DX-2930

DRUG TOLERANCE CHALLENGES IN ENZYME INHIBITOR-BASED NEUTRALIZING ANTIBODY ASSAYS

JUNE 10, 2015

Outline

NAb assays for two inhibitors of plasma kallikrein • Ecallantide: 60 amino acid peptide inhibitor with short half life • DX-2930: human antibody inhibitor with long half life and drug tolerance challenge

Plasma Kallikrein-Kinin System (KKS)

FXII Charged Surface Coagulation Cascade

FXIIa 1-Chain HMWK

Prekallikrein

Active pKal 2-Chain HMWK + Bradykinin

C1-INH Ecallantide, DX-2930

B2R GPCR Activation

Edema / Pain

3

Hereditary Angioedema

•

Types: • Autosomal Dominant (AD) HAE Type 1 – Very low production of C1-INH • AD HAE Type 2 – Normal [C1-INH], but impaired C1-INH function • HAE Type 3 – Normal C1-INH levels, poorly characterized

•

Characterized by intermittent attacks of subcutaneous or submucosal edema affecting the face, larynx, gastrointestinal tract, or limbs

•

Laryngeal swelling can lead to death by asphyxiation (mortality 30% in undiagnosed individuals)

•

Attacks without warning, lasting up to >5 days

•

Orphan disease, prevalence ~ 1/10,000 – 1/50,000 Normal bowel before HAE attack

Swollen bowel during HAE attack

* Photographs courtesy of Dr. Marco Cicardi 4

Plasma Kallikrein Inhibitor: Ecallantide • 60 amino acid recombinant protein produced in Pichia Pastoris

• Kunitz domain • Discovered using phage display • Potent plasma kallikrein inhibitor (Ki = 25 pM) • Approved for treatment of acute attacks of hereditary angioedema • Half-life: ~2 hours • Cmax: ~600 ng/mL CONFIDENTIAL to DYAX CORP.

5

Anti-Ecallantide NAb Assay Principle

1) Pro-Phe-Arg-AMC

2) Pro-Phe-Arg-AMC

3) Pro-Phe-Arg-AMC

pKal

pKal + ecallantide

pKal + Ecallantide bound to NAb

Pro-Phe-Arg + AMC

Pro-Phe-Arg + AMC

Pro-Phe-Arg + AMC

6

Anti-Ecallantide NAb Assay

S

P pKal

NAb positive

Y

(pKal substrate converted to fluorescent product)

serum 7

pKal

+ H

Substrate

ecallantide

S

P

Immobilized biotinylated ecallantide NAb negative pKal ecallantide

(pKal inhibed by DX-88)

7

Anti-Ecallantide NAb Assay: Positive Control

ecallantide

Neutralizing mAb

CM5 sensor chip [ecallantide]

Kd = 56 nM

2.5 M

19.5 nM

ka

kd

KD

8

Anti-Ecallantide NAb Assay

7

Rate (F/s)

6

5

4

3

2 0

200

400

600

800

1000

1200

1400

1600

[NAb Positive Control] (ng/mL)

9

Anti-Ecallantide NAb Assay: Sensitivity 𝑆𝑎𝑚𝑝𝑙𝑒 𝑅𝑎𝑡𝑒 − 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑒𝑑 𝑅𝑎𝑡𝑒 %𝑁𝑒𝑢𝑡𝑟𝑎𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 = ∗ 100 𝑈𝑛𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑒𝑑 𝑅𝑎𝑡𝑒 − 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑒𝑑 𝑅𝑎𝑡𝑒 60

50

%Neutralization

40

• Sensitivity approximately 250 ng/mL 30

• Cut-Point (10.97%) determined using 30 individual human serum samples

20

10

0

0

500

1000

1500

2000

2500

[neutralizing Ab] (ng/mL)

10

Anti-Ecallantide NAb Assay: Drug Tolerance 15 10.97% Cut-point

%Neutralization

10

5

0

-5

-10

-15 0

10000

20000

30000

[Ecallantide] (ng/mL)

Low drug tolerance

11

Plasma Kallikrein Inhibitor: DX-2930 • Human IgG1 produced in CHO cells • Discovered using phage display • Potent plasma kallikrein inhibitor Ki = 125 pM

• In clinical trials for prophylactic treatment of HAE • Half-life: ~2 weeks • Cmax: up to 40 ug/mL (2 x 400 mg SC doses) CONFIDENTIAL to DYAX CORP.

12

Combine SPEAD Step with Enzyme Activity NAb Assay

Y

Plasma Sample

Y

+

nAb 1

7

Biotin DX-2930

7

E F

H

H

Neutralization Plate

Streptavidin Plate

S

P

S

P

pKal DX-2930 pKal DX-2930 NAb positive

NAb negative 13

Combine SPEAD Step with %Neutralization Enzyme Activity Pro-Phe-Arg-AMC

pKal + DX-2930 bound to NAb

Pro-Phe-Arg + AMC

120 500 ng/mL PC 1000 ng/mL PC 2000 ng/mL PC

%Neutralization

100

80

60

40

20

0 No DX-2930

2 ug/mL DX-2930

20 ug/mL DX-2930

SPEAD-enzyme activity assay does not have sufficient drug tolerance 14

Could we justify use of a binding Nab assay? Examine what we know about the structural biology of DX-2930

15

DX-2930 Binds pKal Active Site: Biacore SPR Analysis

80

pKal

Response (RU)

60

40

pKal+AEBSF

20

pKal+PFR-CMK 0

300

prekallikrein 400

500

600

700

800

900

1000

1100

Time (s)

16

pKal:DX-2930 Complex Crystal Structure

• Complex of DX-2930 Fab portion with a deglycosylated pKal variant • X-ray Diffraction to 2.1Å resolution

pKal

HV

• 2,404 Å2 of buried surface at the complex interface – large interaction area promotes specificity and affinity

LV DX-2930 Fab

HC

LC

17

LV-CDR2 Interactions

LV CDR2

E600

HV

E527 K528

pKal

Y555

LV DX-2930 Fab

K50

HC

LC

18

HV-CDR1 Interactions

FR1/HV CDR1

pKal

V410

R416

HV

L418

L439 L412

F27

F29

LV DX-2930 Fab

H434

Cyan = pKal catalytic triad residues

HC

LC

19

HV-CDR3 Interactions

HV CDR3

pKal Y475 S578 H434 D572

D483

HV

W598

R106

LV

R105 P104

DX-2930 Fab

V103

K575 E108

Cyan = pKal catalytic triad residues

HC

LC

20

HV-CDR3 Interactions

pKal

DX-2930 binds at the pKal active site; blocks substrate

* HV

LV *Catalytic serine

pKal : DX-2930

DX-2930 Fab

S 3

S 2

P R

S 1

R

N

HC

LC

S1’

S2’

* D

C

E

2.1Å resolution [Kenniston et al (2014) JBC] 21

DX-2930 and Ecallantide Bind at

pKal Active Site DX-2930 VL

DX-2930 VH

Ecallantide

22

Ecallantide and DX-2930 Interactions DX-2930 contacts Ecallantide contacts (modeled) Shared Contacts

Interactions at the active site 23

Could We Adapt the ADA ECLIA to Detect Nabs? • DX-2930 ADA assay involves a SPEAD step followed by ECLIA • Assay sensitivity: 36.45 ng/mL

• Drug tolerance: 250 µg/mL at 250 ng/mL PC • Principle: Nabs should competition with pKal target for binding to Sulfo-Tagged DX-2930

24

SPEAD-ECLIA NAb Binding Assay

Y

Plasma Sample

Y

+

O/N

nAb 7

1

Acid

Biotin DX-2930

7

E F

H

H

Neutralization Plate

Streptavidin Plate

nAb 7

H

MSD Plate

Sulfo-Tagged DX-2930 +/- pKal

Y

Y

E F

pKal

Y

1

ADA

nAb

NAb negative

NAb positive 25

SPEAD-ECLIA NAb Assay Sensitivity %𝑁𝑒𝑢𝑡𝑟𝑎𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 = 100 1 −

𝑀𝑒𝑎𝑛 𝐸𝐶𝐿 𝑤𝑖𝑡ℎ 𝑝𝐾𝑎𝑙 𝑀𝑒𝑎𝑛 𝐸𝐶𝐿 𝑛𝑜 𝑝𝐾𝑎𝑙

100

%Neutralization

80

positive control human anti-DX-2930 anti-idiotype mAb

60

38.8% Cut-point

40

20

0 0

1000

2000

3000

4000

5000

6000

[NAb] (ng/mL)

26

SPEAD-ECLIA NAb Assay Drug Tolerance

100

%Neutralization

80

60

38.8% Cut-point

40

20

0 0

200

400

600

800

1000

1200

[DX-2930] (µg/mL)

Drug tolerance: 250 μg/mL at 250 ng/mL positive control 27

SPEAD-ECLIA NAb Assay Validation Summary • Cut-point: 38.8% Neutralization (0.1% False Positive Rate) • Sensitivity: 78.92 ng/mL positive control

• Drug tolerance: 250 μg/mL at 250 ng/mL positive control • Intra- and Inter-assay less than 30% (less than 20%) • Short term stability (20 h) and F/T cycles (6) acceptable • Specificity: A non-neutralizing mAb was below the cut-point

28

Summary

• Enzyme based NAb assays were sensitive to drug interference • A structural and biochemical understanding of the interaction leading to the bioactivity supports the use of a binding assay for NAb detection

• A drug tolerant, binding-based NAb assay can be used for an antibody inhibitor of an enzyme

29

Acknowledgements

Ecallantide and DX-2930: Dyax R&D Ecallantide Nab Assay: Tandem labs DX-2930 Structure: Dyax R&D and Berylium DX-2930 ADA and Nab Assay: WIL Research Jeff Sailstad

B2S Consulting