Antibody Structure Antibody Structure and Functionand Function

Keri C. Smith, Ph.D.Keri C. Smith, Ph.D.

January 22, 2008January 22, 2008(or)

Anatomy and Physiology of Antibodies

OverviewOverview

Physical properties of antibodiesPhysical properties of antibodiesStructural and molecular featuresStructural and molecular features

Differences between types of antibodiesDifferences between types of antibodiesFunctional properties of antibodiesFunctional properties of antibodies

Just a reviewJust a review

Antibodies are gammaAntibodies are gamma--globulinsglobulins

Multiple MyelomaMultiple Myeloma

Case Study #19

γ κ

Anatomy of Ig Light and Heavy Anatomy of Ig Light and Heavy ChainsChains

Porter and Porter and NisonoffNisonoff used enzymes to used enzymes to cleave Ig, studied function of isolated cleave Ig, studied function of isolated fragmentsfragmentsEdelman treated Ig with reducing agents to Edelman treated Ig with reducing agents to cleave Scleave S--S bonds, studied the resulting S bonds, studied the resulting polypeptide chainspolypeptide chains

IgIg

Fragmentation and Domain Fragmentation and Domain StructuresStructures

Fab

portion can bind antigenFc

portion can be crystallized

Ig Light ChainsIg Light Chains

Two types of Light chains are found in Ig of Two types of Light chains are found in Ig of all animals, all animals, aaaa sequence differssequence differsKappa chainsKappa chains--Human 60%, mice 95%Human 60%, mice 95%Lambda chainsLambda chains--Human 40%, mice 5%Human 40%, mice 5%

IgIg

Heavy ChainsHeavy Chains

5 classes of H chains in humans5 classes of H chains in humansSimilarities in Similarities in aaaa sequence, but each class has sequence, but each class has a unique sequencea unique sequenceH chains named with Greek letters H chains named with Greek letters corresponding with the class name, IgG, IgA, corresponding with the class name, IgG, IgA, IgM, IgE, IgM, IgE, IgDIgD ((γγ, , αα, , μμ, , εε, , δδ))IgG has 4 subclasses, IgA has 2 subclassesIgG has 4 subclasses, IgA has 2 subclasses

DomainsDomains

Early studies showed regularity of Early studies showed regularity of structure of all the Ig classesstructure of all the Ig classesEach 100Each 100--110 110 aaaa has a 60 has a 60 aaaa SS--S loopS loopV domains code the paratope, binds V domains code the paratope, binds AgAgC domains code regions important C domains code regions important for mediating secondary biological for mediating secondary biological functions, ie binding Complement, functions, ie binding Complement, crossing the placenta.crossing the placenta.

Human Ig Domain FunctionsHuman Ig Domain Functions

Ig Hinge RegionsIg Hinge Regions

Hinge regions on IgG, Hinge regions on IgG, IgA and IgA and IgDIgD are coded are coded by distinct by distinct exonsexonsShort span of Short span of aaaabetween 1between 1stst and 2and 2ndnd C C domainsdomainsRich in Cys and ProRich in Cys and ProProvides for flexibility Provides for flexibility of the moleculeof the moleculeIs readily accessible to Is readily accessible to solvent and enzymessolvent and enzymes

Ig Variable and Hypervariable Ig Variable and Hypervariable RegionsRegions

Amino terminal Amino terminal aaaa sequence was shown to sequence was shown to vary from one L chain to anothervary from one L chain to anotherKabatKabat and Wu developed the Variability Plot and Wu developed the Variability Plot to measure degree of variationto measure degree of variationFound 3 Hypervariable regions in both L and Found 3 Hypervariable regions in both L and H chain V regions.H chain V regions.These are epitope contact These are epitope contact aaaa regions, CDR regions, CDR ((complementaritycomplementarity determining regions)determining regions)

Definition of VariabilityDefinition of Variability

The ratio of the number of different amino The ratio of the number of different amino acids at a given position to the frequency of acids at a given position to the frequency of the most common amino acid at that position the most common amino acid at that position is defined as VARIABILITY.is defined as VARIABILITY.Example: 63 L chains were sequenced and Example: 63 L chains were sequenced and four amino acids were found at position 7. four amino acids were found at position 7. Serine occurred 41 times, ie frequency Serine occurred 41 times, ie frequency 41/63=0.65. Thus 4/0.65=6.15 Variability41/63=0.65. Thus 4/0.65=6.15 Variability

Variability PlotVariability Plot

CDR in 3DCDR in 3D

Ig Classes or IsotypesIg Classes or Isotypes

Result from structural variation in the heavy Result from structural variation in the heavy chainschainsAre 5 major classes or isotypes of Ig in Are 5 major classes or isotypes of Ig in HumansHumansIgG, IgA, IgM, IgE, IgG, IgA, IgM, IgE, IgDIgDSubclasses: Subclasses: IgGIgG (4) and (4) and IgAIgA (2)(2)

Features of Features of IgIg

IsotypesIsotypes

Ig AllotypesIg Allotypes

Allelic variants of Ig Constant regionsAllelic variants of Ig Constant regionsCoCo--dominant autosomal Mendelian genesdominant autosomal Mendelian genesAre allotypic variants of Are allotypic variants of γγ, , αα, and , and κκ chainschainsAllotypes used forensicallyAllotypes used forensicallyAllotype may confer some biological Allotype may confer some biological advantage for some infectious agentsadvantage for some infectious agents

Human Kappa Light Chain Human Kappa Light Chain AllotypesAllotypes

ALLOTYPEALLOTYPE AMINO ACID AMINO ACID DIFFERENCESDIFFERENCES

Km(1)Km(1) Val @ 153, Val @ 153, LeuLeu

@ 191@ 191

Km(1,2)Km(1,2) Ala @ 153, Ala @ 153, LeuLeu

@ 191@ 191

Km(3)Km(3) Ala @ 153, Val @ 191Ala @ 153, Val @ 191

Allotype effects in infectionAllotype effects in infection

Children with G2m(23) allotype had higher Children with G2m(23) allotype had higher levels of immunity to H. levels of immunity to H. influenzaeinfluenzae type B type B than G2m(23)than G2m(23)--negative children and the negative children and the Km(1) allotype was found more effective Km(1) allotype was found more effective than the other kappa chain allotypes.than the other kappa chain allotypes.AmbrosinoAmbrosino, et al. 1985, J. , et al. 1985, J. ClinClin. Invest. 75:1935; . Invest. 75:1935; GranoffGranoff, et al. 1986, J. Inf. Dis. 154:257., et al. 1986, J. Inf. Dis. 154:257.

Ig IdiotypesIg Idiotypes

IdiosIdios: Greek, : Greek, unique for Ab of one specificity.unique for Ab of one specificity.Are epitopes in Ig V regionsAre epitopes in Ig V regionsProvide a natural autoimmune mechanism for Provide a natural autoimmune mechanism for regulating Ab levelsregulating Ab levelsAntiAnti--id antibodies are routinely found and id antibodies are routinely found and downdown--regulate production of Ab.regulate production of Ab.

Two Examples of IdiotypesTwo Examples of Idiotypes

Clinical Relevance of Clinical Relevance of IdiotypesIdiotypes

Active immunotherapy with 1E10 antiActive immunotherapy with 1E10 anti--idiotypeidiotype vaccine in patients with small cell vaccine in patients with small cell lung cancer: report of a phase I trial.lung cancer: report of a phase I trial.

NeningerNeninger et al. Cancer Biol. et al. Cancer Biol. TherTher. 2007. 6:145. 2007. 6:145

IsoIso, , AlloAllo, or , or IdioIdio??????

Ig Structural FeaturesIg Structural Features

IgG Structural FeaturesIgG Structural Features

IgG Structural Features IIIgG Structural Features II

IgGIgG

SubclassesSubclasses

IgG1, IgG2, IgG3, IgG4IgG1, IgG2, IgG3, IgG4Have about 90% homology in their amino Have about 90% homology in their amino acid sequencesacid sequencesDiffer in the numbers and arrangement of the Differ in the numbers and arrangement of the interchaininterchain disulfide bondsdisulfide bondsThis results in differences in their functional This results in differences in their functional propertiesproperties

IgGIgG

Subclasses (2)Subclasses (2)

Important Differences Between Human IgG Subclasses

IgG1 IgG2 IgG3 IgG4

Occurrence (% of total IgG) 70 20 7 3

Half-life 23 23 7 23

Complement binding + + +++ —

Placental passage ++ ± ++ ++

Binding of monocytes +++ + +++ ±

IgGIgG

Subclasses (3)Subclasses (3)

IgGIgG

Biological Properties (1)Biological Properties (1)

Most common Most common IgIg in serumin serumDistributed approximately equally between Distributed approximately equally between intravascular and intravascular and extravascularextravascular spacesspacesLong halfLong half--lifelife

IgGIgG

RecylcingRecylcing

IgG Biological Properties (2)IgG Biological Properties (2)

Agglutination and PrecipitationAgglutination and PrecipitationPlacental passage (G1, G3, G4)Placental passage (G1, G3, G4)OpsonizationOpsonizationAntibody dependent cell mediated Antibody dependent cell mediated cytotoxicity (G1, G3)cytotoxicity (G1, G3)Complement Activation (G1, G2, G3+)Complement Activation (G1, G2, G3+)Toxin/Viral NeutralizationToxin/Viral Neutralization----Passive Passive AbAbinfusioninfusionBacterial immobilizationBacterial immobilization

Three Roles for Three Roles for IgGIgG

Clinical CorrelationClinical Correlation

Hemolytic disease of the fetus/newborn. Hemolytic disease of the fetus/newborn. Maternal IgG antibodies specific for RhD are Maternal IgG antibodies specific for RhD are actively transported across the placenta, actively transported across the placenta, opsonizeopsonize fetal RhD+ RBC for fetal RhD+ RBC for phagocytosisphagocytosisby liver cells, fetal by liver cells, fetal hematocrithematocrit drops to drops to dangerous or fatal levels. Case 45.dangerous or fatal levels. Case 45.

Antibody Dependent CellAntibody Dependent Cell-- Mediated CytotoxicityMediated Cytotoxicity

IgGIgG

ReviewReview

Gamma Globulin, Enzymatic Fragmentation

L & H Chains, V & C Domains, Hinge Regions, Variability and Hypervariable

regions

4 Isotypes

(Classes)

Allotypes, Idiotypes

IgG

Anatomy: γ

H Chains, κ/λ

L Chains

IgG

Physiology: Agglutination, Placental Passage, Opsonization, ADCC, Complement Binding, Toxin/Viral Neutralization, Bacterial Immobilization, Recycling

IgMIgM

Structural FeaturesStructural Features

IgM Biological PropertiesIgM Biological Properties

Efficient bacterial/viral Efficient bacterial/viral agglutinatoragglutinator, , potentially 10 potentially 10 paratopesparatopes/molecule/moleculeIsohemagglutininIsohemagglutinin--naturally present Ab naturally present Ab reactive with A/B blood groups, barrier to reactive with A/B blood groups, barrier to random transfusion & transplantationrandom transfusion & transplantationMost efficient Ig for mediating Complement Most efficient Ig for mediating Complement fixationfixationPrimary antibody response to challengePrimary antibody response to challenge

Affinity and AvidityAffinity and Avidity

Due to itDue to it’’s structure, s structure, IgMIgM has can have the has can have the same same affinityaffinity, but a higher , but a higher avidityavidity for antigenfor antigen

Blood Groups and Blood Groups and IsohemagglutininsIsohemagglutinins

Jessica Jessica SantillanSantillan

Heart transplant patient

Duke University Hospital, Feb. ‘03

Patient was type O, Donor was type A

Kinetics of a Normal Antibody Kinetics of a Normal Antibody ResponseResponse

IgAIgA

Structural FeaturesStructural Features

IgA SubclassesIgA Subclasses

IgA Secretion MechanismIgA Secretion Mechanism

Biological Properties of IgA Biological Properties of IgA AntibodiesAntibodies

IgA is abundant on mucosal surfaces as a IgA is abundant on mucosal surfaces as a ““First Line DefenseFirst Line Defense””..IgA is bactericidal for Gram negative IgA is bactericidal for Gram negative organisms in the presence of Lysozyme.organisms in the presence of Lysozyme.IgA is an efficient viral IgA is an efficient viral agglutinatoragglutinator, , preventing viral attachment to epithelial cell preventing viral attachment to epithelial cell viral receptors.viral receptors.Plays a role in passive immunotherapy Plays a role in passive immunotherapy –– can can be transferred from mother to child in breast be transferred from mother to child in breast milk. milk.

IgDIgD

Structural FeaturesStructural Features

Biological Properties of Biological Properties of IgDIgD AntibodiesAntibodies

IgDIgD is not found in significant amounts in is not found in significant amounts in serum.serum.Principal function involves initial Ag Principal function involves initial Ag triggering of B cells while bound to the triggering of B cells while bound to the membrane on the surface of B cells.membrane on the surface of B cells.

IgEIgE

Structural FeaturesStructural Features

Biological Properties of IgE Biological Properties of IgE AntibodiesAntibodies

IgE mediates Type I hypersensitivity IgE mediates Type I hypersensitivity reactions.reactions.IgE antibodies bind to IgE antibodies bind to FcFcεε receptors on Mast receptors on Mast cells.cells.Ag binding with the IgE antibody induces Ag binding with the IgE antibody induces degranulationdegranulation, secretion of histamine, , secretion of histamine, heparin, and other pharmacologic agents.heparin, and other pharmacologic agents.Are high IgE levels in parasitic infections.Are high IgE levels in parasitic infections.

IgEIgE--FcFcεε

StructureStructure

IgE CrossIgE Cross--linking Leads to Mast linking Leads to Mast Cell TriggeringCell Triggering

Clinical VignettesClinical Vignettes--AllergiesAllergies

Case #32 Case #32 –– Acute Systemic Anaphylaxis: 22 monthAcute Systemic Anaphylaxis: 22 month--old, ate a cookie with peanut butter in it, describes old, ate a cookie with peanut butter in it, describes the symptoms (BP 40/0, norm is 80/60, pulse 185, the symptoms (BP 40/0, norm is 80/60, pulse 185, norm 80norm 80--90, respiration 76, norm 20. Gave 90, respiration 76, norm 20. Gave epinephrine, saline, antiepinephrine, saline, anti--histamine, corticosteroids. histamine, corticosteroids. Case #33 Case #33 –– Allergic Asthma: 14 year old with Allergic Asthma: 14 year old with persistent wheezing, reduced peak flow rate in lungs persistent wheezing, reduced peak flow rate in lungs of 180 liter minof 180 liter min--11 (normal> 350(normal> 350--400 liter min 400 liter min --11) and ) and reduced expiratory volume. Elevated serum reduced expiratory volume. Elevated serum IgEIgEand and eosinophilseosinophils. Treated with inhaled . Treated with inhaled steriodssteriods, oral , oral antihistamine, and antihistamine, and immunotherpeuticimmunotherpeutic antigen antigen injectionsinjections

KISS OF DEATHKISS OF DEATH

Kinetics of a Normal Antibody Kinetics of a Normal Antibody ResponseResponse

Features of Features of IgIg

IsotypesIsotypes

Ig Ig SuperfamilySuperfamily

There are structural similarities to Ig of the There are structural similarities to Ig of the molecules of numerous membrane bound molecules of numerous membrane bound glycoprotein molecules such as the MHC glycoprotein molecules such as the MHC molecules and the T cell receptor molecules. molecules and the T cell receptor molecules. T cell receptors and triggering will be T cell receptors and triggering will be covered in another lecture.covered in another lecture.

Ig Ig SuperfamilySuperfamily——Some MembersSome Members

Antibody EngineeringAntibody Engineering

Single Chain Fv (Single Chain Fv (ScFvScFv))Humanized antibodiesHumanized antibodiesImmunotoxinsImmunotoxins

CodaCoda

The function of antibodies, like the rest of The function of antibodies, like the rest of the body, is only understood with a firm the body, is only understood with a firm foundation in (molecular) anatomy.foundation in (molecular) anatomy.The 5 Ig isotypes each mediate specific The 5 Ig isotypes each mediate specific biological effects, due to different C region biological effects, due to different C region amino acid sequences in their respective H amino acid sequences in their respective H chain.chain.