EBF OPEN SYMPOSIUM, 22-NOV-2018EVA VIESER

HIGH SENSITIVE PK ANALYSIS OF BITE®MOLECULESUSING SIMOA TECHNOLOGY

- Nina Deppisch, Eva Vieser, Michael Lutteropp, Andreas Wolf -

2

Ø Necessity for high sensitive PK assay

Ø Case Study

– 2 assays on 2 platforms: MSD / Simoa

– development & validation

– bridging

– 2-assay strategy for clinical sample analysis

OUTLINE

3

PRINCIPLES OF THE BITE® MODE OF ACTION EXAMPLE: HOW BLINCYTO WORKS

Redirected Lysis

ALLBlast Cell

Cytotoxic T Cell

CD19

CD3

CD25/CD69

T-Cell Activation

Anti-CD19 Antibody

VH

VL

Anti-CD3 Antibody

VH

VL Proliferation of T Cells

Apoptosis

BiTEâ

Blinatumomab

scFv

scFv

Serial Lysis of ALL Blast Cells

4

Target Sensitivity of PK Assay: low pg/mL range low

LOW FIRST-IN-HUMAN (FIH) STARTING DOSE CONSTITUTES A CHALLENGE FOR ASSAY SENSITIVITYFIH starting dose • is based on MABEL approach (Minimal Anticipated Biological Effect Level)• typically in sub-/low [µg/day] or [µg/dose] range• expected serum concentration Cmax < MABEL

canonical BiTE®

- continuous i.v. -half-life-extended BiTE®

- short infusion -

scFc

0 5 1 0 1 5 2 0

0 .0

0 .5

1 .0

1 .5

T im e [D a y s ]

Seru

m C

onc.

Time [Days]

MABEL serum conc.(often in pg/mL range)

0 5 1 0 1 5 2 0

0 .0

0 .5

1 .0

1 .5

T im e [D a y s ]Time [Days]

5

High Sensitive Clinical PK Assay for

• half-life-extended (HLE) BiTE®

• indication: oncology / haematological target

• MABEL concentration: 0.016 ng/mL

• FIH starting dose: 0.05 µg/dose

CASE STUDY

6

1 PROGRAM – 2 ASSAYS ON 2 PLATFORMS

&

MSD / ECL Assay• many BiTE® PK Assays successfully

validated and in-study use• sensitivity often not sufficient

for early clincal dose cohorts

Quanterix / Simoa AssayFeasibility:• superior sensitivity?• full PK profiles in

early dose cohorts?

Development, Validation, Bridging

Plate based Bead based

HD1- Analyzer- IQ/OQ- computer validation

SI2400 / Quickplex- IQ/OQ- computer validation

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SAME ANTIBODY CLONES AS CAPTURE & DETECTION REAGENT ON BOTH PLATFORMS

Cartoons (Simoa) adapted from:Lisa Heiden 2017, GEN Vol.37 No.4

BiTE®

MSD Test Plate

20F4 (α-target binder)

1C1.1-biot. (α-CD3 binder)

Streptavidin –SulfoTag

scFc BiTE®

scFc

20F4 (α-target binder)on

Magnetic Beads

Streptavidin -β-galactosidase

1C1.1-biot. (α-CD3 binder)

MSD SIMOA„Homebrew“

8

Sensitivity in Serumpool (pre-validation): MSD - 0.016 ng/mL Simoa - 0.0005 ng/mL

DYNAMIC RANGE IN HUMAN SERUMPOOLPRE-VALIDATION

MRD = 2 for both assaysCurve Fit: 4-PL fit (weighting 1/y2)

MSDdynamic range(pre-validation)0.016 - 33.4 ng/mL

Simoadynamic range(pre-validation)0.0005- 0.064 ng/mL

0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0 .0 0 1

0 .0 1

0 .1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

1 0 0 0 0 0

1 0 0 0 0 0 0

s e ru m c o n c [n g /m L ]In

str

um

en

t R

es

po

ns

e [

AE

B] In

stru

me

nt R

es

po

ns

e [E

CL

]

M S D A s s a y [E C L ]

L L O Q S IM O A

L L O QM S D

U L O QS IM O A

U L O QM S D

S IM O A A s s a y [A E B ]

9

Simoa assay sensitivity defined by selectivity => LLOQ = 1.5 x 10-3 ng/mL

SIMOA: SELECTIVITY @ LLOQ LEVEL

spike conc. [ng/ml]

> 80% of samples withrecovery ± 25%

0.5 x 10-3 fail L

1.5 x 10-3 pass J

H e a lthy #

1

H e a lthy #

2

H e a lthy #

3

H e a lthy #

4

H e a lthy #

5

d ise a s e d #

1

d ise a s e d #

2

d ise a s e d #

3

d ise a s e d #

4

d ise a s e d #

5

h y p e r lip a em

ic

h a emo ly

t ic

H e a lth

y #6

H e a lthy #

7

H e a lthy #

8

H e a lthy #

9

H e a lthy #

1 01 0 - 4

1 0 - 3

1 0 - 2

BiT

E [

ng

/mL

]

*

*

* n o t d e te rm in e d

0 .5 x 1 0 -3 n g /m L ± 2 5 %p a s s : 9 /1 6

1 .5 x 1 0 -3 n g /m L ± 2 5 %p a s s : 1 3 /1 6

10

METHOD VALIDATION – SELECTED PARAMETERSParameter MSD Simoa

Accuracy & Precision

%BIAS: -5 to 3% %BIAS: -2 to 5%

%CV: 9 to 12% %CV: 5 to 17%

Total Error: 10 to 16% Total Error: 6 to 19%Dynamic Range 0.016 – 33.4 ng/mL 0.0015 – 0.064 ng/mLSpecificity no interference for soluble target levels

up to 1250 pMno interference for soluble target levels up to 1500 pM

Selectivity confirmed for 0.016 ng/mL(incl. healthy, diseased, hyperlipaemic and haemolytic samples)

confirmed for 0.0015 ng/mL(incl. healthy, diseased, hyperlipaemic and haemolytic samples)

Ø Both assays passed validation with comparable A&PØ Simoa with ~10-fold better sensitivity

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MSD Assay Ø primary assay

• long experience with technology, robustness, cost

Simoa Assay Ø secondary assay

• for samples with a primary result< LLOQ in MSD

• to provide full PK profiles in earlyclinical cohorts

2-ASSAY STRATEGY FOR IN-STUDY USE

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Comparability of MSD and Simoa assay confirmed in overlapping dynamic range

BRIDGING OF MSD & SIMOA ASSAY

Ø Samples within overlapping dynamic range wereanalyzed with both assays:0.016 / 0.048 / 0.064 ng/mL

Ø Acceptance Criteria:• Recovery within ±20% of nominal • %Difference ≤ 30%

0 .0 1 6 0 .0 4 8 0 .0 6 4

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

0 .1 0

B r id g in go f p r im a r y (M S D ) a n d s e c o n d a ry (S IM O A ) A s s a y

de

term

ine

d c

on

c[n

g/m

L]

S IM O A M S D

s a m e s p ik e d s a m p le s a n a ly z e d b y

n o m in a l c o n c[n g /m L ]

13

0 2 4 6 8 1 0 1 2

0 .0 1

0 .1

1

T im e [D a y s ]

S u b je c t 3 / d o s e le v e l 3

PK PROFILES FOR EARLY DOSE COHORTS

• Proof of Concept for 2-Assay Strategy Confirmed

0 2 4 6 8 1 0 1 2

0 .0 1

0 .1

1

T im e [D a y s ]

BiT

E s

eru

m c

on

c [

ng

/mL

]

S u b je c t 1 / d o s e le v e l 1

0 2 4 6 8 1 0 1 2

0 .0 1

0 .1

1

T im e [D a y s ]

BiT

E s

eru

m c

on

c [

ng

/mL

]

S u b je c t 2 / d o s e le v e l 2

MSD

Simoa

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Ø PK Assay was developed on the MSD and the Simoa platform to be used for a BiTE program with a clinical starting dose of 0.05 µg/dose

Ø both assays use the same detection reagents and passed validation with comparable results for A&P

Ø 10-fold improvement in sensitivity by Simoa over MSD

Ø successfull bridging of both assays

Ø proof of concept: full PK profiles for early dose cohorts provided by using a2-assay-strategy with MSD as primary and Simoa as secondary assay

SUMMARY

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• Assay Development & Validation (MSD & Simoa)Nina DeppischNadine HerrmannMaren Klein

• Equipment Qualification / Computer ValidationMichael LutteroppRicarda Mayer Jens Bertram Sebastian Grieger

ACKNOWLEDGMENT

Head of PKDM / MunichAndreas Wolf