Antibody Structure and Function

(Part I)

Keri C. Smith, Ph.D.January 14, 2014

Keri.C.Smith@uth.tmc.eduMSB 2.218

(or)

Anatomy and Physiology

of Antibodies

Overview

Physical properties of antibodies Structural and molecular features

Differences between types of antibodies Functional properties of antibodies

To review

Antibodies are gamma-globulins

Anatomy of Ig Light and Heavy Chains

Fab portion can bind antigenFc portion can be crystallized

Ig Light Chains

Two types of L chains are found in Ig of all animals, aa sequence differs

Kappa chains-Human 60%, mice 95% Lambda chains-Human 40%, mice 5%

Ig Heavy Chains

5 classes of H chains in humans Similarities in aa sequence, but each class has a

unique sequence H chains named with Greek letters

corresponding with the class name, IgG, IgA, IgM, IgE, IgD (γ, α, μ, ε, δ)

IgG has 4 subclasses, IgA has 2 subclasses

Specific Domains All classes of Ig Each 100-110 aa has a 60 aa S-S loop V domains code the paratope, binds Ag C domains code regions important for mediating

secondary biological functions

Domain Structure

Building blocks of all immunoglobulin superfamilydomains

Human Ig Domain Functions

Ig Variable and Hypervariable Regions

The V regions of every antibody differ from those of every other Ab

Sequence variability is concentrated in certain segments of both L and H chain V regions

3 Hypervariable (HV) regions in both L and H chain V regions.

These are epitope contact aa regions, CDR (complementarity determining regions)

The ratio of the number of different amino acids at a given position to the frequency of the most common amino acid at that position is defined as VARIABILITY

Formation of the CDR

Regions between HV regions are framework regions (FR)

FR form β sheets, HV regions on outer edges of barrel

Total of 6 HV loops = complementarity-determining region (CDR)

(So-called because the surface that is formed is complementary to the antigen that is bound)

The combination of H and L chain HV regions determines final antigen-specificity

Human Ig Domain Functions

Ig Hinge Regions

Short span of aabetween 1st and 2nd C domains

Rich in Cys and Pro Provides for flexibility

of the molecule

Fc portion

The portion of the Ab that allows for interaction of immune complexes with other cells

Many different forms of Fc receptors (FcR) exist May be regulatory or stimulatory

Fc region, multiple functions

Ig Classes or Isotypes

Result from structural variation in the heavy chains

Are 5 major classes or isotypes of Ig in Humans IgG, IgA, IgM, IgE, IgD Subclasses: IgG (4) and IgA (2)

Features of Ig Isotypes

Ig Allotypes

Allelic variants of Ig Constant regions Co-dominant autosomal Mendelian genes Are allotypic variants of γ, α, and κ chains Allotypes used forensically Allotype may confer some biological

advantage for some infectious agents

Human Kappa Light Chain Allotypes

ALLOTYPE AMINO ACID DIFFERENCES

Km(1) Val @ 153, Leu @ 191

Km(1,2) Ala @ 153, Leu @ 191

Km(3) Ala @ 153, Val @ 191

Allotype effects in infection and autoimmunity

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

Immunoglobulin GM and KM genotypes in Korean patients with systemic lupus erythematosus. Lee et al. Rheumatol Int. 2007 Jan;27(3):219-24

Ig Idiotypes

Idios: Greek, unique for Ab of one specificity. Are epitopes in Ig V regions Provide a natural autoimmune mechanism for

regulating Ab levels Anti-id antibodies are routinely found and down-

regulate production of Ab.

Two Examples of Anti-Idiotypic Ab

Clinical Relevance of Idiotypes

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

Neninger et al. Cancer Biol. Ther. 2007. 6:145

Anti tumor mAb

Anti- Anti tumor mAb

Anti-Anti –anti-tumor mAb

Also reacts with tumor antigen

Iso, Allo, or Idio???

Ig Structural Features

IgG Structural Features

IgG Structural Features II

IgG Subclasses

IgG1, IgG2, IgG3, IgG4 Have about 90% homology in their amino acid

sequences Differ in the numbers and arrangement of the

interchain disulfide bonds This results in differences in their functional

properties

IgG 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 +++ + +++ ±

IgG Subclasses (3)

IgG Biological Properties (1)

Most common Ig in serum Distributed approximately equally between

intravascular and extravascular spaces Long half-life

IgG Recycling

Roopenian & Akilesh Nature Reviews Immunology 7, 715-725 (September 2007)

IgG Biological Properties (2)

Agglutination and Precipitation Placental passage (G1, G3, G4) Opsonization Antibody dependent cell mediated cytotoxicity

(G1, G3) Complement Activation (G1, G2, G3+) Toxin/Viral Neutralization--Passive Ab infusion Bacterial immobilization

Three Roles for IgG

Antibody Dependent Cell-Mediated Cytotoxicity

Clinical Correlation

Hemolytic disease of the newborn. Case #45 Mom is Rh-. Dad is Rh+. 2 normal pregnancies. 3rd pregnancy:

increased indirect Coombs titer (reaction of mom’s Ab with Rh+ red cells)

Increased billirubin in amniotic fluid Low hematocrit in umbilical cord blood

Treatment = Infusion of fetus with type O Rh-RBC

Mechanism of fetal RBC destruction

Maternal IgG antibodies specific for Rh are actively transported acrossthe placenta

Opsonize fetal Rh+ RBC for phagocytosis by liver cells

Fetal hematocrit drops to dangerous or fatal levels. Prophylactic treatment = Rhogam

IvIg

Purified, polyclonal Igpooled from plasma of thousands of donors

In use since the 1980s, originally for immunodeficiency disorders

Also being used in: Solid organ transplantation,

Wegener's granulomatosis,vasculitis, heart failture, spinal chord injury, etc……..

NEJM 2001, Volume 345:747-755

How does IvIg work?

Inhibition of complement activation?

Direct effects on B cells T cells (Treg) Dendritic cells Macrophages

FcRIIb binding?

Trends in Immunology Volume 29, Issue 12, December 2008, Pages 608-615

IgG Review

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 (long half life)