Challenges and Strategies in Designing

MOA Reflective Bioassays for a Bispecific mAb

Xianzhi Zhou, Ph.D

Analytical Biotechnology Development

Bioassays 2014: Scientific Approaches & Regulatory Strategies

March 24 - 25, 2014

Introduction

The emerging of multispecific mAbs

– most of the marketed antibodies are monospecific

– complex diseases involve synergistic action of disease-mediating factors

– blockade of different pathological factors for improved therapeutic efficacy

A bi-functional mAb

– anti- ligand1 backbone

– anti-ligand2 scFv attached at the Fc portion

Bioassay design and considerations for each function

2

(G4S)4 VL VH C N

VL VL

VH VH

S-S

Bioassay Design for Bispecific mAb

Role that Mechanism of Action (MoA) plays in

choosing bioassay format for each function

Strategies for use of cell-based assays and

binding assays at various stages of product

development

– Dual binding ELISA

– For Ligand1 arm:

cell-based assay available

– For Ligand2 arm:

use binding assay at early development stage

develop cell-based assay for later stage

Ligand1

ELISA Plate

His-tag

Ligand2

anti-His-HRP

Ligand1 Reporter Gene Bioassay

NFκB

Luciferase Reporter Gene

Luminescence

NF κB

IκBα

NF κB

α β

IKK

DD DD

R

T

N

FR

T

N

F

TR

AD

DRIP

TRAF2

MEDI3549

TNF-α

TNF-α

IKK pathway

Ligand1 Reporter Gene Bioassay Development

Ligand Titration

1 100 10000 1000000

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

TNF-a (pg/mL)R

ela

tive

Lu

min

es

ce

nce

Un

its

Cell Density

5 15 25 37.5 50 75

0

1000

2000

3000

4000

5000

6000

Cells/well (x1000)

S:N

EC75

[Ligand] (ng/mL) Cells/well

Sig

nal

1 10 100 1000

0

2000000

4000000

6000000

8000000

2 hour

2.5 hour

3 hour

3.5 hour

4 hour

4.5 hour

[MEDI3549] ng/mL

Re

lati

ve

Lu

min

es

ce

nce

Un

its

(R

LU

)

Reporter Gene Bioassay Time Courses

6

Incubation of Cells with mAb/ligand1

mAb Pre-Incubation With Ligand1

0.1 1 10 100 1000 10000

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 Minutes

15 Minutes

30 Minutes

45 Minutes

60 Minutes

90 Minutes

MEDI3549 (ng/mL)

Re

lati

ve

Lu

min

es

ce

nce

Un

its

(R

LU

)

[mAb] (ng/mL)

[mAb] (ng/mL)

Sig

nal

Sig

nal

No Plate Positional Effect

7

row A/H

row B/G

row C/F

row D/E

[mAb] (ng/mL)

RL

U

Pre-incubate

Drug/Ligand

60±15 min @RT

Add 50 µL mixture

to 50 µL cells

4-4.5 hr @37ºC

Add 100 µL

SteadyGlo

Measure

Luminescence

30-60 min @RT

Specificity, Linearity, Stability Indication

Lu

min

es

ce

nce

L

um

ine

sc

en

ce

[mAb]

Accuracy

103%

Intermediate

Precision

4.8%

0 8 12

Rela

tive

Po

ten

cy

Time

5C

40C

Bioassay Design for Ligand2 Arm

Ligand2 plays complex role

Unclear signaling pathway

– Ligand2 can act as an agonist or antagonist depending on

cell type and experimental context

– The exact molecular mechanism underlying Ligand2

actions are not known

Stage-based bioassay approach

– Binding assay for early-stage development

– Cell-based assay for late-stage development

9

Competitive Binding Assay Using SPR

10

Sensor Surface

Receptor

Ligand2 mAb mAb

Ligand2

Sensor Surface

Receptor

11

Accuracy Expected Potency Level

50% 100% 150% AC

Assay 1 96 101 101 99%

Assay 2 98 100 103 99%

Assay 3 94 100 105 99%

Average 96 100 103 99

CV (%) 2.1 0.6 1.6 0.0

Repeatability

Sample ID Assay 1 Assay 2 Assay 3 Average RP

(%)

CV

(%)

Sample 1 99 97 98 98 1.0

Sample 2 101 100 100 100 0.6

Sample 3 100 102 99 100 1.5

Average RP

(%) 100 100 99

Overall

Accuracy 100%

CV (%) 1.0 2.5 1.0 Overall CV 1.3%

SPR Binding Assay Performance

Principle of Receptor Phosphorylation Assay

Ligand2 SC region mediate

homo-oligomerization to

tetrameric and higher-order

oligomers

Binding of the multimeric

ligand2 clusters the receptor

Ligand2 binding induces

phosphorylation of the receptor

ELISA to measure

pReceptor

Barton et al., (2006) Nature Structural Biology Vol. 13:524

Ligand2

Receptor

13

Day1

Day2

Y R P

Capture Ab: a-R

Detection Ab: a- pR

Seed cells overnight

Treat cells with drug/Ligand2 Mixture

Lyse cells /Transfer lysate to ELISA Plate

Measure pReceptor by ELISA

Phosphorylation Assay Procedure

Receptor Phosphorylation Upon Stimulation

CCM vs. Serum Free assay media

Reaches maximum at 10 minutes

14

30 min

20 min

15 min

10 min

5 min

0 min

CCM Serum Free Media

[Ligand2] [Ligand2]

Sig

nal

Sig

nal

Receptor Phosphorylation Assay Optimization

Cells

– Cell Line

– Growth Media optimization

– Assay media: CCM vs. serum free

– Cell number titration

– Passage comparison

Ligand2/mAb

– Pre-incubation time course

– Stimulation Time course

– Ligand2 titration

– Ligand2 lot comparison

– Tetramer vs. rhLigand2

Lysis

– Aspiration vs. direct addition

– Base buffers

– Benzonase concentration

– Lysis Time course

15

Capture Ab

– Coating concentration

– Coating condition

Detection Ab

– Anti-pTYR clone comparison

– Titration

– Detection Time course

– Buffers

Other

– ELISA readout

– Wash buffer conditions

Cell Titration and Ligand2 Titration

100K

67K

44K

30K

20K

13K

EC80

40K cells/well Sig

nal

[Ligand2]

Lysis Buffer Optimization

Lysis buffer

Pierce Cell Lysis Buffer

17

1 10 100 1000 10000

0

100000

200000

300000

400000

500000

6000000 min

5 min

10 min

20 min

30 min

40 min

MEDI3617 (ng/mL)

Flu

ore

sce

nce

Aspiration Step Direct Addition

0

2

4

6

8

10

12Pierce RIPA

CST RIPA

Pierce Cell Lysis Buffer

S:N

Lysis time Course

10-20 minutes

[mAb]

Sig

nal

18

Antibody Optimization: Capture/Detection Ab

Capture Ab: 5 µg/mL

Detection Ab 50 ng/mL

0 1.31 3.28 8.19 20.48 51.2 128 320 800 2000

0

2

4

6

8

10

12

12

4

1.333333333

0.444444444

0.148148148

0.049382716

0

[biotin-anti-hTIE2] ug/mL

Eu-anti-pTYR Titration vs. Biotin-anti-TIE2 Titration

[EU-PY20] ng/ml

S:N

Capture Ab (mg/mL)

Detction Ab (ng/mL)

19

Phosphorylation Assay Development

Low throughput

– 6 well plate

Variable

19 19

Research Assay

High throughput

– 96 well plate

Variable

Early-stage

1E-3 0.01 0.1 1 10 100 0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Late-Stage

Increased sensitivity

Improved assay

performance

Enzyme Fragment Complementation Assay

20

β-gal

Fragment1 β-gal

β-gal substrate

Signal

Receptor

Ligand2

SH2

β-gal

Fragment2

SH2

21

Expected

Potency

Measured

Potency

50% 54%

75% 77%

100% 102%

125% 128%

150% 148%

DiscoveRx Assay Performance

Effects of mAb Oxidation on Two Functions

22

The Impact of Met- and Trp-oxidation on mAb Activity

Samples %Met-Ox

(M261)

%Met-Ox

(M436)

%Trp-Ox

(W94)

% RP L1

Bioassay

%RP L2

Bioassay

%RP L2

SPR

Ctrl 6.1 2.8 0.0 98 108 96

Photo-Exposed 45.4 35.2 28.4 45 86 77

Ctrl+0.3% H2O2 100.0 100.0 1.6 109 118 106

Ctrl+0.3% H2O2

(w/photo-exposure) 92.2 89.5 30.1 41 88 75

Excipient Screening for Photo-Protection

23

Sample Description %RP L1

Bioassay

%RP L2

Bioassay

%RP L2

SPR

W94

Oxidation

Dark Control 98 108 96 0

Photo-Exposed 45 86 77 28.4

Photo-Exposed/10 mM A 54 77 79 26.8

Photo-Exposed/10 mM B 40 79 75 29.6

Photo-Exposed/10 mM C 84 90 111 5.8

Photo-Exposed/10 mM D 50 83 97 41

Photo-Exposed/10 mM E 85 99 115 1.6

Summary

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For ligand1:

– cell based assay available and performed well

– used from the beginning of product development

For ligand2

– SPR binding assay used at early stage

– Receptor phosphorylation assay used as characterization assay

– Continuous effort in developing robust cell based assay

Bioassays for each arm of the bispecific antibody:

– Necessary for investigating the two functions

– Reveals the two arms have different photosensitivity

– Correlates with each other

Acknowledgements

Vicky Bushman

Ashley Mullan

Morgan Wilson

Sarah Ronan

Ken Miller

Julian Relton

Xu-Rong Jiang

Michael Washabaugh

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