Mark Tornetta

Antibodies to Challenging Receptor Targets through NGS and Cell-Based Antibody Phage Panning

Points to convey regarding receptor targets

▪ Antigen diversity- cells and engineered ECDs

▪ Be creative with your selections

▪ NGS can ‘screen’ virtually the whole selected population

▪ NGS along with appropriate analysis tools does help withcandidate selection

2

NGS and phage display: proof of concept

▪ Library QC

– Deep sequencing using 454 of natural and synthetic antibody libraries

▪ Matochko, W.L. et al. (2009), Glanville, J. et al. (2009)

▪ Phage panning

– Clone enrichment vs. ELISA screening

▪ Matochko, W.L. et al. (2012), Ravn, U. (2010), Ravn, U. (2013)

3

Where NGS may help most

4

Cell-based panning

Homologous proteins

Rare/novel epitopesConformation

specificity

5

Janssen Antibody Phage Display Libraries

Library V2.0 combines VH diversity with the germline VL scaffolds

Library V3.0 includes diversity within the VL scaffolds.

Diversified KappaGermline Kappa

169 323 551

Heavy Chain Libraries

X

V2.0 V3.0

Vκ3-20 Vκ4-1 Vκ3-11 Vκ1-39 Vκ3-20 Vκ4-1 Vκ3-11 Vκ1-39

6

NGS analysis process

• Upload fastq or fasta files

• ORF determination based on germ line V-region ‘templates’

• Perform sequence analysis (mostly based on HCDR3)• Number of sequences captured

• Germline distribution

• Candidate frequency

• Developability risks

• HCDR3 lineup

• Dynamic subtraction to determine candidate uniqueness between panning groups

In-house Sequence analysis software

7

Isolating antibodies to a GPCR

Target- GPCRc: Class C, 27aa N-terminal peptide, Extracellular loops – 9, 23 & 14 amino acids long

Peptide based panning followed by ELISA screen

▪ N-terminal peptide panning

– 12 hits, 9 cell binders – all with some non-specific cell binding

Cell-based panning followed by NGS

▪ Negative and competitive type selections

– Generated many target specific candidates

8

Challenges of cell-based phage panning

Dynamic, charged, complex mixture

GPCR

Small exposed peptide loops in membrane milieu

How does Fab-phage get access?

How are non-specific Fabs removed?

Standard cell panning: negative selection

10

Phage library

Parental Cells

Target cells

Parental Cells

Target cells

NGS NGSTarget selection

Parental cell subtraction

Amplify bound phage

Amplify unbound phage

2X

Final roundCollect both

FACS-based panning: competitive selection

11

Phage library

Target CellsDye-

labelled

Parental Cells

Target cells

Target cell phage

Target CellsDye-

labelled

Parental Cells

Sort target cells

Parental Cells

Target cells

NGS

NGS

Final Round

Collect both

Sort target cells

Panning against parental cells

Parental Cells

3X

NGS on round 3 phage

To create a list of Fab sequences generated to the “background” of parental cells

VH NGS Sequence processing

13

GPCRc cells HEK cells

Panning technique Standard FACS Standard

Total DNA sequences 553,858 497,205 2,104,535

HCDR3 sequences 13640 23351 72006

Dynamic subtraction: HCDR3 unique

600 848

Sequence comparison shows large overlap

between target and parental HEK cells

Comparison to parental cell panning

FACS(Total target cell sequences: 1,079,235)

V2.1 STD(Total target cell sequences: 252,158)

V3.0 STD (Total target cell sequences: 359,659)

0

200

400

600

v2.1 Std

vs

v2.1HEK

v3.0 Std

vs

v3.0HEK

FACS vs

v2.1HEK

FACS vs

v3.0HEK

Nu

mb

er o

f clo

nes

that

have >

50

co

pie

s

Dynamic Subtraction

Unique to

target cell

Fab-phage sequences from parental cell panning

Distribution of H-CDR3 lengths

15

0

20000

40000

60000

80000

100000

120000

140000

7 8 9 10 11 12 13 14

HCDR3 Length

Nu

mb

er o

f seq

uen

ces

STDv2.1

STDv3.0

FACSv2.1/3.00

20406080

100120140160180

7 8 9 10 11 12 13 14

HCDR3 Length

Nu

mb

er o

f seq

uen

ces

STDv2.1

STDv3.0

FACSv2.1/3.0

Total V-regions Unique CDR3 sequences

• Broad diversity of antibody sequences

• Shift in HCDR3 diversity from total to unique V-regions following dynamic subtraction

0

500000

VLk3-20

0

500000

VLk4-1

0

500000

VLk3-11

0

100000

200000

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

GC…

VLk1-39

63/87 bound specifically to

target cells

What do you get out of ELISA screening?

▪ Cell binding assay with Fabs from E. coli is sub-optimal

▪ Screen each panning experiment for Fab expression and sequence positive clones

– 56 Fab sequences identified from 300 picked colonies

– 31/56 were found in the NGS analysis

– 7 of the confirmed NGS binders identified in this ELISA screening format

17

mAb functionality: T-cell re-direction GPCRc/CD3 DuoBody Abs

18

T c e ll m e d ia te d c y to to x ic ity a s s a yM M IR (4 8 h rs )

D o n o r ID : M 6 4 5 7

0 .00 0 1

0 .01 1

1 0 0

1 0 0 0 0

0

2 0

4 0

6 0

8 0

1 0 0

G C D B 1 8

G C D B 1 9

G C D B 2 0

G C D B 2 1

G C D B 2 2

G C D B 2 3

G C D B 2 4

G C D B 2 5

C o n c e n tra t io n (n M )

% C

yto

tox

icit

y

T c e ll m e d ia te d c y to to x ic ity a s s a yM M IR (4 8 h rs )

D o n o r ID : M 6 4 5 7

0 .00 0 1

0 .01 1

1 0 0

1 0 0 0 0

0

2 0

4 0

6 0

8 0

1 0 0

G C D B 2 6

G C D B 2 7

G C D B 2 8

G C D B 2 9

G C D B 3 0

G C D B 3 1

G C D B 3 2

G C D B 3 3

C o n c e n tra t io n (n M )

% C

yto

tox

icit

y

T c e ll m e d ia te d c y to to x ic ity a s s a yM M IR (4 8 h rs )

D o n o r ID : M 6 4 5 7

0 .00 0 1

0 .01 1

1 0 0

1 0 0 0 0

0

2 0

4 0

6 0

8 0

1 0 0

G C D B 3 4

G C D B 3 5

G C D B 3 6

G C D B 3 7

G C D B 3 8

G C D B 3 9

G C D B 4 0

G C D B 4 1

C o n c e n tra t io n (n M )

% C

yto

tox

icit

y

T c e ll m e d ia te d c y to to x ic ity a s s a yM M IR (4 8 h rs )

D o n o r ID : M 6 4 5 7

0 .00 0 1

0 .01 1

1 0 0

1 0 0 0 0

0

2 0

4 0

6 0

8 0

1 0 0

G C D B 4 3

G C D B 4 4

G C D B 4 5

C N T O 7 0 0 8

C o n c e n tra t io n (n M )

% C

yto

tox

icit

y

Null arm (DB18-31)

CD3 arm (DB32-45)

CD3 arm

Cell panning advantages for challenging receptor targets

▪ Affinity maturation using cells

– Antibody to a single pass receptor bound to recombinant protein in uMrange, with cell-binding at ~2nM

▪ Cell panning with NGS isolated highly enriched set of mutations

▪ Cell binding improved into pM range

▪ Glycosylated TNFR-family single domain receptor

– Recombinant protein highly glycosylated

▪ Standard phage panning yielded no binders

– Cell-based panning: 50% of selected VH genes show positive cell binding

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Cell panning summary

▪ Cell panning methods were effective in isolating GPCR antibodies

▪ In-house NGS analysis application effectively identified candidates to test in downstream assays

▪ NGS significantly expanded the number of viable candidates

▪ These methods have demonstrated our ability to generate multiple antibodies to a GPCR

▪ This provides confidence to include cell panning as another means for generating antibodies to membrane targets

20

Where NGS may help most

21

Cell-based panning

Homologous proteins

Rare/novel epitopesConformation

specificity

Protein selections utilizing NGS

22

Receptor with and without bound ligand

Domain specific panning

D1 D3D2

D3D2

D1 D2

R1ECD: Function-specific antibodies

23

NGS groups

3X

3X

Dynamic substraction

293 385101

779*

* 126 lost in analysis, probably in the “both”

category

Captured 3 possible functional groups

R1ECD Results▪ Selection method and NGS improved our efficiency regarding candidate selection,

functional testing, and manage the affinity maturation process.

▪ 26/66 clones bound specifically to target cells

▪ NGS allowed us to obtain candidates with agonist, antagonist, and enhancer activities

24

Ab 1 Ab 2

Ab 3

R2ECD: Domain specific antibodies

▪ Protein panning– 44 Vh sequence unique and domain specific hits– Vast majority confirmed NGS analysis with binding specificity assays– Vast majority bound specifically to target cells

25

D1 D3D2

D3D2

D1 D2

Protein Variant Selections

NGS and Dynamic

substraction

Domain specific antibody groups

D1

D2

D3

~50% of hits

~25% of hits

~25% of hits

*Additional candidates with different binding profiles were obtained from cell panning

Summary

▪ Next generation sequencing is a critical tool for screening cell-based phage selections

▪ Successfully applied to multiple receptors for discovery and affinity maturation

▪ Combining diverse panning strategies, NGS, and dynamic sequence analysis is an efficient way to choose candidates confidently for downstream characterization within leaddiscovery.

26

Acknowledgements

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John WheelerRam PillarisettiSuzanne EdavettalMatt HusovskyJin LuRay BrittinghamJohn KehoeBrian WhitakerRama Reddy