B-Cell Epitopes

Chapter 10

ClausLundegaard

Antibodies

Antibodies. What are they?

• Virtually any substance can elicit an antibody response. • Clear extra cellular pathogens• neutralizing antibodies

• Antibody repertoire• > 1011 in humans

• How is this possible?• ~ 30.000 genes in the humans genome!• Immunoglobulin gene rearrangement

Antibody Effect. Neutralizing Antibodies

Virus or Toxin

Neutralizing Antibodies Inhibit cellular infection

Clear pathogen infection

Antibody - Antigen interaction

Fab (fragment antigen binding)

Antigen

Epitope

Paratope

Antibody

B-Cells. How are they made?

B-lymphocytes each displaying a unique B-cell receptor

Stem Cell

Precurser B-lymphocytes

Gene rearrangements

Gene Shuffling

Number of gene segments

The 12/23 rule of recombinationrecombination signal sequence (RSS)

{

Only combined 12 RSS to 23 RSS

Mechanism of gene rearrangement

RAG proteins (recombination-activating genes)

Addition of P and N nucleotides

TdT: terminal deoxynucleotidyl transferase

Antibody variable regions, CDR’s (Complementarity-determining regions)

Variable regionsAlpha-carbon trace of the structure of the heavy chain and light chain variable regions of a typical antibody. The framework regions of both chains are shown in grey whilst the complementarity determining regions (CDRs) are coloured individually, i.e.

Heavy chainCDR 1 = Light blueCDR 2 = CeriseCDR 3 = Yellow Light ChainCDR 1 = RedCDR 2 = GreenCDR 3 = Blue

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

CDR Regions

CDR = complementarity determining region

http://212.219.234.139/html/anti_alpha.html

Identifying CDR regions

• The Kabat definition is based on sequence variability and is the most commonly used• The Chothia definition is based on the location of the structural loop regions• The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software• The contact definition has been recently introduced by us and is based on an analysis of the available complex crystal structures.

Identification of CDR’s (II)

CDR-H1Start Approx residue 26 (always 4 after a Cys) [Chothia / AbM defintion];Kabat definition starts 5 residues laterResidues before always Cys-XXX-XXX-XXXResidues after always a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-AlaLength 10 to 12 residues [AbM definition];Chothia definition excludes the last 4 residues

CDR-H2Start always 15 residues after the end of Kabat / AbM definition) of CDR-H1Residues before typically Leu-Glu-Trp-Ile-Gly, but a number of variationsResidues afterLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/AlaLength Kabat definition 16 to 19 residues;AbM (and recent Chothia) definition ends 7 residues earlier

CDR-H3Start always 33 residues after end of CDR-H2 (always 2 after a Cys)Residues before always Cys-XXX-XXX (typically Cys-Ala-Arg)Residues after always Trp-Gly-XXX-GlyLength 3 to 25(!) residues

Example

>BU02A02.1GVQCEVHLLESGGGLVQPGGSLRLSCAASGFTFYSYAMSWVRQAPGKGLEWVSANSGSGGSTYYADSVRGRFTISRDNSKNTLYLQMNSLSAEDTAVYFCAKAPGYYYYYGMDVWGQGTTVTVSSGKNGHSRAFV

15 amino acids after end of CDR1

Somatic hypermutations

B-Cell Activation (Proliferation depends on affinity) No

AffinityLow Affinity

No Affinity

High Affinity

Somatic Hypermutations

Memory B-cells

Plasma cells

B-Cell Activation

B Cell

T Helper Cell

Class II MHC

Bound Peptide

TCR

Cartoon by Eric Reits

Controversial issues

• Is the 11/23 rule always obeyed?• Can you have multiple D genes?

• Can D genes be inserted backwards?• I.e can both D and the inverted D genes be used?

• Does V, D and J palindrom segments exits?

What did we find?

• Issue of next talk