biased antibody repertoires: from concept to implementation department of biological sciences...
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Biased Antibody Repertoires:From Concept to Implementation
Department of Biological SciencesFlorida International University
Miami, Florida
May - July 2005
Juan C. Almagro, Ph.D
1. Statement of the problem
3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens
Plan of the talk
4. Design and Validation of Topography-Biased Antibody Libraries
2. Structure-Function relationships in antibodies
However, we cannot predict the specificity of a given antibody sequence or structure.
Hence, Our understanding of the evolution of the antibody repertoire is limited and
antibodies cannot be designed de novo.
0
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1998
2000
year
Num
ber
of s
truc
ture
s
cumulative
releases per year
• Today: ~ 600 structures are available at PDB (ABG; Antibody: Structure-function web site and IMGT)
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5000
10000
15000
20000
25000
1965
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year
Num
ber
of s
eque
nces
Cumulative
Sequences per year
• Today: ~ 40 K sequences are available in databases, including the complete repertoire of germline genes from several species (IMGT)
Statement of the problem
1. Statement of the problem
3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens
Plan of the talk
4. Design and Validation of Topography-Biased Antibody Libraries
2. Structure-Function relationships in antibodies
Taken from Andreas Plückthun’s home page, with permission
Canonical structures(Chothia and Lesk, J. Mol. Biol. 196: 901, ‘87)
Type 1
Type 3
L1
~ 4,000 antibody sequences
Complete sequences
VL:VH dimmers
Canonical structure in L1, L2, L3, H1 and H2
381 VL:VH sequences
Predicting the Specificity of Antibody Sequences Based on the Structure of the Antigen-Binding Site
Found 10
0
5
10
15
20
25
1-2-
2-1-
1
1-2-
4-1-
1
1-3-
4-1-
1
1-3-
2-1-
1
1-2-
1-1-
1
1-4-
3-1-
1
1-4-
4-1-
1
1-1-
4-1-
1
1-1-
2-1-
1
1-2-
3-1-
1
othe
rs
ExpectedL1: 5
L2: 1
L3: 5 x
H1: 3
H2: 4 x25 12= 300x
Canonical structure classes in the known sequences
Vargas-Madrazo et al., J. Mol. Biol. 254: 497, ‘95
Only L1 and H2 contribute
to the structural diversity
Canonical Structure Classes
make ~ 90% of the sample
Canonical Structure class
H1-H2-L1-L2-L3 Frequency
(%)
Protein(169)
Surface Antigen(22)
Polysaccharide(17)
Nucleic acid(42)
Peptide(19)
Hapten(112)
1-1-2-1-1 3.2 56d 0 0 25 0 19
1-1-4-1-1 3.7 10 0 33 13 0 44
1-2-1-1-1 7.1 5 5 80 5 0 4
1-2-2-1-1 24.5 16 44 0 18 0 23
1-2-3-1-1 2.9 57 43 0 0 0 0
1-2-4-1-1 14.2 11 4 5 24 52 4
1-3-2-1-1 7.9 15 26 0 31 20 8
1-3-4-1-1 10 43 0 14 11 25 6
1-4-3-1-1 6.8 11 0 0 0 0 89
1-4-4-1-1
others
6.6
13.1
4
26
0
17
0
17
41
6
0
21
55
13
Canonical structure classes
Antigen size
classified in gross specificities
Vargas-Madrazo et al., J. Mol. Biol. 254: 497, ‘95
Anti-protein Anti-peptide Anti-hapten
Topography-specificity relationship
Model to correlate loop lengths (in particular L1 and H2) with the specificity
Predicting the Specificity of Antibody Sequences Based on the Structure of the Antigen-Binding Site
59 unique antibody structures
19 anti-protein
18 anti-peptide
22 anti-hapten
Determine residues in contact
~ 300 structures
0.0
0.5
1.0
Protein
Peptide
Hapten
Residues in contact with proteins, peptides and haptens
L1 L2 L3
SD
R u
sage
0.0
0.5
1.0
Protein
Peptide
Hapten
SD
R u
sage
H1 H2 H3Almagro. J. Mol. Recognit. 17:132, ‘04
Some positions in the antigen-binding siteinteract with the antigen very often (> 70%of the antibodies).
Others do so with a frequency between 30% and 70%.
A third group interacts with the antigen infrequently (<30%).
The frequency of contacts differs depending upon the type of antigen with which the antibody interacts.
Anti-protein Anti-peptide Anti-hapten
Contact usage - specificity relationship
Model to create diversity in the antigen-binding site as a function of the specificity
Conclusions I
1. Model to correlate the structure of the antigen binding site
with its specificity.
2. Guide for tailoring the antigen-binding site diversity depending upon the type of antigen
the antibody interacts with.
1. Statement of the problem
3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens
Plan of the talk
4. Design and Validation of Topography-Biased Antibody Libraries
2. Structure-Function relationships in antibodies
Full diversity: 20 aa + 1 amber codon in positions often found in contact with
proteins and peptides
Limited Diversity: YDAS (Felluose et al., PNAS 34, 12467, ‘04)
R/K
To simplify the diversity in positions of medium
usage while avoiding stop codons
To explore all amino acid variants in
positions with high contact usage
All the germline genes have R at
this position, except dp47 that has K
Dp47 scaffold
VH Repertoire with Tailored Diversity
Theoretical diversity: 720 x 94 x 2 = 6.7 x 1014 variants
Construction of the VH repertoire
1 10 20 30 40 50 60 70 80 90 100 110 |...|....|....|....|....|....|....|....|....|....|..a..|....|....|....|....|....|..abc..|....|....|a....|....|....|.. 1 3 5 7 9 Leader |||||||<<<<||||||| |||||||<<<<<<||||||||||||||<<<<<<<<<<||||||| |||||||<<<||||||| |||||||<<<<<||||||| |||||||<<<LLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFOOOOMOWVRQAPGKGLEWVSOIOOOOGOTOYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAOOOOOYFDYWGQGTLVTVSSGGG>>>>>>>>>||||||| |||||||>>>>||||||| |||||||||||||| |||||||>>>>||||||| |||||||>>>||||||| |||||||>|||||||linker
2 4 6 8 10
Size:
3 x 108
members
The repertoire was synthesized by overlapping PCR (Stemmer et al., Gene. 164:49, ‘95)
in a single-step PCR reaction by using 10 internal oligonucleotides and two amplification primers
pIT
D1.3-VLVH repertoire
*(His)5linker
Selection
0.00
1.00
2.00
3.00
4.00
1.00E+10 1.00E+11 1.00E+12 1.00E+13 1.00E+14
Titer (pfu/mL)
OD
(450
nm
)
KM13
D1.3
Round 1
Round 2
HEL-coated immunotubes
Validation of the VH repertoire
Polyclonal ELISA
Chimeric library
Washed away
non-bound Ф
Trypsin to elute
bound Ф
Characterization
In Round 2, the ELISA signal was similar to D1.3 displayed on the phage
Frequency of scFv’s (hit rate)
0.000
0.500
1.000
1.500
2.000
2.500
1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161
Clone number
OD
(45
0 nm
)
Clone Frequency OD
H130 35| . . . . |
H250 55| . . a . | . . . .
H3 100. | a b c
D1.3 1.230 T G Y G V N M I W - G D G N T D R E R D Y RJCAV-II-HEL-C6-1 1 0.434 A A S Y M S S I A S Y D G A T D R E D V Y YJCAV-II-HEL-E3-1 1 0.597 Y N D S . A . . Y . S Y . D . D K . G A M .JCAV-II-HEL-B3-1 1 0.597 . H D A . A T . H A . S . S . D . . E . F .JCAV-II-HEL-C7-1 2 1.254 . G . S . . Y . G A S S . D . D . . K P Q .JCAV-II-HEL-F8-1 1 1.657 . S A A . A . . Y . S Y . D . S . . G A M .JCAV-II-HEL-B8-1 2 1.779 D D . D . . Y . G Y . Y . D . A . . R A T .JCAV-II-HEL-B7-1 5 1.747 S K A A . A . . S A S S . D . D . . M P T .JCAV-II-HEL-F8-2 3 1.925 Y N . A . D G . L . D S . D . Y . . E A T .JCAV-II-HEL-E8-2 1 1.984 . F D S . . . . G A A A . D . S . . M P L .JCAV-II-HEL-H6-1 3 2.276 S T D A . A D . T A D S . D . A . . R P L .
select clones
Grow in 2xTY
IPTG
Test in ELISA
Expression and relative affinity
0
0.25
0.5
0.75
1
1 10 100 1000
Log (Dilution)
Rel
ativ
e Sc
ale* D1.3
HELII-B7
HELII-H6
*Relative Scale:
The chimeric scFvs are 7-9 times better expressed than D1.3 as suggested by the ED50
1- (Max-O.D. / Max - Min)
The affinity for HEL may be similar to D1.3 as suggested by the slope of the curves
HEL-D1.3 affinity 5 nM ( Foote and Winter, J. Mol. Biol. 224: 487, ‘92)
Conclusions II
4. The scFvs dominating the population are well expressed in E. coli and may have affinities in the nM range.
1. A VH repertoire with tailored diversity to recognize proteins and peptides was designed and constructed
3. After the second round of selection on HEL-coated Immunotubes, diverse scFvs against HEL were obtained, thus validating the library
as source of VH domains.
2. It was cloned with the VL chain chain of D1.3 to yield a chimeric library of 3 x108 members.
1. Statement of the problem
3. Design and Validation of a VH Repertoire with Tailored Diversity for Protein and Peptide Antigens
Plan of the talk
4. Design and Validation of Topography-Biased Antibody Libraries
2. Structure-Function relationships in antibodies
Dp47 scaffold with tailored diversity for proteins and peptides (Almagro et al., J. Mol. Biol. Submitted)
Topography biased antibody libraries
Invariant VL chain with a long L1 Invariant VL chain with a short L1
Theoretical diversity: 2.1 x 1010
Invariant VL chains
Short L1
0
10
20
30
0 5 10 15 20 25 30 35 40 45
A27
Use
Fre
quen
cy (
%)
Human Germline Genes (IGMT)
The difference between repertoires is reduced to one insertion of 5 amino acids at the tip of L1 and 5 mutations in L1,
positions: 28, 29, 30, 30a and 31
Long L1
Numbering 30 ..|....|abcdef....|A27 CRASQSVSS-----SYLAW | |||| ||||1-4 (B3)CKSSQSVLYSSNNKNYLAW |||||||||||||||||||A27md CKSSQSVLYSSNNKNYLAW
Graft L1 of B3 in A27
Combination with different invariant VL chains
Size:
3.6 x 108
memberspIT
A27/Jk1VH repertoire
*(His)5linker
Anti-protein repertoire
Size:
6 x 107
memberspITA27/Jk1modVH repertoire
*(His)5linker
Anti-peptide repertoire
Insertion of 5 aminoacids at L1
Panel of Selectors
V3 loop gp120(V3)
14 aa; 1.6 KDa
Hen Egg White Lysozyme(HEL)
129 aa; 14.3 KDa
Bovine Serum Albumin(BSA)
583 aa; 66.4 KDa
V3-BSA conjugate ND
Selections conducted as described for VH-D1.3
Polyclonal ELISA after Round 3V3 selections
V3-BSA selections
BSA selections
HEL selections
0.000
1.000
2.000
3.000
4.000
1.0E+07 1.0E+09 1.0E+11 1.0E+13Titer (cfu/mL)
O.D
. 450
nm
0.000
1.000
2.000
3.000
4.000
1.0E+07 1.0E+09 1.0E+11 1.0E+13
Titer (cfu/mL)
O.D
. 45
0 nm
0.000
1.000
2.000
3.000
4.000
1.0E+07 1.0E+09 1.0E+11 1.0E+13
Titer (cfu/mL)
O.D
. 450
nm
0.000
1.000
2.000
3.000
4.000
1.0E+07 1.0E+09 1.0E+11 1.0E+13
Titer (cfu/mL)
O.D
. 450 n
m
Red: Anti-peptide library Blue: Anti-protein library
Frequency of positive clones
32/96
42/192
0/960/96
91/96 92/96
64/192
3/96
Anti-peptide library yields more scFvs for V3 and V3-BSA than for proteins
Anti-protein library yields more scFvs for proteins than forV3 or V3-BSA
0
20
40
60
80
100
V3 V3-BSA BSA HEL
Fre
quen
cy (
%)
LL1 library
SL1 library
select clones
Grow in 2xTY
IPTG
Test in ELISA
Unique scFvsDetermined by DNA sequencing
Library Selector Unique / Totalpep V3 1/6pep V3-BSA 1/6pep BSA -pep HEL 2/5pro V3 -pro V3-BSA 1/3pro BSA 2/5pro HEL 2/6
Specificity of unique clones O
.D. 4
5 0 n
m
O.D
. 45 0
nm
V3 and V3-BSA selections
BSA selections
HEL selections
V3 V3-BSA BSA HEL
0.000
1.000
2.000
LL1-B2
LL1-B1
O.D
. 45 0
nm
V3 V3-BSA BSA HEL
0.000
1.000
2.000 SL1-H1
SL1-H2
LL1-H1
LL1-H2
The scFv selected from the anti-peptide lib. on V3 is specific for V3 and V3-BSA
The scFv selected from the anti-protein lib. on V3-BSA is specific for the carrier
ScFvs selected on proteins are specifics
V3 V3-BSA BSA HEL
0.000
1.000
2.000
SL1-VB1
LL1-V1
Expression and Relative Affinity
HEL selectionsO
.D. 4
5 0 n
m
0.00
0.50
1.00
1.50
2.00
2.50
3.00
1 10 100 1000
SL1-H1
SL1-H2
LL1-H1
LL1-H2
D1.3
Different dynamic ranges, better slopes and higher ED50 indicating differences in binding (different epitopes?)
ScFvs from SL1 (anti-protein library) look better than the those isolated from LL1 (anti-peptide library)
General Conclusions
1. Anti-protein library produced diverse specific scFvs against two protein models: BSA and HEL.
2. Anti-protein library did not produce scFvs against the peptide model, free or conjugated. Only against the carrier.
3. Anti-peptide library produced specific scFvs against the peptide.
5. Anti-peptide library did not produce scFvs against BSA and against HEL produced less binders than the anti-protein library.
6. Together, these results suggest that antibody libraries can be biased toward the recognition of different kinds of antigens based on structural principles
4. Anti-peptide library produced less binders against HEL than the anti-protein library.
AcknowledgmentsFlorida International UniversityAlvaro Velandia
Matt Osentoski
Sylvia L Smith
National University of MexicoLuisa Fernadez
Alejandra Blancas
Enesto Ortiz
Baltazar Beceril
Lourival Possani
Alejandro Alagon
This work was supported by:
•Grant 1R03AI057752-01 from NIH/NIAID •Sub-Contract DAAD13-03-C-0065 from CBD/USF.
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