B cell Epitopes CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS. Technical University of Denmark - DTU Department of systems biology

Transcription

1 B cell Epitopes

2 Antibody Effect Virus or Toxin Antibodies

3 Antibody Effect Virus or Toxin Antibodies

4 Antibodies

5 Antibodies

6 Antibody - Antigen interaction Antigen Antibody

7 Antibody - Antigen interaction Antigen Fab Antibody

8 Antibody - Antigen interaction Antigen Epitope Fab Antibody

9 Antibody - Antigen interaction Antigen Paratope Fab Antibody

10 B-Cells Gene rearrangements Stem Cell Precurser B-lymphocytes B-lymphocytes each displaying a unique B-cell receptor

11 Figure 4-2

12 Figure 4-3

13 Figure 4-4

14 Figure 4-5

15 Figure 4-6

16 P-nucleotide introduction

17 N-nucleotide introduction

18 Somatic Hypermutations 2001 by Garland Publishing

19 B-Cell Activation No Affinity Somatic Hypermutations High Affinity Memory B-cells Low Affinity No Affinity Plasma cells

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

21 Cartoon by Eric Reits

22 Monoclonal Abs Hybridomas Phage Display

23 Hybridomas

24 Phage Display

25 Phage Display

26 Structural Epitopes

27 Discontinuous Epitopes

28 Discontinuous Epitopes

29 Discontinuous Paratopes

30 CDR Regions 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

31 Identification of germ-line genes Why? As bookkeeping and reporting tool For detection of potentially important residues in binding affinity.

32 Joinsolver

33 Vbase

34 Vquest

35 VDJsolver

36 Questions to be addressed Can multiple D genes be inserted? Violation of 12/23 rule Can D genes be inserted backwards? Is there a D gene preference? Is there a reading frame preference for D genes? If yes, is it part of the gene rearrangement?

37 Data sets 6329 clonally unrelated rearrangements un-mutated functional 3707 mutated functional 274 un-mutated non-functional 380 mutated non-functional

38 P nucleotides Sequences Permutated sequences Distance from heptamer to gene end No. of seq No. with P % with P VH gene No. of seq No. with P % with P p-value < JH gene < end of D gene < end of D gene

39 P nucleotides Sequences Permutated sequences Distance from heptamer to gene end No. of seq No. with P % with P VH gene No. of seq No. with P % with P p-value < JH gene < end of D gene < end of D gene

40 P nucleotides Sequences Permutated sequences Distance from heptamer to gene end No. of seq No. with P % with P VH gene No. of seq No. with P % with P p-value < JH gene < end of D gene < end of D gene

41 How many types of D genes? Conventional D genes Identified in 81% of sequences unmutated sequences, 64% of mutated sequences Inverted D genes Long inverted D genes can not be excluded Two or more D genes

42 D gene usage 27 conventional D genes, 34 known alleles

43 D-gene usage and JH gene JH proximal D genes more often recombined to JH4 than JH6 and JH distal D genes more often to JH6

44 Inverted (palindrom) D genes

45 Inverted (palindrom) D genes Inverted D genes are not used! (or used extremely infrequent)

46 Multiple D genes V-gene Longest-D Shortest-D J-gene 65 sequences with two D genes Average length of shortest D genes: 11.6bp Average length of longest D genes: 18.8bp Average length of D genes in permuted sequences: 11.3bp Average length of D genes in normal sequences: 17.8bp => multiple D genes are not present!!!

47 D gene reading frames Reading Frame Stop Hydrophilic Hydrophobic Gene P NP P NP P NP D2-2*01 RIL**YQLLC (1) GYCSSTSCYA (2) DIVVVPAAM (3) D2-2*02 RIL**YQLLY (1) GYCSSTSCYT (2) DIVVVPAAI (3) D2-2*03 WIL**YQLLC (1) GYCSSTSCYA (2) DIVVVPAAM (3) D2-8*01 RILY*WCMLY (1) GYCTNGVCYT (2) DIVLMVYAI (3) D2-8*02 RILYWWCMLY (1) GYCTGGVCYT (2) DIVLVVYAI (3) D2-15*01 RIL*WW*LLL (1) GYCSGGSCYS (2) DIVVVVAAT (3) D2-21*01 SILWW*LLF (1) AYCGGDCYS (2) HIVVVIAI (3) D2-21*02 SILWW*LLF (1) AYCGGDCYS (2) HIVVVTAI (3) Total The recombination mechanism utilizes each D gene reading frame at same frequency

48 D gene reading frames Reading Frame Stop Hydrophilic Hydrophobic Gene P NP P NP P NP D2-2*01 RIL**YQLLC (1) GYCSSTSCYA (2) DIVVVPAAM (3) D2-2*02 RIL**YQLLY (1) GYCSSTSCYT (2) DIVVVPAAI (3) D2-2*03 WIL**YQLLC (1) GYCSSTSCYA (2) DIVVVPAAM (3) D2-8*01 RILY*WCMLY (1) GYCTNGVCYT (2) DIVLMVYAI (3) D2-8*02 RILYWWCMLY (1) GYCTGGVCYT (2) DIVLVVYAI (3) D2-15*01 RIL*WW*LLL (1) GYCSGGSCYS (2) DIVVVVAAT (3) D2-21*01 SILWW*LLF (1) AYCGGDCYS (2) HIVVVIAI (3) D2-21*02 SILWW*LLF (1) AYCGGDCYS (2) HIVVVTAI (3) Total The recombination mechanism utilizes each D gene reading frame at same frequency

49 VDJsolver performance Unmutated sequences #: p<0.01 : P<0.001 Mutated sequences

50 Results regarding recombination and diversity and open questions DIR, OR15, multiple D genes and VH replacements are not used at a significant rate Inverted D genes are used rarely All D genes not used at same frequency What determines if a D genes is used? D gene usage somewhat dependent on JH gene Does multiple D-J recombination steps take place? All D gene reading frames used at equal rate at the recombination step

51 Results regarding recombination and diversity and open questions (cont.) N addition not random but dependent on end nucleotide Does nucleotide availability or the specificity of TdT determine the N addition? Trimming not random but dependent on gene and sequence What enzyme(s) is responsible for the trimming?

52 Numbering Schemes The Kabat numbering scheme is a widely adopted standard for numbering the residues in an antibody in a consistent manner. However the scheme has problems! The Chothia numbering scheme is identical to the Kabat scheme, but places the insertions in CDR-L1 and CDR-H1 at the structurally correct positions. This means that topologically equivalent residues in these loops do get the same label (unlike the Kabat scheme). The IMGT unique numbering for all IG and TR V-REGIONs of all species relies on the high conservation of the structure of the variable region. This numbering, set up after aligning more than sequences, takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR), structural data from X-ray diffraction studies, and the characterization of the hypervariable loops.

53 Identification of CDR regions CDR-L1 Start Approx residue 24 Residue before is always C Residue after is always W. Typically WYQ, but also, WLQ, WFQ, WYL Length 10 to 17 residues CDR-L2 Start always 16 residues after the end of CDR-L1 Residues before generally IY, but also, VY, IK, IF Length always 7 residues CDR-L3 Start always 33 residues after end of CDR-L2 Residue before is always C Residues after always FGXG Length 7 to 11 residues CDR-H1 Start Approximately residue 31 (always 9 after a C) (Chothia/AbM defintion starts 5 residues earlier) Residues before always CXXXXXXXX Residues after always W. Typically WV, but also WI, WA Length 5 to 7 residues (Kabat definition); 7 to 9 residues (Chothia definition); 10 to 12 residues (AbM definition) CDR-H2 Start always 15 residues after the end of Kabat/AbM definition of CDR-H1 Residues before typically LEWIG, but a number of variations Residues after K[RL]IVFT[AT]SIA (where residues in square brackets are alternatives at that position) Length Kabat definition 16 to 19 residues (AbM definition and most recent Chothia definition ends 7 residues earlier; earlier Chothia definition starts 2 residues later and ends 9 earlier) CDR-H3 Start always 33 residues after end of CDR-H2 (always 3 after a C) Residues before always CXX (typically CAR)