Therapeutic Factor IX (FIX) Activity After Single

Treatment With AMT-060 (AAV5-FIX) In Hemophilia B

Patients With Pre-existing Anti-AAV5 Humoral

Immunity

Anna Majowicz, Bart Nijmeijer, Margit Lampen, Lisa Spronck, Martin de Haan,

Harald Petry, Sander van Deventer, Christian Meyer, Marco Tangelder,

Valerie Ferreira

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▪ 1:5 anti-AAV8 NAB titer totally blocks in vivo liver transduction in Non-

human Primates (NHP) with AAV8-hFIX (Jiang et al., Blood 2006)

▪ 1:17 titer of pre-existing anti-AAV2 NAB is described to “modulate linear

dose response” in humans to AAV2-hFIX treatment:

1:17 mean hFIX activity of 3%

1:2 mean hFIX activity of 11%

(Manno et al., Nature Medicine 2006)

▪ 1:1 titer of bioengineerd AAV-Spark100 capsid was reported to be

associated with reduced therapeutic efficacy (George et al., NEJM 2017)

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AAV5

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Patient # FIX activity (basal) Pre-existing anti-AAV5

NAB titer, GFP-based

assay

Total pre-existing anti-AAV5

(ELISA)

Low dose cohort (5e12 gc/kg)

1 < 1% Negative Negative

2 < 1% Negative Negative

3 < 1% Negative Positive (87)

4 1.5% Negative Positive (256)

5 < 1% Negative Negative

High dose cohort (2e13 gc/kg)

6 < 1% Negative Negative

7 < 1% Negative Negative

8 1% Negative Negative

9 1% Negative Negative

10 1% Negative Negative

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n= 50 healthy donors

GFP-based assay

1/50 tested positiveLUC-based assay

32/50 tested positive

an

ti-A

AV

5 N

AB

tit

er

1 0 1

1 0 2

1 0 3

1 0 4

2

Inh

ibit

ion

of

tra

ns

du

cti

on

(% r

ela

tiv

e t

o c

on

tro

l)

-6 0

-4 0

-2 0

0

2 0

4 0

T o ta l a n t ib o d ie s a g a in s t A A V 5 - O D (4 5 0 n m )

an

ti-A

AV

5 N

AB

tit

er

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4 1 .6 1 .8 2 .0 2 .2 2 .4 2 .6 2 .8 3 .0 3 .2

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

r=0.85 (p<0.0001)

****

Total anti-AAV5 (ELISA) and anti-AAV5 NAB measured

with a LUC-based assay show strong correlation

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Patient # * FIX activity

(basal)

* Median FIX acivity post

treatment (min-max)

* Pre-existing

anti-AAV5 NAB

titer, GFP-

based assay

Pre-existing

anti-AAV5

NAB titer,

LUC-based

assay

* Total pre-existing anti-AAV5 (ELISA)

Low dose cohort (5e12 gc/kg)

1 < 1% 6.6% (5.2-8.8) Negative < 4 Negative

2 < 1% 4.9% (4.0-7.2) Negative < 4 Negative

3 < 1% 1.3% (1.1-1.4) Negative 210 Positive (87)

4 1.5% 7.0% (5.6-11.5) Negative 340 Positive (256)

5 < 1% 3.1% (2.1-4.5) Negative 21 Negative

High dose cohort (2e13 gc/kg)

6 < 1% 11.9% (10.3-14.4) Negative < 4 Negative

7 < 1% 6.6% (3.2-10) Negative < 4 Negative

8 1% 7.8% (4.3-9.8) Negative < 4 Negative

9 1% 3.3% (2.7-5.5) Negative < 4 Negative

10 1% 6% (4.9-8.7) Negative < 4 Negative

siblings

* Leekbeek et.al, 2017 American Society of Hematology

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0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

21 0 4

41 0 4

61 0 4

81 0 4

11 0 5

1 .21 0 5

w e e k

To

tal

an

ti-A

AV

5 a

b

Ig G

Ig M

NAB: -

TAB: -

Patient 1 Patient 4

NAB: 210

TAB: + (87)

NAB: 340

TAB: + (256)

Patient 3

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

21 0 4

41 0 4

61 0 4

81 0 4

11 0 5

1 .21 0 5

w e e k

To

tal

an

ti-A

AV

5 a

b

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

21 0 4

41 0 4

61 0 4

81 0 4

11 0 5

1 .21 0 5

w e e k

To

tal

an

ti-A

AV

5 a

b

Patient 5

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

21 0 4

41 0 4

61 0 4

81 0 4

11 0 5

1 .21 0 5

w e e k

To

tal

an

ti-A

AV

5 a

b

NAB: 21

TAB: -

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▪ Anti-AAV5 NAB titer as high as 340 did not impair the

efficacy of AAV5-FIX (AMT-060) therapy in human.

▪ The patient with the highest anti-AAV5 NAB titer had the

highest FIX activity in his dose cohort.

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GROUPS IV injection of AAV5-FIX [gc/kg]

1. n=3 5 e11

2. n=5 5 e12

3. n=3 2.5 e13

4.n=3 9 e13

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No inhibition of liver transduction by AAV5 in NHP with pre-existing

anti-AAV5 NAB up to titer of 1030

% o

f h

FIX

in

th

e p

las

ma

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▪ Anti-AAV5 NAB titer as high as 340 did not impair the

efficacy of AAV5-FIX (AMT-060) therapy in human.

▪ Anti-AAV5 NAB titer as high as 1030 did not impair the

efficacy of AAV5-FIX (AMT-060) liver transduction in

NHP.

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?

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97%

< 3 4 0 > 3 4 0

0

2 0

4 0

6 0

8 0

1 0 0

a n ti-A A V 5 (1 6 0 ) N A B t ite r

Nu

mb

er o

f s

am

ple

s

9 2 %

8 %

n = 1 0 0

< 1 0 3 0 > 1 0 3 0

0

2 0

4 0

6 0

8 0

1 0 0

a n ti-A A V 5 (1 6 0 ) N A B t ite r

Nu

mb

er o

f s

am

ple

s

9 7 %

3 %

n = 1 0 0

anti-AAV5 NAB titer in healthy donor sera

92% eligible

1 00

1 01

1 02

1 03

1 04

an

ti-

AA

V5

NA

B t

ite

r

1 0 3 0

3 4 0

2

n=100

53 donors (53%) <2

anti-AAV5 NAB 340 cut-off

97% eligible

anti-AAV5 NAB 1030 cut-off anti-AAV5 NAB >247%, median titer = 27

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anti-AAV5 titer

NAB >2: 47%

median titer = 27

mean titer= 453.9

anti-AAV8 titer

NAB >2: 37%

median titer = 87

mean titer= 805.6

anti-AAV2 titer

NAB >2: 49%

median titer = 97

mean titer= 4504

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anti-AAV5 titer

NAB >2: 47%

median titer = 27

mean titer= 453.9

anti-AAV8 titer

NAB >2: 37%

median titer = 87

mean titer= 805.6

anti-AAV2 titer

NAB >2: 49%

median titer = 97

mean titer= 4504

*

**

*Jiang et al., Blood 2006

** Manno et al., Nature Medicine 2006

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▪ 97% of all individuals can benefit from AAV5-based gene therapy

▪ 37% of individuals would be excluded from AAV8-based studies;

91.9% of these can still benefit from AAV5-based gene therapy

▪ 49% of individuals would be excluded from AAV2-based studies;

93.9% of these can still benefit from AAV5-based gene therapy

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