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Designing clinical trials with BiTE®

antibody constructs by leveraging from nonclinical dataBenno RattelBiologics Congress Berlin, 2015

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BiTE® Antibody Contructs are Designed to Function asa Bridge between T Cells and Cancer Cells

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(1) Strictly target cell-dependent activation of resting T cells by BiTE®

(2) Highly potent and complete lysis of target cell by BiTE® activated T cells

(3) Lysis of dividing as well as non-dividing target cells

(4) Serial lysis by BiTE®-activated T cells

(5) Sustained proliferation of BiTE®-activated T cells

(6) Does not require MHC Class I andpeptide antigen for recognition by T cell

(7) Does not require T cell clone with specificT cell receptor

Key Hallmarks of BiTE® Mode of Action

Hoffmann, P. et al. Intl J Cancer 2005; 115: 98-104

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• CD19-CD3 bispecific BiTE antibody construct• Clinically developed for treatment of r/r ALL• Received breakthrough designation from the US FDA

in July 2014• BLA submitted to FDA in Sept 2014 and to EMA in Oct

2014

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FDA Approved BLINCYTOTM on December 3rd, 2014

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The New BiTE® Platform is NHP Cross‐reactive

BLINCYTOTM New Platforms v3.0

Target Human and Chimpanzee Human and NHP

T Cells (CD3) Human and Chimpanzee Human and NHP

Pharmacological Characterization

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• Side-by-side comparison of pharmacology in human and toxicology species systems

For bispecific binding– binding affinity for human and Cyno target antigens– epitope mapping– specificity of binding

In Vitro Pharmacology Package for First in Human (FiH) Study

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In Vitro Pharmacology Package for FiH Study Continued

For redirected lysis– dose-response of target cell lysis with various target cells

• correlation of EC50 of lysis with target expression level• specificity of target cell lysis

– kinetics of lysis– dependence on effector to target ratios– target cell lysis by various T cell populations– PBMC / T cell donor variability– redirected lysis of patient-derived malignant cells by

autologous T cells (ex vivo PoC)

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In Vitro: Blinatumomab‐Redirected Lysis

Amgen Report No. 103-PCD-0061

MT103 [ng/mL]

Spec

ific

Lysi

s [%

]

NCEB-1

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

GRANTA-519

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

HBL-2

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

A

C

E

EHEB

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

MEC-1

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

Karpas-422

-20

0

20

40

60

80

100

10-4 10-2 100 1020

B

D

F

MT103 [ng/mL]

Spec

ific

Lysi

s [%

]

NCEB-1

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

GRANTA-519

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

HBL-2

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

A

C

E

EHEB

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

MEC-1

-20

0

20

40

60

80

100

10-3 10-2 10-1 100 101 1020

Karpas-422

-20

0

20

40

60

80

100

10-4 10-2 100 1020

B

D

F

Redirected Lysis (EC90 ) of Human B Lymphoma Lines at approx.0.5 ng/ml

Redirected Lysis (EC90 ) of Human B Lymphoma Lines at approx.0.5 ng/ml

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Estimation of a Therapeutic Dose Based on In Vitro PD Data

• EC90 of BLINCYTOTM -mediated redirected lysis: ~ 0.5 ng/mL

• In the clinic, anti-tumor activity was observed at a c.i.v

dose of 15 µg/m2/d

• Blinatumomab steady state serum concentration at 15

µg/m2/d: 0.62 ng/mL (range 0.18-1.0 ng/mL)

In vitro EC90 data predicted the human exposure required for anti-tumorefficacy

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• Sample derived from B-CLL Patient with very low E:T cell ratio (1:48)

Ex vivo:  Elimination of Lymphoma Cells byBlinatumomab Analogue

Löffler et al. Leukemia 2003 17:900-909

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Example: Ex vivo Elimination of AML Blasts by AMG 330

Kupka, C. et al. Blood 2014 Jan 16;123(3):356-65

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Example: Ex Vivo Depletion of CD33+ Cells from Cyno Bone Marrow by AMG 330

0 24 48 72 96 120 144

0

20

40

60

80

100

w/o AMG 330with AMG 330

Time [h]

CD

33+ [%

of v

iabl

e ce

lls]

0 24 48 72 96 120 144

0

20

40

60

80

100 with AMG 330w/o AMG 330

Time [h]

T-ce

lls [%

of a

ll ce

lls]

Friedrich, M. et al., Mol Cancer Ther. 2014 Jun;13(6):1549-57.13(6)

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In Vitro Pharmacology Package for FiH Study Continued

• For dose- and target cell-dependent T cell activation– induction of surface markers for early and late T cell

activation– increase in T cell size and induction of proliferation– specificity of T cell activation – expression of granzyme and perforin in BiTE®

antibody-activated CD8+ and CD4+ T cells– time- and dose-dependency of cytokine release by T

cells

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Example: In Vitro Induction of T CellProliferation by BLINCYTOTM

-20

0

20

40

60

80

100

120

140

63 6.3 0.63 0.063 0.0063 0 MT102 PHA/ IL2

MT103 Concentration [ng/mL]

Prol

ifera

tion

[%]

CD19-depleted PBMCPBMC

Amgen Report No. 103-PCD-0065

Blinatumomab Concentration [ng/mL]

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Ensuing T Cell Expansion in Patients

T C

ells

[103 /

l]

Baseli

ne

Maxim

um

0.0

0.5

1.0

1.5

2.0

p = 0.0002

T C

ells

[103 /

l]

Baseli

ne

Maxim

um

0.0

0.5

1.0

1.5

2.0

Klinger et al. Blood. 2012;119:6226-33

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Example: In Vitro Target Cell‐dependent Inductionof Activation Markers on T Cells by BLINCYTOTM

No Target Cells CHO (10%) CHO-CD19 (1%) CHO-CD19 (5%) CHO-CD19 (10%)

10

20

30

40

50

CD

69- o

r CD

25-p

ositi

veT

Cel

ls [%

]

CD69 - - + + - - + + - - + + - - + + - - + +CD25 + + - - + + - - + + - - + + - - + + - -CD4 + - + - + - + - + - + - + - + - + - + -CD8 - + - + - + - + - + - + - + - + - + - +

Brischwein , K. et al. J Immunother. 2007;30:798-807

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T‐Cell Activation Marker CD69 on CD8+ and CD4+T Cells is Transiently Upregulated

• The T-cell activation marker CD69 was investigated for 6 patients and is transiently upregulated on CD8+ and CD4+ T cells after infusion start in cycle 1.

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BLINCYTOTM‐induced Cytokine Release In Vitro

Cytokines

Cyt

okin

e [p

g/m

L]

IFN- TNF IL-10 IL-6 IL-4 IL-20

200

400

600

800

1500200025003000 Cytotox Assay with 1 µg/mL MT103

Cytotox Assay w/o MT103

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Cycle 2

Time Point [Days]

Mea

n C

ytok

ine

Con

cent

ratio

n [p

g/m

l]

00.1

10.1

70.3

30.5

9 1 2 7 14 21 28

0

100

200

300

400

500

600

700

800

900 IL-2IL-6IL-10IFN-TNF-

B

Cytokine Release by BLINCYTOTM is Transient and Modest

Mean Peak Cytokine Levels in Serum of all Assessable Patients

Klinger et al. Blood. 2012;119:6226-33

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Cytokine Profiles of Individual Patients after 60 µg/m2/day BLINCYTOTM given as First Dose or After Double Dose Steps

• Cytokine elevation was reduced after a stepwise dosing approach to a target dose of 60 µg/m2/day compared to a starting dose of 60 µg/m2/day

Note: Data < LOD were set to 10 pg/mL ( ½ LOD).

N= 9N= 21

60 µg/m2/day5 15 60 µg/m2/day

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In Vivo: Activity of BLINCYTOTM

Amgen Report No. 103-PCD-0098 and R20130026

Raji Burkitt‘s Lymphoma Xenograft Granta-519 Mantle Cell Lymphoma Xenograft

0 5 10 15 20 25 300

250

500

750

1000

1250

1500

1750

2000 Gr.1 Vehicle w/o PBMCGr.2 VehicleGr.3 AMG 103 (0.5 mg/kg)Gr.4 AMG 103 (0.05 mg/kg)Gr.5 AMG 103 (0.005 mg/kg)Gr.6 AMG 103 (0.0005 mg/kg)

Days after Tumor Cell Injection

Tum

or V

olum

e [m

m3 ]

Treatment(days 1-5)

0 20 40 60 80 1000

20

40

60

80

100

Treatmentq1dx26

1 Vehicle control w/o T cells

2 Vehicle control

3 AMG 103 (0.267 mg/kg/day); **

4 AMG 103 (0.027 mg/kg/day); **

5 AMG 103 (0.003 mg/kg/day); ***

6 AMG 103 (0.133 mg/kg/day); ***

Day of study

Surv

ival

[%]

• Efficacy in a panel of xenograft models

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Peripheral B‐Cells are Depleted and T Cells Redistribute During BLINCYTOTM Treatment

• B- and T-cell counts varied widely between patients before infusion start.

• For all but one patient (nonresponder), peripheral B cells were depleted rapidly (< 1 week for most patients) and remained undetectable throughout treatment.

• There was a swift redistribution of T cells after treatment start (day 1) and dose step (day 8); T-cell counts recovered to baseline within several days, where they remained stable in most patients.

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10 -1

10 -2

10 -3

10 -4

MRD pos.

MRD neg.

07-07 09-07 11-07 01-08 03-08 05-08 07-08 09-08 11-08

Cons. 1 Cons. 2 Cons. 3 Cons. 4Induction

Chemotherapy GMALL (Elderly) Protocol

Blinatumomab

Study PopulationB-precursor ALL patients in complete hematological remission with molecular failure or molecular relapse at any time after chemotherapy

Leukemia

Phase 2 Study with Blinatumomab inPatients with Residual B‐ALL in Bone Marrow

Treatment4 week continuous infusionDose: 15 g/m²/d

Patient #109-002

Nonclinical Safety Evaluation

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• Study of target-related risk – TCR studies and RNA expression profiles– Comparative target distribution between human and cyno

tissue panels

• Comparative in vitro pharmacology in human and cynoco-culture systems incl. determination of MABEL

• Studies in non-human primates– Exploratory dose range-finding in cyno monkeys – Toxicity study in cyno monkeys including detailed

hematology, toxicokinetics, immunogenicity and safety pharmacology assessments

Toxicology Package for FiH Study

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Example : Determination of MABEL

0

20

40

60

80

100

10- 4 10- 2 100 102 1040MT112/BAY2010112 [ng/mL]

Spec

ific

Lysi

s [%

]

Effector cells: human PBMC (n=20)

Amgen Report No. 112-NCD-0007

MABEL is the minimum anticipated biological effect level Selection of the Most

Sensitive Test System Cell line with highest

expression of TAA surface molecules

Detemination of the most sensitive endpoint:redirected lysisT cell activationcytokine release

BiTE [ng/mL]

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• Transient elevation of body temperature • Transient cytokine release• Hematologic changes

– transient reduction in LYMPH and MONO (recovered by Day 8)

– increased CD4 and CD8 cell activation (CD69) transient increase in NEUT

• Acute phase response elevated CRP, reduced albumin, increased globulin and

fibrinogen

Common Effects Observed in Cynos within the First 48 hrs of Treatment 

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Example: PD Endpoints in Cyno StudiesControl BiTE®-treated Group

Day 7BloodFACS

BoneMarrowFACS

Prestudy Day 7

Bone marrow

(sternum)

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Cyno Model Relevant and Translates to Clinic• Cyno is a pharmacologically-relevant model PD effects present indicators of BiTE® activity observed signs of toxicity dependent on tumor-target expression on

normal tissues

• BiTE® effects in cyno mimic those observed clinically– body temperature changes = fever/chills cytokine release hematological changes acute inflammatory response

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• Amgen– Matthias Friedrich– Oliver Thomas– Petra Deegen– Anja Henn– Alexander Sternjak– Grit Lorenczewski– Patrick A. Bäuerle– Andreas Wolf– and the ARM technical staff

Acknowledgements

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AMGEN Research (Munich) GmbH