New Therapies for Hemophilia: Options and Assays...Factor VIII Activity at 14 Days in the Two Dose Groups. 40 20 10 5 3 1 100 10 1 rFVIII Mean Plasma FVIII:C BIVV001 (IU/dl) Percent

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©2020 MFMER | slide-1©2020 MFMER | slide-1

New Therapies for Hemophilia: Options and AssaysRajiv K. Pruthi, M.B.B.S.Consultant I Associate Professor of MedicineDirector, Mayo Comprehensive Hemophilia Center Co-Director, Special Coagulation and Molecular Hematopathology Laboratories, Chair, Coagulation Diseases Oriented Group (Hematology)Mayo Clinic, Rochester, MN

A Case-based Workshop: Clinical and Laboratory Aspects of Hemophilia and Thrombosis Dec 4th, [email protected]

©2020 Mayo Foundation for Medical Education and Research | slide-1


Relevant Financial Relationship(s)

• Consulting honoraria (advisory boards): • CSL Behring; Genentech Inc; Bayer Healthcare

AG; HEMA biologics; Instrumentation Laboratories.

Off Label Usage:

• Most products mentioned are FDA approved.

• Selected ones are not FDA approved.

DISCLOSURES

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LEARNING OBJECTIVE

Overview of evolution of options for therapy for bleeding disorders

Currently available options for management of hemophilia

Impact on hemophilia centers and coagulation laboratories

©2020 MFMER | slide-4

Illustrative case• A 12 month old male was evaluated for prolonged epistaxis

• Family history: no known bleeding disorders

• Laboratory data:• Complete blood count: normal (normal platelet count)• PT: normal• aPTT: prolonged (corrects on 1:1 mixing study with normal pooled

plasma)• Factor assays: FVIII:C:

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• 1900 to 1940s: Whole blood

• 1950-60s: Plasma

• Mid-1960s: cryoprecipitate

• Late 1960s: plasma derived (pd) factor VIII

• Mid 1980s: purified/viral inactivation process

• 1990s: recombinant FVIII & FIX

• Early 2000s: pdVWF and Initial (ongoing) gene

• therapy trials

• 2014: extended half life (t ½ ) factors

• 2015: recombinant VWF

• 2017: Non-factor therapeutic options• FDA approved:emicizumab• Clinical trials:

• Fitusiran, concizumab, • BAY 1093884, PF-06741086

Laboratory Assays:FVIII/FIX:One stage assay (OSA)VWF functional assay:Ristocetin cofactor assay

Advances: • Chromogenic factor assays

• Alternate VWF functional assays

Evolution of Therapies for Hemophilia


Goals in management of bleeding disorders

• Prophylaxis:• Reduction in annualized bleeding rate (ABR)

• Preservation of joints over long term• Reduce frequency of infusion

• Treatment of bleeds:• Effective with minimal infusions• Reduction of recurrent bleeds

• Individualized pharmacokinetics• Reliable factor assays

• Options: factor concentrates, non-factor concentrates, Gene therapy

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Major advance: extended half life concentrates

• Fusion of factor VIII and IX molecule with linkers that extend half-life

• Fc Fusion• Fragment crystallizable (Fc) domain of human immunoglobulin

• Pegylation• Polyethylene glycol (PEG) polymers • Variable pegylation and location on molecule

• Albumin fusion

• Disadvantage of FVIII extended half-life products• Relies on FVIII binding to VWF

• Emerging FVIII molecule: BIVV001 (rFVIIIFc-VWF-XTEN• rFVIII fused to dimeric Fc, D’D3 domain of VWF, two XTEN polypeptides • ‘Uncouples’ FVIII reliance on VWF half life


100

0Time (hours)

10

1

rFVIIIrFVIIIFc

FVIII

:C (I

U/d

L)

3%

1%20 40 60 80 100 120

Mahlangu et al Blood 2014

Extended t ½ FVIII and FIX

rFIXrFIXFc

0Time (hours)

FIX:

C(IU

/dL)

3%

1%

100

10

060 120 180 240 300

Powell et al NEJM 2014

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Critical review of long t ½ FVIII products

0.5 1.0 1.5 2.0 2.5 3.0

0.5 1.0 1.5 2.0 2.5 3.0

AUC ratio (mean and 90% CI)

AUC ratio (mean and 95% CI)

rFVIII product/Study

BAX 855/ NCT01736475BAY 94-9027/NCT01184820, high dose

BAY 81-8973/NCT01029340

rFVIII-SingleChain/NCT01486927

Human-cl rhFVIII/NCT00989196

rFVIIIFc/NCT01181128

N8-GP/NCT01205724

2.17

1.44

1.19

1.35

0.98

1.69

1.49

Reproduced from Mahalangu et al Haemophilia 2018; 24:348


Factor VIII Activity at 14 Days in the Two Dose Groups.

40

20

10

5

3

1

100

10

1

rFVIII

BIVV001

Mea

n Pl

asm

a FV

III:C

(IU/d

l)

Perc

ent P

lasm

a FV

III:C

Act

ivity

Days after infusion

Dose: 65 IU/kg

Reproduced from Konkle BA et al. N Engl J Med 2020;383:1018-1027

1412107543210

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Illustrative case

•A 12 month old severe hemophila:•Subcutaneous port placed•Started regular IV dosing of extended half life•One year later, port had to be removed due to infection

•Parents are asking about alternatives•Poor peripheral IV access•Are there subcutaneous medications options


EmicizumabBispecific antibodyAdministered subcutaneously (once weekly, once every two weeks or once a month)

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©2020 MFMER | slide-13Oldenberg J et al. NEJM 2017; 377(9):809-18

FVIII inhibitor

Thrombingeneration


Emicizumab in hemophila A patients without inhibitors

05

101520253035404550

Annualized Bleeding Rates

Emicizumab weeklyEmicizumab q2 weeksNo prophylaxis

2.41.6 – 3.9

2.61.6 – 4.3

47.628.5 – 79.6

Mahlangu, J et al N Engl J Med 2018; 379:811-822

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Study Study population Dosing Efficacy SafetyHAVEN1 HA with inhibitors

(n=109)LD: 3 mg/kg/wkk x 4 MD: 1.5 mg/kg/wk

Prophy vs no prophy87% reduction in ABR

TMA:3Thromb: 2

HAVEN2 Pediatric HA with inhibitors (n=60)

LD: 3 mg/kg/wk x 4 MD: 1.5 mg/kg/wk; 3 mg/kg every 2wk; 6 mg/kg every 4wk

Prophy vs no prophy99% reduction in ABR

No thromb

HAVEN3 HA without inhibitors(n=152)

LD: 3 mg/kg/wk x 4 MD: 1.5 mg/kg/wk; 3 mg/kg every 2wk;

Episodic therapy vs Prophy96 & 97% reduction in ABR

No major safety issues

HAVEN4 HA without inhibitors(n=48)

LD: 3 mg/kg/wk x 4 MD: 6 mg/kg every 4wk

Similar to HAVEN 1,2,3

No major safety issues

|3146613-15


Non-factor therapies: FDA approval

Drug Hemophilia A with inhibitors

Hemophilia A with NO inhibitor

Potential diseasetarget

Enhancing Procoagulant cascadeEmcizumab Yes Yes Hemophilia ASuppressing anticoagulant cascadeConcizumab No No Hemophilia A&BFitusiran No No Hemophilia A&B

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©2020 MFMER | slide-17CP1106730-1

Factor IXa

Thrombin (IIa)

Factor IX

Ca2+

Factor XCa2+ PL

Factor VIIIFactor Xa

Factor XIaFactor XI

Surface

Thrombin

Intrinsic Pathway

Factor VIIaTissue factor

Ca2+

Ca2+

Factor IX

Factor VIITissue factor

Factor XaThrombinFactor VIIaFactor IXa

Extrinsic Pathway

Vascular injury

Tissue factor

Factor VII

Factor X

Factor Xa

Factor V

Fibrinogen Fibrin

Fibrin (cross-linked)

Factor XIII

Factor XIIIa

Ca2+Fibrin

Actions of Protein C, S and Antithrombin

Ca2+ PL

Factor Va

Protein CProtein S

Factor VIIIaCa2+ PL

Antithrombin

Prothrombin (II)

Tissue factorPathway inhibitor


Role of TFPI: prevent generation of Xa

TFPI

TFPI: tissue factor pathway inhibitor

VIIa X

TF

Phospholipd

VIIa XTF

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Concizumab: mechanism of action

TFPI

TFPI: tissue factor pathway inhibitor; TF: tissue factor

anti-TFPI (concizumab)VIIa X

TF Xa


Concizumab: Phase I study

3146613-20Eicher, H et al JTH 2018; 16: 2184

First dose Last dose

1000

100

10

10 10 20 30 40 50 60 70 80

0.8 mg/kg0.5 mg/kg0.25 mg/kg

Con

cizu

mab

in p

lasm

a(n

g/m

L

Time (days)

Placebo-20 -10 0 10 20 30 40 50 60 70 80

020406080

100120

Time (days)

First dose Last dose

Unb

ound

TFP

I(n

g/m

L

Placebo0.25 mg/kg 0.5 mg/kg

0.8 mg/kgConcizumab dose

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RNA translation to protein: action of fitusiran

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fitusiran

tRNA

tRNAtRNA

tRNA

aminoacid

aminoacid

aminoacid

aminoacid

mRNAribosome

tRNA bound to amino acidEmpty tRNA

Growing peptide chain


Relationship between Antithrombin Level and Thrombin Generation.

Pasi KJ et al. N Engl J Med ;377:819-828

Peak

thro

mbi

n le

vel (

nM)

Antithrombin level (%)0 20 40 60 80 100 120 140

250

200

150

100

50

0

Hemophilia AHemophilia BNormal

Pasi KJ et al. N Engl J Med ;377:819-828

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Assays to monitor emerging agents

•Fitusiran: measurement of antithrombin levels•TFPI inhibitors: TFPI levels•Emerging assays:

•Global assays •thrombin generation•thromboelastography

©2020 MFMER | slide-24Reproduced from: Pasi KJ et al. N Engl J Med 2020;382:29-40

FVIII

act

ivity

IU/d

LO

ne s

tage

FVI

IIC

Median Factor VIII

Levels:Yr 1: 88.6%Yr 2: 45.7%Yr 3: 29.8%

Annu

aliz

ed

Ble

edin

gRat

e(e

piso

des/

yr)

Before infusion

1 year 2 year 3 year

After infusion

Median16.5

Mean 16.3

15

20

5

10

00.9

00.2

0 00.7

96% reduction in mean ABRNo of participants: 6

360

320

280

240

200

160

120

80

40

00 8 16 24 32 40 48 52 65 78 91 104

7 7 7 6 7 7 7 7 7 7 7 7 7 7 7Number of participants

117 130 143 156

Week

AAV5-hFVIII-SQ Gene therapy for hemophilia A

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Factor IX Activity after One Peripheral Infusion of SPK-9001 in the Participants Who Did Not Have an Adeno-Associated Viral Capsid-Directed Immune Response.

Reproduced from George LA et al. N Engl J Med ;377:2215-2227

0 4 8 12 16 20 24 28 32 36 40 44 48 52 70 78

60

50

40

30

20

10

0

Week after vector infusion

4 43

12

0 0 0 0 0 0 0 0 0 0 0 0

10

32

2

48

Participant number1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

60

40

20

0

1 yr pre-infusion Post-infusion

Participants (n=10)


Coagulation factor assays

•One stage•Most commonly used

•Chromogenic assays•Less used but increasingly common

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Factors influencing one stage factor assays: contact activators, phospholipid & calibrators

Activator Phospholipid (PL) Calibrators Instrumentation

Ellagic acid basedSynthetic

Plasma derived standards

Mechanical methods

Ellagic acid

Polyphenolic acid (ellagicacid like) CephalinSilica based

Colloidal silica SoyaChromogenic standards

Optical methodsMicronized silica Vegetable/PlantSilica dioxide Bovine

Concentrate specificstandardsSulfatides and silica Rabbit brain

Kaolin Porcine and chicken


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Acceptable activators for EHL concentratesProduct OSA:silica OSA:ellagic CSA

BAX 855 (Adynovate) √1 √ √rFVIII:Fc (Elocatate) √ √ √rFVIII:SC (Afstyla) √2 X √rFVIII;(N8-GP) √1 √ √rFVIII; (BAY 94-9027) √3 √ √rFIXFc (Alprolix)* √ √ √1r-FIX-FP (Idelvion)* √ X1 XN9-GP(Rebinyn)* X √ √1

3146613-29

1, exceptions; 2, multiply OSA result by 2; 3, unknown; * avoid kaolin based reagents; X, unacceptable


Intrinsic Extrinsic

VIIXII

XIIX

VIII

XVII

Fibrinogen

aPTT PT

Contact activator:+Ca +Phospholipid

One stage FVIII assay

Fibrin clotPatient plasma + FVIII deficient plasma

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FVIII assay: role of the calibrator (native factor vs concentrate)

3146613-31

200

100

60

40

20

5 10 20 30 50 100

Calibrator

FVIII Activity

Clo

tting

tim

e (s

)

Patient plasma

Kitchen et al Quality in Lab Hemost & Thromb 2009


0

20

40

60

80

100

120

140

0 1 3 10 30 100

300

1000 1 10 10

0

APTT

(s)

Human FVIII deficient plasmaWith FVIII inhibitorsWithout FVIII inhibitors

ACE910 (nM) rhFVIII U/dL

Muto A. et al JTH 2014; 12:206

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1000

1200

0 20 40 60 80 100 1200

200

400

600

800

Emicizumab ug/mL

One stage assay

FVIII activity

120

100

80

60

40

20

00 50 100 150 200

Emicizumab ug/mL

Chromogenic assay

Human X & IXa

Bovine X & IXa

FVIII activity

One stage assay and chromogenic assay with human substrate: false FVIII result in presence of emicizumab

One stage assay and chromogenic assay with human substrateResults in a false FVIII result in presence of emicizumab


Currently available options for management of hemophilia: Ever expanding

•Standard half-life factor concentrates

•Extended half-life concentrates•Non-factor based therapies•Modified factor concentrates and non-factor options impact laboratory testing

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Impact on hemophilia centers and coagulation laboratories•HTCs: Knowing which reagents laboratories utilize•Laboratories: Knowing which product patient is on

•There are acceptable and non-acceptable reagents for each product

•Strategy to understand performance characteristics of reagents:

•Field studies•Package inserts


Implications for FVIII inhibitor Bethesda assay

•Using one stage assays and chromogenic assays with human substrate

•False negative or lower estimates of Bethesda titers•Performance of Bethesda assay using chromogenic FVIII assays with bovine substrates is required

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[email protected]

Documents

New Therapies for Hemophilia: Options and Assays...Factor VIII Activity at 14 Days in the Two Dose Groups. 40 20 10 5 3 1 100 10 1 rFVIII Mean Plasma FVIII:C BIVV001 (IU/dl) Percent