1. B-Cell Epitopes
Chapter 10
Claus
Lundegaard

2. Antibodies

3. 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?
~ genes in the humans genome!
Immunoglobulin gene rearrangement

4. Antibody Effect. Neutralizing Antibodies
Virus or Toxin
Neutralizing Antibodies
Inhibit cellular infection
Clear pathogen infection

5. Antibody - Antigen interaction
Paratope
Fab (fragment antigen binding)
Epitope
Antibody

6. B-Cells. How are they made?
Stem Cell
Precurser B-lymphocytes
Gene rearrangements
B-lymphocytes each displaying a unique B-cell receptor

7. Gene Shuffling

8. Number of gene segments

9. The 12/23 rule of recombination
recombination signal sequence (RSS) {
Only combined 12 RSS to 23 RSS

10. Mechanism of gene rearrangement

11. RAG proteins (recombination-activating genes)

12. Addition of P and N nucleotides
TdT: terminal deoxynucleotidyl transferase

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

15. CDR Regions CDR = complementarity determining region
Variable regions
Alpha-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 chain
CDR 1 = Light blue
CDR 2 = Cerise
CDR 3 = Yellow
Light Chain
CDR 1 = Red
CDR 2 = Green
CDR 3 = Blue
CDR = complementarity determining region

16. 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.

17. Identification of CDR’s (II)
CDR-H1
Start Approx residue 26 (always 4 after a Cys) [Chothia / AbM defintion];
Kabat definition starts 5 residues later
Residues before always Cys-XXX-XXX-XXX
Residues after always a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-Ala
Length 10 to 12 residues [AbM definition];
Chothia definition excludes the last 4 residues
CDR-H2
Start always 15 residues after the end of Kabat / AbM definition) of CDR-H1
Residues before typically Leu-Glu-Trp-Ile-Gly, but a number of variations
Residues after Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala
Length Kabat definition 16 to 19 residues;
AbM (and recent Chothia) definition ends 7 residues earlier
CDR-H3
Start 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-Gly
Length 3 to 25(!) residues

18. Example
>BU02A02.1
GVQCEVHLLESGGGLVQPGGSLRLSCAASGFTF YSYAMSWVRQAPGKGLEWVSANSGSGGSTY YADSVRGRFTISRDNSKNTLYLQMNSLSAEDT AVYFCAKAPGYYYYYGMDVWGQGTTVTVSSG KNGHSRAFV
15 amino acids after end of CDR1

19. Somatic hypermutations

20. B-Cell Activation (Proliferation depends on affinity)
No Affinity
No Affinity
Low Affinity
Somatic Hypermutations
High Affinity
Plasma cells
Memory B-cells

21. B-Cell Activation
T Helper Cell
TCR
B Cell
Class II MHC
Bound Peptide

22. Cartoon by Eric Reits

23. Is the 11/23 rule always obeyed? Can D genes be inserted backwards?
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?

24. What did we find?
Issue of next talk