High-Throughput Screening for Antibody Discovery …...High-Throughput Screening for Antibody...

High-Throughput Screening for Antibody Discovery Using Mirrorball Christyne Kane Senior Scientist I AbbVie Bioresearch Center

Antibody Engineering & Therapeutics December 12, 2017

Disclaimer

Christyne Kane is an employee of AbbVie.

Assessment of equipment is the opinion of the author.

AbbVie does not endorse any of the platforms described in this presentation.

High-Throughput Screening for Antibody Discovery using Mirrorball | Date 12.12.17 | © 2017 2

Overview

Antibody Discovery:

Mirrorball HTS

Cell Killing Assay

Cell Surface Binding • Singleplex

•Multiplex

Isotype Determination: Bead Color Discrimination

ELISA Alternative: Bead Size Discrimination


Antibody Screening Assays

High-Throughput Screening for Antibody Discovery


• Soluble Target Binding • Cell Surface Antigen Binding • Species Cross-reactivity • Target Specificity • Functionality • Epitope Binning • Isotype

Use of Mirrorball Technology

Desired Antibody Properties

20-40

5 x 104

Numbers In

Viv

o An

tibod

y Di

scov

ery

Wor

kflo

w

5 x 107

Immunizations

Hybridoma Generation

(Fusion)

Screening

Sub-cloning

Purification & Characterization

Mirrorball Technology

• HOMOGENEOUS ASSAY - No wash steps

• Ability to MULTIPLEX for multiple antigens, cross-reactivity, or counter-screening

• Compatible with CONDITIONED MEDIA

• SENSITIVITY (adequate for low concentration supernatants)

• +/- HIT ASSESSMENT, flexible data analysis

• Integrates with AUTOMATION, miniaturization for HTS

• TIME SENSITIVE (24-hour turnaround)

• PLUG-AND-PLAY assay for multiple targets


Mirrorball Instrument

3 LED lasers • 405nm • 488nm • 640nm

4 Fluorescent Channels • FL-1 (420-488nm) • FL-2 (488-540nm) • FL-3 (560-610nm) • FL-4 (650-690nm)

1 Scatter Channel (SC-1)

Automation: Thermo Scientific Orbitor RS

Benefits of Mirrorball Mirrorball Instrument Configuration

Case Study: Target A


• TNF family member

• Transmembrane glycoprotein

• Forms a homotrimer

• Autoimmune indications

Desired Binding Properties

• Binding to specific epitope

• Binding to cell surface protein

Goal: To generate a panel of antibodies binding cell-surface Target A.

Target Biology

• Peptide immunizations in mice

Immunization Strategy

Membrane Extracellular

7μm Bead

Multiplexed Screening Enabled by Bead Size Discrimination

16μm Bead

Non-specific Antigen (optional)

Beads are Distinguishable

Specific Binding Detectable

Mix-and-Read Assay Setup

BV421 Anti-Species

Target-Specific Ab

Target Antigen

SA/Biotin

• 384 well plate format • No washing steps • Shorter incubation times • Smaller volumes of reagents • Multiplex-able

Compare to ELISA

16μm

bea

ds

7μm

bea

ds

BV421

Bead Size


Hybridoma HTS by Mirrorball Completely Correlates with ELISA


0.0 0.1 0.2 0.3 0.40

2000

4000

6000

0.4 0.5 0.6 0.7 0.8 0.9 1.0

Hit by Mirrorball& ELISAHit by neither

Mir

rorb

all

(Med

ian

FL2

Mea

n)Mirrorball vs. ELISA

ELISA(OD 450nm)

• Singleplex assay using 7μm beads

• No false positives • No false negatives

100% Correlation Threshold

Screening Methods are in Agreement for Hit Identification


Screening Assay Number of mAbs

Primary Screening (ELISA) 153

Secondary Screening

ELISA 38

Flow 38

Mirrorball 38

Binding to Target A

• Bead-based binding assay correlated with ELISA methods 100% for early supernatant screening.

• 8 antibodies identified bound specifically to Target A by flow cytometry.

Summary

0.01 0.1 1 10 100 10000.0

0.5

1.0

1.5

Binding to Target A Peptide (ELISA)

nM

OD

450

mAb 1mAb 2mAb 3mAb 4mAb 6mAb 7mAb 8

Neg. ControlmAb 5

0.0001 0.01 1 100 100000

50

100

150

200

Binding to Target A Cells (Flow)

nMGeo

Mea

n R

atio

TN

F-29

3H/w

t-29

3-H

mAb 1mAb 2mAb 3mAb 4mAb 6mAb 7mAb 8

Neg. ControlmAb 5

Case Study: Target B


• Soluble protein

• Neurology and cardiovascular indications

Desired Binding Properties

• Binding to target B protein

• +/- Binding to target protein family members

Goal: To generate a panel of antibodies binding Target B.

Target Biology

• Protein immunizations in mice



Direct Isotype Determination Enabled by Sixplex Bead-based Assay


sol-R2 sol-R3 sol-R4 sol-R5

SA/Biotin

Beads are Distinguishable

AF488

FL-4

sol-R

5

sol-R

4

sol-R

3

sol-R

2


IgG2b IgG3 IgG2a IgG1

AF488 Anti-Species Light Chain

Anti- Species HC

Test Antibody

AF405/BV421 *LC-specificity

(optional)


HC & LC Isotypes are Clearly Detected in Hybridoma Supernatant


Kappa Lambda Antibody HC Isotype LC Isotype

mAb 1 IgG3 Kappa

mAb 2 IgG1 Kappa

mAb 3 IgG2a Kappa

mAb 4 IgG2a Kappa

mAb 5 IgG1 Lambda

mAb 6 IgG2a Kappa

mAb 7 IgG2a Kappa

mAb 8 IgG2a Kappa

mAb 9 IgG2a Kappa

mAb 10 IgG3 Kappa

Able to detect multiple isotypes per sample

IgG

2b

IgG

3

IgG

2a

IgG

1

IgG

2b

IgG

3

IgG

2a

IgG

1

IgG

2b

IgG

3

IgG

2a

IgG

1

IgG

2b

IgG

3

IgG

2a

IgG

1

IgG

2b

IgG

3

IgG

1

IgG

2b

IgG

3

IgG

2a

IgG

1

HT Isotype Determination Improves Upon Traditional ELISA


ELISA Isotyping Mirrorball Isotyping

Isotype Antibody Isotype

IgG2a mAb 1 IgG3

IgG2a mAb 2 IgG2a

IgG2a mAb 3 IgG2a

IgG2a mAb 4 IgG2a

IgG1 mAb 5 IgG3

IgG2a mAb 6 IgG2a

IgG2a mAb 7 IgG2a

IgG2a mAb 8 IgG2a

IgG2a mAb 9 IgG2a

IgG3 mAb 10 IgG3

0

0.5

1

IgG1 IgG2a IgG2b IgG3

OD

(450

nm)

0

0.5

1

IgG1 IgG2a IgG2b IgG3

OD

(450

nm)

Non-specific signals have potential to dilute or overpower specific signals, leading to incorrect isotype conclusions.

IgG

1

IgG

2a

IgG

2b

IgG

3

IgG

1

IgG

2a

IgG

2b

IgG

3

Specific signals are greater than all background levels, allowing for accurate determination of isotype results.

Case Study: Target C


• 7-Transmembrane GPCR with short extracellular loops.

• Small molecule antagonists and agonists are available.

• Unprecedented for targeting by biologics

NH2

COOH

M1

M2

M3

M4

M5

M6

EL1 EL2 EL3

Extr

acel

lula

r

M7

Mem

bran

e

Desired Binding Properties • Binding to cell surface • Cross reactivity to cyno • Cross reactivity to rat

Desired Biological Activity • Cell Killing

Goal: To generate a panel of antibodies binding cell surface Target C which can be internalized.

Target Biology

• cDNA immunizations in rats


Singleplex Cell Binding Assay Enables HT Supernatant Screening


AF647 Anti-Species

Target - Expressing

Cell Line

(GFP+)

Target- Specific Ab Target

Antigen

GFP

AF647

Merge

GFP+ Cells are Detectable



(-) Binding (+) Binding AF647

GFP

Target C Campaign Enabled by Mirrorball Antibody Screening


Mirrorball Number of mAbs

Target C Species

Human 65

Cyno 59

Rat 39

Cell Killing 22/28 tested

Rat

nM0.000

10.0

01 0.01 0.1 1 10 10

010

000

2

4

6

8

10

GM

FI R

atio

(Tra

ns/W

T)

Cyno

nM0.000

10.0

01 0.01 0.1 1 10 10

010

000

5

10

15

GM

FI R

atio

(Tra

ns/W

T)

Human

nM

GM

FI R

atio

(Tra

ns/W

T)

0.000

10.0

01 0.01 0.1 1 10 10

010

000

5

10

15

20

Flow Cytometry Binding to Target C

Bio-activity

• Single-plex cell binding assay enabled all stages of supernatant screening.

• Antibodies identified bound specifically to Target C by flow cytometry and had a range of functional potencies.

Summary

Case Study: Target D


• TNF family member

• Transmembrane glycoprotein

• Forms a homotrimer

Desired Binding Properties • Binding to cell-surface protein • Cross-reactivity to Cyno

Desired Biological Activity • Block ligand/receptor interactions

Goal: To generate a panel of antibodies binding Target D which blocks interaction with receptor.

Target Biology

• Whole cell immunizations in mice



Multiplexed Cell Binding Assay Enables HT Supernatant Screening

• 384 well plate format • No washing steps • Shorter incubation times • Smaller volumes of reagents • Fewer cells required • Multiplex-able

Cell Types are Distinguishable Mix-and-Read Assay Setup

AF488

Far Red

AF488 Anti-Species

Target- Expressing

Cell Line


Antigen

Parental Cell Line

100nM Far Red

1000nM Far Red

Compare to Flow Cytometry

Pare

ntal

Tran

sfec

ted



Hybridoma HTS by Mirrorball: 89% Correlation with Flow Cytometry


0 20 40 60 80 1000

100

200

300

400

500

1000 2000 3000

500

1000

1500

2000Hit by Mirrorball& Flow CytometryHit by Flow Cytometry onlyHit by neither

Mir

rorb

all

(Tra

ns M

edia

n FL

2 M

ean/

WT

Med

ian

FL2

Mea

n)Mirrorball vs. Flow Cytometry

Flow Cytometry(GMFI Ratio: Trans/WT)

• Mirrorball missed 5/45 hits by Flow Cytometry (false negatives)

• No false positives

89% Correlation

Multiplexed Cell Binding Assay is Implemented for Sub-clone Screening


Binding & Blocking Assays

• Multiplexed cell binding assay demonstrated an 89% correlation with Flow cytometry methods.

• All downstream sub-clone screening was performed using the Mirrorball multiplexed cell binding assay, enabling the delivery of 8 antibodies.

Summary

0.0001 0.01 1 1000

200

400

600

800

Binding to Target D Cells

Conc. (nM)

GM

FI (T

rans

/WT)

mAb 1mAb 2mAb 3mAb 4mAb 5mAb 6mAb 7mAb 8Neg.Control

0.001 0.01 0.1 1 10 1000

20000

40000

60000

Target D Ligand Blocking (Flow)

Conc. (nM)

GM

FI

mAb 1

mAb 3mAb 4mAb 5mAb 6mAb 7mAb 8

mAb 2

Neg ControlPos Control

Screening Assay Number of mAbs

Primary Screening (Flow) 95

Secondary Screening

Flow 56

Blocking (Flow) 16

Case Study: Target E


• GPI-linked, glycosylated, membrane protein.

• Modulates T cell and macrophage activation.

• Oncology indications

Desired Binding Properties • Binding to cell surface target E • Cross-reactivity to Cyno • Cross-reactivity to Mouse

Desired Biological Activity • Internalization

Goal: To generate a panel of antibodies binding cell surface Target E which can be internalized.

Target Biology

• cDNA immunizations in mice


GPI

GPI


High Throughput Cell Killing Assay


Detection Live Apoptotic* Dead

Hoechst Annexin V-AF488* TO-PRO-3

SMi Anti-Species Ab

Target- Expressing

Cell Line


Antigen

~72-96hrs *Optional


Cells & Media Cells & Ab Complex

Well View

Target-specific Cell Killing is Detectable


Cell Killing

Conc. (nM)

% V

iabi

lity

0.000

10.0

01 0.01 0.1 1 10

0

20

40

60

80

100

Cells & Media

Neg. ControlAnti-Target C mAb

Example Data Plate View

100 - # Dead Cells + # Apoptotic Cells

# Total Cells

• Target-specific killing is detectable. • No non-specific killing is observed.

Summary % Viability =

Cell Killing Assay Enables HT Detection of Internalizing Antibodies


Platform Advantages • Compatible with early hybridoma screening. • Automated for high-throughput assay set-up and data reporting. • Streamlined process to identify functional leads.

0

20

40

60

80

100

Cell

Viab

ility

10% SN 2% SN 0.4% SN 0.08% SN

(+) (-) Hybridoma clones

Screening of Hybridoma Supernatants

Antibody Screening Assays: Saving Time, Labor & Reagents

Antibody Screening

Assays

ELISA Alternative: Bead Size Discrimination • Plug-and-play assay is easily established, requiring smaller amounts of reagents

and significantly less time, compared to traditional ELISA.

Isotype Determination: Bead Color Discrimination • Color-coded sol-R beads enable the establishment of a robust multiplex

assay able to identify 4-8 unique antibody properties with increased sensitivity compared to traditional methods.

• Format may be applied to cross-reactivity and specificity assays.

Cell Surface Binding • No wash assays require 1/10th the cells of traditional flow cytometry

screening and may be automated at all steps, including data processing.


Antibody Discovery: Mirrorball HTS

Cell Killing • High throughput cell killing assay allows for functional screening of hybridoma

supernatants using a small amount of material and a variety of secondary-conjugated toxins.

Acknowledgements

Amanda Horowitz Kwasi Ofori

Mary Abdalla Jane Seagal

William Stine

Julie Hugunin Shawn Jennings

Chung-Ming Hsieh

Diana Caracino Richard Kim Paul Wylie Ben Schenker Doug Mann Aline Cagwin Daniela Ionescu Jenny Buttaro Mirrorball Support


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