bf]

Avidity: refers to the interaction betweenan entire antibody (which can bindat least two epitopes) with an entireantigen (which can have thousandsof epitopes). Avidity is also calledfunctional affinity (the binding ofone epitope to one paratope is thencalled intrinsic affinity).

What properties of epitopes doantibodies recognize?

1. Composition

2. Conformation

Change antigen composition or conformation - lose binding affinity

Immunodominant epitopes

• Most antigens contain multiple potential epitopes

• Some epitopes are recognized by antibodies far more often than others

• These epitopes are termed immunodominant• In protein antigens, immunodominant

epitopes often contain a charged group surrounded by nonpolar groups

Immunodominant epitope recognized by two different antibodies

Antigen = Influenza virus hemagglutinin

Green and yellowareas representAb contact points

(small differencein these areas)

Ab1 bindsto greenarea

Ab2 bindsto yellowarea

Glutamic acid residue(surrounded by mostlynonpolar groups)

Specificity, Shared Epitopes, Cross-Reactivity

• Specificity - antibodies bind to specific 3D surfaces, and small changes in these surfaces can lead to greatly diminished binding

• Shared epitopes - occasionally different proteins contain nearly identical 3D surfaces (proteins may be related)

• Cross-reactivity - binding of an antibody or antisera to multiple proteins (can be caused by shared epitopes)

Specificity: structural discrimination between twonear-identical mite allergens by an antibody

7A1 epitope sites on two mite antigens blue = common amino acids, red = different amino acids

MAb 7A1 binds MAb 7A1 does not bind

Changing 2 amino acids in the epitope abolishes MAb binding

Cross-reactivity and disease

• Some pathogen proteins contain an epitope that is very similar to an epitope present on a host protein

• Some of the antibodies raised against the pathogen may cross-react and bind to the host protein

• These cross-reacting antibodies can lead to autoimmune diseases

Cross-reactivity and disease

• Myocarditis

• Encephalomyelitis/ multiple sclerosis

• Myasthenia gravis

• Chlamydia outer membrane protein/ cardiac myosin heavy chain

• Hepatitis B viral protein/myelin basic protein

• Herpesvirus protein/ nicotinic acetylcholine receptor

An unusual antibody structure

• 2G12 is a human antibody capable of neutralizing HIV-1 by binding to a dense patch of carbohydrates on the envelope protein gp 120

• 2G12 neutralizes a broad range of HIV-1 isolates and recognizes the disaccharide Mannose alpha 1-2 Mannose with a Kd in the nanomolar range

• Carbohydrates can be used to mask immunogenic protein surfaces, and these carbohydrates are usually poor immunogens

The solution: the heavy chain variable domains switch positionsand form an interlocking surface at the top of the immunoglobulin

Proline 115causesheavy chainto flip overand interactwith otherlight chain

HIVgp120

Red = carbohydrates

2G12Ab

Monoclonal antibodies• Created in 1975 by Georges Kohler in Cesar

Milstein’s lab (while trying to make an immortal B cell line)

• Created by fusing spleen cells from an immunized mouse with a mutant mouse myeloma cell line

• Fused cells (hybridomas) are selected by culture in HAT media

• Hybridomas that secrete the desired antibody are selected by further screening

• Worldwide market for antibodies in 2005 was $15 billion dollars

Small molecule precursors

(blocked by aminopterin)

nucleotides

2 ways to make nucleotides1. De novo synthesis 2. Salvage pathways

Thymidine Hypoxanthine

TK HGPRT(missing in myeloma cell line)

nucleotides

HAT media contains hypoxanthine, aminopterin, and thymidineDe novo synthesis is blocked, cells must use salvage pathways

1. Myeloma cells die(do not have salvage pathway)

2. Spleen cells die(primary cells don’t live in culture)

3. Hybridoma cells survive(have salvage pathway enzymes from spleen cellsand culture survival ability from myeloma cells)

After a week in HAT media….

Antibody-dependent techniques

• Immunoassays - ELISA and RIA• Immunoprecipitation• Flow cytometry and fluorescence-activated

cell sorting (FACS)• Western blotting• Immunofluorescence and

immunohistochemistry• Surface plasmon resonance

Summary

• Basic antibody structure• IgA, IgD, IgE, IgG, IgM• Isotypes, allotypes, idiotypes• Antigenicity and immunogenicity• Interactions with antigens• Affinity and avidity• Specificity and cross reactivity• Monoclonal antibodies