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Biased Antibody Repertoires:
From Concept to Implementation
Juan C. Almagro, Ph.D
Department of Biological Sciences
Florida International University
Miami, Florida
May - July
2005
4. Design and Validation of
Topography-Biased Antibody Libraries
year
2000
1998
1996
1994
1992
1990
0
1988
1999
1997
1995
1993
1991
1989
1987
1985
1983
1981
1979
1977
1975
1973
1971
1969
1967
0
100
1986
5000
releases per year
200
1984
10000
1982
15000
cumulative
300
1980
Sequences per year
400
1978
Cumulative
20000
1976
Number of structures
25000
1965
Number of sequences
Statement of the problem
year
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.
4. Design and Validation of
Topography-Biased Antibody Libraries
Canonical structures
(Chothia and Lesk, J. Mol. Biol. 196: 901, ‘87)
Taken from Andreas Plückthun’s home page, with permission
Type 3
L1
Type 1
Predicting the Specificity of Antibody Sequences
Based on the Structure of the Antigen-Binding Site
~ 4,000 antibody sequences
Complete sequences
VL:VH dimmers
Canonical structure in
L1, L2, L3, H1 and H2
381 VL:VH sequences
Canonical structure classes
in the known sequences
L1: 5
Expected
L2: 1
x
L3: 5
x
H1: 3
25
H2: 4 x
12
= 300
25
10
20
15
Canonical Structure Classes
10
make ~ 90% of the sample
5
others
1-2-3-1-1
1-1-2-1-1
1-1-4-1-1
1-4-4-1-1
1-4-3-1-1
1-2-1-1-1
1-3-2-1-1
1-3-4-1-1
to the structural diversity
1-2-4-1-1
0
Only L1 and H2 contribute
1-2-2-1-1
Found
Vargas-Madrazo et al., J. Mol. Biol. 254: 497, ‘95
Canonical structure classes
classified in gross specificities
Canonical Structure class
H1-H2-L1-L2-L3
Frequency
Surface Antigen
(22)
Polysaccharide
(17)
Nucleic acid
(42)
Peptide
(19)
(%)
Protein
(169)
Hapten
(112)
1-1-2-1-1
3.2
56
d
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
6.6
4
0
0
41
0
55
others
13.1
26
17
17
6
21
13
Antigen size
Vargas-Madrazo et al., J. Mol. Biol. 254: 497, ‘95
Topography-specificity relationship
Anti-protein
Anti-peptide
Anti-hapten
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
~ 300 structures
59 unique antibody structures
19 anti-protein
18 anti-peptide
22 anti-hapten
Determine residues in contact
Residues in contact with proteins,
peptides and haptens
1.0
SDR usage
Protein
Some positions in the antigen-binding site
interact with the antigen very often (> 70%
of the antibodies).
Peptide
Hapten
0.5
Others do so with a frequency between 30%
and 70%.
0.0
L1
L2
L3
A third group interacts with the antigen
infrequently (<30%).
1.0
Protein
The frequency of contacts differs depending
upon the type of antigen with which the
antibody interacts.
SDR usage
Peptide
Hapten
0.5
0.0
H1
H2
H3
Almagro. J. Mol. Recognit. 17:132, ‘04
Contact usage - specificity relationship
Anti-protein
Anti-peptide
Anti-hapten
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.
4. Design and Validation of
Topography-Biased Antibody Libraries
VH Repertoire with Tailored Diversity
Dp47 scaffold
Full diversity: 20 aa
+ 1 amber codon in positions
often found in contact with
proteins and peptides
R/K
All the germline genes have R at this
To explore all amino acid variants in
position, except dp47 that has K
positions with high contact usage
Limited Diversity: YDAS
(Felluose et al., PNAS
34, 12467, ‘04)
To simplify the diversity in positions of medium
usage while avoiding stop codons
Theoretical diversity:
720 x 94 x 2 = 6.7 x 1014 variants
Construction of the VH repertoire
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
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
D1.3-VL
VH repertoire
linker
pIT
*
(His)5
Size:
3 x 108
members
Validation of the VH repertoire
Chimeric
library
Washed away
Trypsin to elute
non-bound Ф
bound Ф
Characterization
Selection
HEL-coated immunotubes
KM13
4.00
Polyclonal ELISA
OD (450 nm)
D1.3
3.00
Round 1
2.00
Round 2
1.00
0.00
1.00E+10
1.00E+11
1.00E+12
1.00E+13
1.00E+14
Titer (pfu/mL)
In Round 2, the ELISA signal was similar to D1.3 displayed on the phage
Frequency of scFv’s (hit rate)
2.500
select clones
OD (450 nm)
2.000
1.500
1.000
0.500
Grow in 2xTY
0.000
1
11
21
31
41
51
61
71
81
91 101 111 121 131 141 151 161
Clone number
IPTG
Test in ELISA
Clone
D1.3
JCAV-II-HEL-C6-1
JCAV-II-HEL-E3-1
JCAV-II-HEL-B3-1
JCAV-II-HEL-C7-1
JCAV-II-HEL-F8-1
JCAV-II-HEL-B8-1
JCAV-II-HEL-B7-1
JCAV-II-HEL-F8-2
JCAV-II-HEL-E8-2
JCAV-II-HEL-H6-1
Frequency
OD
1
1
1
2
1
2
5
3
1
3
1.230
0.434
0.597
0.597
1.254
1.657
1.779
1.747
1.925
1.984
2.276
H1
30
35
| . . . . |
H2
50
55
| . . a . | . . . .
H3
100
. | a b c
T
A
Y
.
.
.
D
S
Y
.
S
M
S
.
T
Y
.
Y
.
G
.
D
R
R
K
.
.
.
.
.
.
.
.
G
A
N
H
G
S
D
K
N
F
T
Y
S
D
D
.
A
.
A
.
D
D
G
Y
S
A
S
A
D
A
A
S
A
V
M
.
.
.
.
.
.
.
.
.
N
S
A
A
.
A
.
A
D
.
A
I
I
.
.
.
.
.
.
.
.
.
W
A
Y
H
G
Y
G
S
L
G
T
S
.
A
A
.
Y
A
.
A
A
G
Y
S
.
S
S
.
S
D
A
D
D
D
Y
S
S
Y
Y
S
S
A
S
G
G
.
.
.
.
.
.
.
.
.
N
A
D
S
D
D
D
D
D
D
D
T
T
.
.
.
.
.
.
.
.
.
D
D
D
D
D
S
A
D
Y
S
A
E
E
.
.
.
.
.
.
.
.
.
R
D
G
E
K
G
R
M
E
M
R
D
V
A
.
P
A
A
P
A
P
P
Y
Y
M
F
Q
M
T
T
T
L
L
R
Y
.
.
.
.
.
.
.
.
.
Expression and relative affinity
Relative Scale*
1
0.75
D1.3
HELII-B7
0.5
HELII-H6
0.25
*Relative Scale:
1- (Max-O.D. / Max - Min)
0
1
10
100
1000
Log (Dilution)
The chimeric scFvs are 7-9 times better expressed than D1.3
as suggested by the ED50
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
1. A VH repertoire with tailored diversity
to recognize proteins and peptides was designed and constructed
2. It was cloned with the VL chain chain of D1.3 to yield
a chimeric library of 3 x108 members.
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.
4. The scFvs dominating the population are well expressed in E. coli and
may have affinities in the nM range.
4. Design and Validation of
Topography-Biased Antibody Libraries
Topography biased antibody libraries
Dp47 scaffold with tailored diversity
for proteins and peptides (Almagro et al., J. Mol. Biol. Submitted)
Theoretical diversity:
2.1 x 1010
Invariant VL chain with a long L1
Invariant VL chain with a short L1
Invariant VL chains
Use Frequency (%)
Short L1
Long L1
30
A27
20
10
0
0
5
10
15
20
25
30
35
40
45
Numbering
30
..|....|abcdef....|
A27
CRASQSVSS-----SYLAW
| ||||
||||
1-4 (B3)CKSSQSVLYSSNNKNYLAW
|||||||||||||||||||
A27md
CKSSQSVLYSSNNKNYLAW
Human Germline Genes (IGMT)
Graft L1 of B3 in A27
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
Combination with different
invariant VL chains
linker A27/Jk1
VH repertoire
*
(His)5
pIT
Anti-protein repertoire
Size:
3.6 x 108
members
Insertion of 5 aminoacids at L1
VH repertoire
linker A27/Jk1mod
pIT
Anti-peptide repertoire
*
(His)5
Size:
6 x 107
members
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 3
V3 selections
BSA selections
4.000
O.D. 450 nm
O.D. 450 nm
4.000
3.000
2.000
1.000
3.000
2.000
1.000
0.000
0.000
1.0E+07
1.0E+09
1.0E+11
1.0E+13
1.0E+07
1.0E+09
1.0E+11
1.0E+13
Titer (cfu/mL)
Titer (cfu/mL)
HEL selections
4.000
4.000
3.000
3.000
O.D. 450 nm
O.D. 450 nm
V3-BSA selections
2.000
1.000
0.000
2.000
1.000
0.000
1.0E+07
1.0E+09
1.0E+11
Titer (cfu/mL)
Red: Anti-peptide library
1.0E+13
1.0E+07
1.0E+09
1.0E+11
Titer (cfu/mL)
Blue: Anti-protein library
1.0E+13
Frequency of positive clones
91/96
100
select clones
Frequency (%)
Grow in 2xTY
80
60
40
92/96
LL1 library
SL1 library
32/96
64/192
42/192
20
0/96
3/96
0/96
0
IPTG
Test in ELISA
V3
V3-BSA
BSA
HEL
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
Unique scFvs
Determined by DNA sequencing
Library
pep
pep
pep
pep
pro
pro
pro
pro
Selector
V3
V3-BSA
BSA
HEL
V3
V3-BSA
BSA
HEL
Unique / Total
1/6
1/6
2/5
1/3
2/5
2/6
Specificity of unique clones
V3 and V3-BSA
selections
BSA selections
2.000
2.000
SL1-VB1
LL1-B2
LL1-V1
LL1-B1
1.000
1.000
0.000
V3
V3-BSA
BSA
0.000
HEL
The scFv selected from the anti-peptide
lib. on V3 is specific for V3 and V3-BSA
V3
V3-BSA
BSA
HEL
HEL selections
2.000
SL1-H1
SL1-H2
The scFv selected from the anti-protein lib.
on V3-BSA is specific for the carrier
LL1-H1
LL1-H2
1.000
ScFvs selected on proteins
are specifics
0.000
V3
V3-BSA
BSA
HEL
Expression and Relative Affinity
HEL selections
SL1-H1
3.00
SL1-H2
2.50
LL1-H1
2.00
LL1-H2
1.50
D1.3
1.00
0.50
0.00
1
10
100
1000
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.
4. Anti-peptide library produced less binders against HEL than the anti-protein
library.
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
Acknowledgments
Florida International University
Alvaro Velandia
Matt Osentoski
Sylvia L Smith
National University of Mexico
Luisa 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.