Efficacy of a Vectorized Anti-Tau Antibody Using Systemic

© 2021 Voyager Therapeutics, Inc. – Not for Distribution.1

Efficacy of a Vectorized Anti-Tau

Antibody Using Systemic Dosing of a

Blood Brain Barrier Penetrant AAV

Capsid in Mouse Models of TauopathiesWencheng Liu, PhD

ASGCT 2021

Abstract No. 105

ASGCT 2021© 2021 Voyager Therapeutics, Inc. – Not for Distribution.

Disclosure

• Wencheng Liu is a full-time employee of Voyager Therapeutics,

Cambridge, MA, USA

| 2 |


Antibody Vectorization:Monoclonal Antibody Delivery via AAV Gene Therapy

• Modular Antibody Vectorization Cassettes with cell-

specific or general expression

• In-house PoC for serum delivery through muscle

expression of full-length Abs

• Active research in drugging intracellular proteome

through vectorized nanobodies, degraders, and other

innovative platforms

Voyager’s SolutionsChallenges Today

• Delivery to CNS with passive immunotherapy is

very limited (i.e., 0.1% of Abs pass through BBB)

• Inability to target the intracellular proteome

• Delivery of antibody fragments remains a challenge

due to PK liabilities

• Potential for unspecific or toxic off-target effects

• Prohibitive manufacturing volumes & costs

Vectorized

Antibodies

Targeted antibody therapies have been revolutionary solutions for medicine (e.g.,

oncology, inflammation), but similar efforts in neurological indications (e.g., AD,

tauopathies, synucleinopathies) have been met with significant challenges

3


Tauopathies: Progressive neurodegenerative diseases defined by the presence of tau

aggregates in the brain

• Alzheimer’s Disease

• Frontotemporal Dementia (includes MAPT mutations)

• Progressive Supranuclear Palsy

• Corticobasal Syndrome

Current Treatments Are Symptomatic only

• Abnormal tau accumulation is a hallmark of AD, FTD, PSP, CBS (including neurofibrillary tangles in AD)

• Tau pathology closely correlates with disease progression and cognitive decline in AD

• Imaging agents are now available to visualize pathological proteinopathies

Spreading Mechanism Hypothesis May Provide Opportunity for Therapeutic Antibody Intervention – Tauopathies/AD

Distribution of Tau Pathology by Stage

AD Braak

Staging:

Significant limitations to passive

immunization, including:

• Modest (20-40%) reductions in

tau pathology in animal models

• Requirement for high doses of

antibody to be administered

weekly or monthly

• Very low levels of antibody

achieved in the brain that may

limit efficacy

• Limited intraneuronal antibody

exposure

• Antibody fragments cannot be

used due to pharmacokinetic

half-life liabilities

Correlates with Neuroanatomical Connectivity

Adapted from Jouanne M, Rault S, Voisin-Chiret AS.

Eur J Med Chem. 2017 Oct 20;139:153-167.

I, II III, IV V, VI


Two Potential Pathogenic Mechanisms

• Can deliver non-secreted intrabodies with either cell-

type specific or pan-cell type capsids/promoters

• Voyager exploring use of intrabody efforts are early

stage

Cell AutonomousIntracellular Therapeutic Activity

• Can deliver as secreted antibody

• Tau and alpha-synuclein programs represent basic PoC

for rodent delivery and antibody expression in CNS

• Could include both prion-like spread as well as other

extracellular cell-to-cell mechanisms

Cell Non-AutonomousExtracellular Therapeutic Activity

Adapted from McEwan WA, et al. Proc Natl Acad Sci

USA. 2017 Jan 17;114(3):574-579.

Adapted from Marciniuk K, Ryan Taschuk R, Napper

S. Clin. Develop. Immunol. 2013, Id. 473706

Extracellular tau assembly

Seeded aggregation

Native protein

Uptake of tau assembly

TRIM21

Neutralization of seeding

Extracellular antibody:tau assembly complex


AAVrh.10PHF1 Treatment Reduces Insoluble p-Tau in the Hippocampus of P301S Mice

Light

ChainIgk

Heavy

ChainIgG1

Capsid

Furin 2A polyA

Heavy

Chain

Light

ChainITR

PromoterVectorized PHF1

Antibody

PHF1 Distribution in Hippocampus

Insoluble PHF LevelsTotal Tau Levels

Efficacy Against Insoluble PHF

6

ITR/y

Veh

icle

0

500

1000

1500

Re

lative

AT

8 IR

0

500

1000

2000

Re

lative

To

tal T

au

IR

1500

****0

500

1000

2500

Rela

tive A

T1

00

IR

1500

2000

AAVIg

G-P

301S

AAVPHF1-

P30

1S

Unt

reat

ed 2

m-P

301SW

T

Unt

reat

ed 6

m-P

301S

AAVIg

G-P

301S

AAVPHF1-

P30

1S

Unt

reat

ed 2

m-P

301SW

T

Unt

reat

ed 6

m-P

301S

AAVIg

G-P

301S

AAVPHF1-

P30

1S

****


Optimization of Vectorized Antibodies

7

Promoters Evaluated Components for Optimization

Intron

Signal peptide sequences

Ab H- and L-chain order

Ab H- and L-chain codons

2A or IRES site

Cleavage site

Poly-A

Stuffer sequence (if needed)

Promoter

CAG

CBA

GFAP

Synapsin

Expected Expression

Ubiquitous

Ubiquitous

Astrocyte Specific

Neuron Specific

Light

ChainIgk

Heavy

ChainIgG1

Capsid

Furin 2A polyA

Heavy

Chain

Light

ChainITR

Promoter

Vectorized

Antibody

Example

ITR/y


• Dose: 1.4E13 vg/kg, ssAAV101.anti-tau Ab

• Route: IV bolus

• In-life duration: 4 weeks

• (Anti-IgG IHC for anti-tau Ab expression)

Vectorized Anti-Tau Antibody Delivery Results in Promoter-Driven, Cell-Specific Expression in Mouse CNS

8

Vehicle GFAP-A GFAP-B Syn-A Syn-B

Vehicle CAG-A CAG-B CBA-A CBA-B

Low magnification (2X) of brain hemispheres from mice dosed

with different promoters/constructs showing stronger staining

with Construct A

GFAP-A

Syn-A

CAG-A

CBA-A

Cortex Hippocampus Thalamus

Higher Magnification (40X) showing cell-specific expression in

multiple brain regions

Ubiquitous

Promoter

Astrocytes

Neurons

Source: Wencheng Liu, Oral Presentation, ASGCT 2019

Yellow arrows:

Expression in neurons

Blue arrows:

Expression in astrocytes

Experiment

Design


0 10 20 30

0.1

1

10

100

1000

10000

Time Post-dose (Days)

Antibod

y (

ng/g

Cort

ex)

CBA

GFAP

SYN

0 10 20 30

0.1

1

10

100

1000

10000


An

tibody (

ng/g

Hip

pocam

pus)

GFAP

SYN

CBA

0 10 20 30

100

1000

10000

100000

1000000


Antib

ody (

ng

/mL S

eru

m) CBA

GFAP

SYN

0 10 20 30

1

10

100

1000


Antibod

y (

ng/m

l C

SF

)

CBA

GFAP

SYN

0 10 20 30

0.1

1

10

100

1000

10000


VG

/Cell

GFAP

SYN

CBA

• Dose: 1.4E13 vg/kg, ssAAV101.anti-tau Ab

• Route: IV bolus

• In-life duration: 1, 2, 3, 7, 14 and 28 days

• Vectors: CBA-, GFAP, and SYN anti-tau Ab

IV Dosing with Full-Length Anti-Tau Ab Vectors Achieves High Levels of the Antibody Expression in Mouse CNS

Kinetics of Antibody Expression Kinetics of VG in Hippocampus

9

Experiment

Design

Results• Antibody expression detected

as early as 2 days post-dose,

approaches maximum levels at

7 days, and plateaus at 14-28

days following IV administration

• Concentrations of antibody

stabilized at 2750 ng/g tissue

(CBA and GFAP promoters)

and 450 ng/g tissue (SYN

promoter); AAV biodistribution

stabilized at 30 vg/cell in

hippocampus

Cortex Hippocampus Hippocampus

Serum CSF


Evaluation of Vectorized Tau Antibodies in Mouse Models of Tauopathies

• Tau is an abundant soluble cytoplasmic protein that binds to microtubules to promote microtubule stability and function

• In AD and other tauopathies, tau aggregates become hyperphosphorylated and form insoluble NFTs.

• Passive immunization targeting tau has emerged as one of the more promising approaches for reducing or preventing

tau pathology

– N-terminal epitopes: have failed to show beneficial effects on patients in several clinical trials with PSP (AbbVie and Biogen) and AD (Roche)

– Mid-domain epitopes: have emerged as one of major targeted domains (Biogen, J&J, UCB/Genentech/Roche, AD/PD 2021)

• Vectorized anti-tau antibodies targeting two different phosphorylated tau domains: Mid domain and C-terminal

• Used mouse BBB-penetrant capsid to deliver vectorized antibodies to evaluate efficacy in animal models

10

N-term 1N 2N Mid Domain R1 R3 R4R2 C-term

PS

20

2, P

T2

05

PT

21

2, P

S2

14

PS

39

6P

S4

04

PS

40

9

23

4-2

46

pT

21

7

pS

19

9

pT

18

1

12

5-1

31

Mid-domain Epitopes C-terminal Epitopes

30 60 250100 350 380 441


Mouse Tauopathy Models for Efficacy Studies

11

Hippocampus, PBS Inj.

Hippocampus, AD Tau Seed Inj.

P301S Hippocampal Seeding Model• FTD Mutant Tau TG Animals

• Seeding into Hippocampus

• Vector IV dosed -2 weeks (anti-seeding)

• Experiment terminated +6 weeks

• Critical Readout: AT100 stained cells in Ipsilateral

Hippocampus (contra also examined)

P301S Intrinsic Model• FTD Mutant Tau TG Animals

• No seeding

• Vector IV dosed at age weeks


• Critical Readout: AT8 ELISA or AT100 stained

cells in Hippocampus

Path. Tau Hp Inj.Vx. Tau Ab IV Inj.

X


wks

8

wks

14

wks

20

wks

24

0

500

1000

1500

Rela

tive A

T8 IR

Hippocampus

CTX (Not include PC)

ATA ELISA


wks

8

wks

14

wks

20

wks

24

0

500

1000

1500

Rela

tive A

T8 IR

Hippocampus


ATA ELISA


12










• No seeding






X



Vectorized Mid-domain Ab Demonstrates Dose-Dependent Expression and Efficacy

13

Mid-domain Ab Level at Seeding • Expression of Mid-domain Ab

can be detected, in dose

dependent manner, at the time

of seeding and termination

• 46% significant reduction of

AT100 positive area is

observed in the ipsilateral

hippocampus of seeding model

that were treated with 1.4e13

Vg/kg of vectorized mid-domain

Ab by CBA Promoter

Efficacy Measured by AT100 IR

Brain

IHCP301S

age

Necropsy

14

wks

6 wks

IV AAV-Ab

Seeding

AD lysate HC

or Necropsy

8 wks

• Vector: VOY101.CBA.Mid-domain Ab

• Route: Vector IV dosed at age of 6 weeks

• Live phase: 8 weeks

• 2 doses: 5e12 or 1.4e13 Vg/Kg

• Critical readout: AT100 IHC

P301S Seeding

Model

Experiment

Design


Results

Ipsilateral Contratetral

Veh

icle

5e12

1.4e

13

0

1000

2000

3000

4000

ng A

b A

/g C

ort

ex

Veh

icle

5e12

1.4e

13

0

1000

2000

3000

4000

ng A

b A

/g H

ippocam

pus

Veh

icle

5e12

1.4e

13

0

500

1000

1500

Antib

ody A

(ng/m

L C

SF

)

Veh

icle

5e12

VG/K

g

1.4e

13 V

G/K

g

0.00

0.05

0.10

0.15

AT

100 (

IR / a

rea)

Ipsila

tera

l * p=0.03

Veh

icle

5e12

VG/K

g

1.4e

13 V

G/K

g

0.00

0.05

0.10

0.15

AT

100 (

IR / a

rea)

Contr

ate

ral

* p=0.06

Cortex Hippocampus CSF


Dose-Dependent Expression and High-Dose Efficacy Using Vectorized C-Terminal Ab

14

C-term Ab Level at Seeding • Expression of C-term. Ab can

be detected, in dose dependent

manner, at the time of seeding

and termination

• 46% reduction of AT100

positive area is observed in the

seeding model that were

treated with 1.4e14 Vg/kg of

vectorized C-term. Ab driven by

CBA promoter

Efficacy Measured by AT100 IR

• Vector: VOY101.CBA.Mid-domain Ab



• 2 doses: 1.4e13 or 1.4e14 Vg/Kg

• Critical readout: AT100 IHC

P301S Seeding

Model

Experiment

Design

Results

Brain

IHCP301S

age

Necropsy

14

wks

6 wks

IV AAV-Ab

Seeding

AD lysate HC

or Necropsy

8 wks


Vehicle

1.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0

1000

2000

3000

4000

ng A

ntibody B

/g C

ort

ex

Vehicle

1.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0

1000

2000

3000

4000

ng A

ntibody B

/g H

ippocam

pus

Vehicle

1.4e

13 V

G/K

g

1.4e

14 V

G/K

g

1000

10000

100000

1000000

ng A

ntibody B

/mL S

eru

m

No-

seed

P30

1S

Veh

icle

1.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0.00

0.05

0.10

0.15

AT

100 I(I

R / a

rea)

Ipsila

tera

l

p=0.02*

p=0.06

No-

seed

P30

1S

Vehicle

1.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0.00

0.02

0.04

0.06

0.08

0.10

AT

100 (

IR / a

rea)

Contr

ate

ral

Cortex Hippocampus CSF Ipsilateral Contratetral



15










• No seeding






X


wks

8

wks

14

wks

20

wks

24

0

500

1000

1500

Rela

tive A

T8 IR

Hippocampus


ATA ELISA


Dose-Dependent Expression and High-Dose Efficacy Using Vectorized C-Terminal Ab

16


XAb

InfusionBrain

IHC

ELISAP301S

2

Months

old

Necropsy

Day

91

Day

1

• No seeding

• Vector: VOY101.CBA.C-term. Ab



• 3 doses: 1.4e13, 4.4e13, or 1.4e14 Vg/Kg


cells in Hippocampus and Cortex

P301S Intrinsic

Model

Experiment

Design

Results• Significant reduction of AT8 IR

at cortex of all three doses

• Numbers in blue on the top of

each column on the graph A

and B represent antibody levels

in cortex or CSF, respectively

• The % of AT8 IR of treatment

groups relative to vehicle

control is shown on the top of

each column in graph C

C-term Ab Level at Termination Efficacy Measured by AT8 ELISA

Cortex CSF

Vehicle

1.4e

13 V

G/K

g

4.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0

500

1000

1500

Antibod

y (

ng/g

Cort

ex)

333

793

1036

Vehicle

1.4e

13 V

G/K

g

4.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0

200

400

600

Antib

od

y (

ng

/mL C

SF

)

0

177.1

409.8

497.4

Veh

icle

1.4e

13 V

G/K

g

4.4e

13 V

G/K

g

1.4e

14 V

G/K

g

0

200

400

600

Rela

tive A

T8 IR

41%41.3%55%

*

** **

Cortical Tau Pathology by AT8 IR


Vectorized Antibodies Demonstrate Efficacy in Multiple Animal Tauopathy Models

• AAV vectorized antibodies can delivery high levels of antibody to the CNS

• Both Tau Mid-domain and C-terminal antibodies show efficacy in vectorized studies when driven by a

ubiquitous CBA promoter:

– P301S Seeding model: Significant reduction in pathological tau following seeding observed with both Mid- and C-

terminal domain antibodies

– P301S Intrinsic model: Significant reduction in pathological tau observed with C-terminal domain antibody. Mid-

domain analyses in progress

• Future Investigations

– Evaluation of Mid-domain antibody in Intrinsic model

– Evaluation of antibodies in efficacy models using GFAP or SYN promoters to drive cell-specific expression

• Multiplexing antibody delivery?

– Potential to vectorized multiple fragment antibodies in a single AAV vector

– Could targeting multiple epitopes increase efficacy?

17


Acknowledgements

18

Jerrah Holth

Maneesha Paranjpe

Xiao-Qin Ren

Yanqun Shu

Giridhar Murlidharan

Charlotte Chung

Alex Powers

Emalee Peterson

Abigail Ecker

Usman Hameedi

Kyle Grant

Vinodh Kurella

Dillon Kavanagh

Omar Khwaja

Jay Hou

Steve Paul

Kelly Bales

Todd Carter

Documents

Efficacy of a Vectorized Anti-Tau Antibody Using Systemic