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 <a.gerard@hifibio.com>

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