ALTHEA Gold Libraries™ Novel Antibody Libraries for Therapeutic Antibody
Discovery
Abstract
Philippe Valadon1, Renae Nelson1
1 Antibody Design Labs, San Diego, CA 92121
Philippe Valadon1, Sonia M. Pérez-Tapia2, Renae S. Nelson1, Omar U. Guzmán-Bringas2, Hugo I. Arrieta-Oliva2, Keyla M. Gómez-Castellano2, Mary Ann Pohl3, Juan C. Almagro4
1 Antibody Design Labs, San Diego, CA 92121 3 Tri-Institutional Therapeutics Discovery Institute, New York, NY 10021 2 UDIBI, ENCB, Instituto Politécnico Nacional, México 2009-2013 4 GlobalBio Inc., Cambridge, MA 02138
Multi-stage Antibody Library Construction
ALTHEA Gold libraries™
ALTHEA Gold Libraries™ are highly diversified scFv libraries (> 1010 cfu each) enriched for developable antibodies resulting from the combination of in
vitro selected scaffolds with in vivo validated CDR-H3 diversity.
Flat scaffold for protein targets
Grooved scaffold for peptide targets
3-23/3-20 PDB: 5I1i
3-23/4-1 PDB: 5I1d
Alternative Topographies to Bind Protein and Peptide Targets
The shape of the antigen-binding site determines the type of the antigen the antibody interacts with.
Finlay & Almagro. Front Immunol. 3:342, 2012
ALTHEA Gold Libraries™ scaffolds provide two alternative antigen-binding site topographies: One flat suitable for protein antigens and the
other grooved biased towards the recognition of peptides.
Antigen-binding site
Protein-A
The VH scaffolds binds Protein-A and enables filtration of well-folded antibodies. One of the VL binds Protein-L and enables assay
development and identification of variants coming out of the anti-peptide libraries when used combined with the anti-protein library
Also…
Biopanning with Protein Models
Quality Control of ALTHEA Gold Libraries™
Summary
Easy to analyze
Highly Diverse
Developable antibodies
>1010 members
Two alternative topographies +
CDR-H3 with a Gaussian distribution of lengths from 1 to 20
Highly stable (Tm >700C)
One universal VH scaffold Two alternative VL scaffolds
Known structures Protein-A/L binding
~70% (>7 x 109) unique & functional antibody fragments
Philippe Valadon, M.D., Ph.D. [email protected] Tel: +1.858.480.6213 www.abdesignlabs.com
Juan C. Almagro, Ph.D. [email protected] Tel: +1.617.710.4487 www.globalbioinc.com
For Information, please contact:
Each set of ALTHEA Gold Libraries™ is unique, combining carefully designed human scaffolds of improved developability with natural CDR-H3 diversity. Please direct your inquiry for ownership to Antibody Design Labs or GlobalBio, Inc.
Protein-A : scFv complex PDB: 1dee
Protein-L : scFv complex PDB: 1mhh
Deep Sequencing of Over One Million of H3J fragments
0
2
4
6
8
10
12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Anti-Protein
Anti-Peptide
The potential of ALTHEA Gold Libraries™ to generate specific and diverse antibodies has successfully been validated with
several undisclosed targets, including a soluble target, a peptide/protein complex and a membrane protein
After reshuffling natural H3J diversity from the pool of 200 donors into selected scFvs for high stability in the primary libraries, all the clones are unique, with
~70% being highly stable
Frequency (%)
CDR-H3 length (Kabat’s definition)
0
10
20
30
40
Anti-Protein
Anti-Peptide
Human IGHJ germline gene usage
Frequency (%)
We present here, the design, implementation and validation of ALTHEA (from the Greek “to heal”) Gold Libraries™. ALTHEA Gold Libraries™ are semisynthetic scFv libraries built on synthetic human well-known VH and VL germline genes combined with natural H3J fragments obtained from a pool of 200 human donors. The three germline genes provide a universal VH scaffold paired with two distinct VL scaffolds. The VL scaffolds offer two alternative antigen-binding site topographies to bind protein or peptide targets. The antigen-binding site (except the CDR-H3) diversity is designed in positions identified as in contact with protein and peptide antigens in the known antigen-antibody complex structures. The diversification regimes are designed based on the amino acid frequencies of the repertoire of human germline genes and known human antibody sequences. Through a proprietary process comprising the selection for thermostable scFv variants, followed by shuffling of the natural H3J fragments, ALTHEA Gold Libraries™ provide a highly diverse, functional and developable repertoire of human antibody fragments suitable for antibody therapeutic discovery and development. The potential of ALTHEA Gold Libraries™ to generate specific antibody fragments has been assessed with multiple targets including protein models (Hen Egg White Lysozyme; HEL, Human Serum Albumin; HSA), several therapeutic targets including TNF, a protein/peptide complex, and membrane proteins. In all case studies, diverse and specific antibodies were obtained with Kd in the low nM range.
Carefully designed scaffolds with neutral CDR-H3 fragments,
displayed as scFvs
Heat shock
Highly stable scFv library
Rescue with Protein A
200 donors Age < 40 years
100 males & 100 females
RT-PCR
with specific
primers
10 min @ 700C
Diverse H3J fragments
Primary Libraries Natural H3J fragments
Sfi I
VH (3-23)
CDR-H3 +
Joining
Linker GS19 Vk (3-20/4-1)
PelB
Sfi I
Neutral/Natural
Diversity
~ 100 bp
Synthetic
~ 800 bp
Tags Designed Diversity Designed Diversity
Diversity: 106 Diversity: 106
Protein-L
Antigen-binding site
HEL-coated
Immunotubes
100 ug/mL
TDI libraries
Washed away
non-bound Ф
bound Ф
Eluted with Trypsin
Rescued
with helper Ф
Anti-Protein
or
Anti-Peptide
(1012 Ф) ~100x coverage
Plated in 2xYT/Carb
Human Serum Albumin ALTHEA Gold Libraries™ SL2 by Direct Capture
Yield specific scFvs against multiple targets using diverse
biopanning strategies Highly functional
Ph
ag
eo
utp
ut
(UFC
/ml )
x 10 5
0
100
200
300
400
500
600
700
800
Round 1 Round 2 Round 3 Round 4
0
200
400
600
800
1000
1200
1400
1600
Round 1 Round 2 Round 3 Round 4
Lysozyme Parallel Biopanning of the Anti-protein and Anti-peptide Libraries and Trypsin Elution
Protein-A binding after 720C (10 min)
Carefully Designed Diversity
Identified positions in contact with protein and peptide targets and diversified based on human germline genes and known antibody sequences amino acid frequencies
0%
15%
30%
45%
1.20 - 1.49 1.50 - 1.99 2.00 - 2.49 2.50 - 3.00
RESOLUTION
> 50% below 3.0 Å
Macaca mulatta
Rattus norvegicus
Humanized
Chimeric
Mus musculus
Homo sapiens
SPECIES
Anti-Protein
Anti-Peptide
GENERAL SPECIFITIES
Studied over 2,500 antibodies known structure to design ALTHEA Gold Libraries™
CDR-H3
VL
900
VH
0%
20%
40%
60%
80%
100%
ǁ A
1
ǁ B
1
ǁ C
1
ǁ D
1
ǁ E1
# F
1
ǁ G
1
ǁ H
1
ǁ A
2
ǁ B
2
ǁ C
2
ǁ D
2
ǁ E2
ǁ F2
ǁ G
2
# H
2
A3
ǁ B
3 C3
ǁ D
3
ǁ E3
ǁ F3
ǁ G
3
# H
3
ǁ A
4
ǁ B
4
ǁ C
4
ǁ D
4
ǁ E4
ǁ F4
ǁ G
4
H4
A5
ǁ B
5
ǁ C
5
ǁ D
5
ǁ E5 F5
ǁ G
5
ǁ H
5
ǁ A
6
B6
ǁ C
6
ǁ D
6
3-2
0/3
-23
3-2
0/3
-23
TB
S
TB
S
= In-frame
0%
20%
40%
60%
80%
100%
ǁ A
1
ǁ B
1
ǁ C
1
ǁ D
1 E1
ǁ F1
ǁ G
1
H1
A2
ǁ B
2
ǁ C
2
ǁ D
2
ǁ E2
ǁ F2
ǁ G
2
# H
2
ǁ A
3
ǁ B
3
ǁ C
3
ǁ D
3
# E
3
ǁ F3
ǁ G
3
ǁ H
3
ǁ A
4
ǁ B
4
ǁ C
4
ǁ D
4
ǁ E4
ǁ F4 G
4
# H
4
ǁ A
5
ǁ B
5
ǁ C
5
ǁ D
5
ǁ E5
# F
5
ǁ G
5
ǁ H
5
ǁ A
6
ǁ B
6
ǁ C
6
D6
4-0
1/3
-…
4-0
1/3
-…
TB
S
TB
S
Biopanning and Screening
Selection
Specific Clones Amplification
Further Characterization
RAM kit™ if needed
Positive Clones Screening
ALTHEA Gold Libraries™
L1 L2 L3
L1 L2 L3
Diversified Synthetic Scaffolds Sequence Diversity
H1 H2
H1 H2
In-frame
indels
0
0.2
0.4
0.6
0.8
1
1.2
4.00E+08 4.00E+09 4.00E+10 4.00E+11
R1
R2
R3
R4
3-20/3-23 (-)
0
0.2
0.4
0.6
0.8
1
1.2
4.00E+08 4.00E+09 4.00E+10 4.00E+11
R1
R2
R3
R4
3-20/3-23 (-)
cfu x 105 cfu x 105
OD
Φ/mL
OD
Anti-protein
Anti-peptide
Anti-peptide
Anti-protein
Summary of the Biopanning Results: Hit rate 80% after 3 rounds
BSA HEL
Titre Polyclonal ELISA
Amplicon 2 SL1 % SL2 %
Total sequences 1,077,746 100.00 1,227,176 100.00
Accepted sequences 853,327 79.0 990,864 80.7
Unique sequences 851,681 99.8 989,005 99.8
Unique CDR-H3 sequences 522,939 61.3 592,317 59.8
0.048 0.599 0.045 0.045 0.045 0.045 0.81 0.873 0.868 0.85 0.742 0.68
0.041 0.041 0.041 0.041 0.546 0.042 0.783 0.87 0.882 0.851 0.583 0.69
0.05 0.059 0.046 0.047 0.055 0.047 0.826 0.902 1.03 0.865 0.753 0.723
0.047 0.045 0.045 0.045 0.561 0.743 0.819 0.92 0.919 0.841 0.571 0.726
0.048 0.046 0.045 0.045 0.044 0.717 0.601 0.933 0.894 0.851 0.795 0.727
0.042 0.04 0.691 0.039 0.041 0.04 0.838 0.919 0.751 0.851 0.806 0.782
0.17 0.044 0.044 0.044 0.044 0.045 0.239 0.917 0.892 0.826 0.818 0.801
0.044 0.042 0.043 0.043 0.048 0.295 0.982 0.951 0.914 0.907 0.853 0.275
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
1 2 3 4 5 6 7 8 9 10 11 12
Clone 11
Clone 5
Clone 3
(-)
Dilutions
3 clones EC50 40-100 nM by ELISA
Best clones HSA-B3 Kd 3.3 nM as Fab
