a demonstration of the hifibio celligotm platform...
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Antibody Discovery by Deep Mining of Immune Repertoires: A demonstration of the HiFiBiO CelliGOTM Platform versatility and
robustness with Tetanus Toxoid antigen as a case studyPresentation: Annabelle Gérard, PhD - Project & Group Leader <[email protected]>
EAC. November 13-16/2016. Basel
Acknowledgements - HiFiBiO labs Paris (ESPCI) & Cambridge (Lab Central), HiFiBiO consul-tants (Paris, LBC-ESPCI), its collaborators (Paris, LCMD-ESPCI), Pierre Bruhns & team for animal hosting and immunizations, HiFiBiO Founders (Harvard, Broad Inst., ESPCI) and HiFiBiO customers.
HiFiBiO Paris site: ESPCI. 10 rue Vauquelin, Paris 75005. France.HiFiBiO Cambridge site: Lab Central. 700 Main St., Cambridge MA 02139.
www.hifibio.com
Detection of antigen-specific Antibody secretion in droplets
Diversity of Paired Sequences
High Throughput Cloning and Expression of Ab candidates
3- Identi�cation of functional mAbs
1- Droplet based Single cell VH/VL pairing
1- Single-cell droplet based bio-assays
Rapid, Automated Paired VH/VL Cloning
Heavy Light
Microfluidic Channel
Aggregate of Magnetic particles
Fluorescence readout
Sig
nal (
Vol
ts)
AntigensAntibodies
Time (ms)
2- Identi�cation of mAb speci�c to soluble antigen
The CelliGOTM
technology is a fully integrated antibody discovery engine that is based on single drop-let microfluidics, to achieve high throughput, single cell, function-based screening of antibody-secret-ing primary B cell populations.
We present a case study using B cells derived from a tetanus toxoid (TT) immunized mouse. By apply-ing the CelliGO platform, we easily generated several hundreds of highly diverse anti TT specific anti-bodies. The recovered antibody repertoire covers more than 10 V-genes families, and includes several examples of clonal epansion. Antibodies have been cloned and transiently expressed as human IgG. More than 90% of the expressed antibodies showed specificity to TT. Our efficient and streamlined workflow can isolate and confirm specificity towards a diverse set of target specific antibody reper-toires within 4-5 weeks.
We have designed, implemented and validated droplet based microfluidics bio-assays to detect single cell monoclonal antibody secretion and select the droplets containing cells expressing antibodies specific to the antigen of interest. The droplet volume is compatible with cell survival for extended time and achievement of high secretion rate within minutes of incubations. Our plateform is compatible with any primary cell species.
Our bio-assays uses combination of fluorescently labeled reagents to identify, analyse and sort droplets con-taining antigen and function-specific antibody secreting cells (internalization, agonist/antagonist activities, etc...)
We have developped our proprietary single cell bar-codes for the recovery of naturally paired VH and VL sequences.
Phylogenetic tree of paired se-quences V-genes families shows recovery of > 10 V-genes families; for which some paires highlight co-mi-grating heavy and light chain variable sequences.
These clusters reflect the effi-ciency and the sensitivity of our plateform for deep mining into the B cell repertoire.
Heavy and Light chain sequences co-migration
Dial-out based PCR of paired sequences from the sequencing library and subsequent in frame cloning into expression vector are automated to achieve high throughput cloning and expression of antibody candidates into human IgG1 backbone.
Fluorescence readout
AntibodiesReporter cellB cellMembrane embeded Antigen
Micro�uidic channel
DetectionLaser line
mAbs
mAbs producing cell
Reporter cell
mAbsdetection
mAbs detection
Membrane boundtarget assay
Reporter cell
Beadline
Micro�uidic channel
DetectionLaser line
Antigen
mAbs
mAbs capture
mAbs producing cell
Antigen
mAbsdetection
mAbs detection
Light-Chaincapture assay
Time (ms)
2- Clonal expansion
Characterization and ValidationFrom >1000 distincts antibodies recovered from 2 ani-mals, compressed into > 300 unique antibodies (based on VDJ recombination), 41 have been selected for rapid cloning and ELISA.Above 90% of tested mAbs confirmed binding.
1- Sorted cell viability/speci�city
HiFiBiO CelliGOTM Platform versatility supports discovery of antibodies targeting a diversity of epitopes
Affinity of mIgG TT17 (is muTT7) is 8.7 nM from this assay. Published showed 8.01 nM for huTT7.
4- BiaCore SPR
2- A�nity maturation
3- Binding validation (ELISA)
~100 sorted cells were analyzed by ELISpot, in wells pre-coated with either α-mouse IgG, TT antigen or irrele-vant antigen.
0
20
40
60
80
100
120
Irrelevant AntigenTTIgG
% v
iabl
e se
creti
ng c
ells
TT17
CelliGO accommodates a wide range of flexible in-droplet bioassays based on interaction with recombinant antigens, bacterial and cell transmembrane targets, and on functional assays (identification of internalizing, agonist and antigonist antibodies). Our platform is able to rapidly identify potent antibodies, to efficiently mine immune repertoires of wild-type and transgenic rodents, successfully select and recov-er target specific cross species reactive antibodies.
We thank Ginger Shen (Pfizer, Cambridge, MA) for the Biacore SPR analysis of the anti-TT antibodies.
15 mAbs isolated from CelliGO have been analyzed by Biacore SPR.
Our antibodies have undergone affinity matu-ration into the secondary lymphoid organs of the animal.
Antigen-based ELISA was used to asses specificity of the cloned anti-bodies to the target.
ELISpot coating
Antibody-secreting cellReporter cell
Droplet
The data show that Tetanus Toxoid can bind to most of the anti-TT a n t i b o d i e s with high, medium and low affinities.
Single cell VH/VL barcoded sequencing library pool
(1)(2)
(3)(4) (4)
(1,2,3)
IrrelevantAntigen
TT
IgG
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
O.D
. (a.
u.)
Serial dilution of CelliGO anti-TT mAbs supernatant
- +
TTS5Ab
1TTS
5Ab2
TTS5Ab
3TTS
5Ab4
TTS5Ab
6TTS
5Ab7
TTS5Ab
8TTS
5Ab12
TTS5Ab
14TTS
5Ab16
TTS5Ab
17TTS
5Ab19
TTS5Ab
20TTS
5Ab21
TTS5Ab
24TTS
5Ab25
TTS5Ab
18TTS
5Ab11
TTS5Ab
9TTS
5Ab15
TTS5Ab
22TTS
5Ab13
TTS6Ab
1TTS
6Ab2
TTS6Ab
4CLTTS
6Ab5
TTS6Ab
6TTS
6Ab7
TTS6Ab
8TTS
6Ab9
TTS6Ab
10TTS
6Ab33
TTS6Ab
63TTS
6Ab66
TTS6Ab
73TTS
6Ab78
TTS6Ab
57TTS
6Ab3_M
UTTS
6Ab_CL
TTS6Ab
69TTS
6Ab77
Sig
nal (
Vol
ts)
Number of mutations (nucleotides) into the variable heavy and light chain sequences.
VHVL
0 10 20 30
Num
ber o
f seq
uenc
es
0
10
20
30
40
We monitored viability, antibody secretion and anti-body specificity of sorted cells by ELISpot.
ELIS
pot C
oatin
g