Tolerogenic ImmTOR™ Nanoparticles Enhance Vector ... · Aparajita Chowdhury, Sheldon Leung, Takashi Kei Kishimoto Selecta Biosciences, Watertown, MA USA A. SEAP expression in serum

Page 1

7 11 18 33 46 63 700

5000

10000

15000

Time after AAV prime (d)

RL

U

AAV-SEAP AAV-SEAP + ImmTOR, 100 g

d56

100 366 100 407 1.0 1.6 1.2 2.0

100 294 100 326 100 288 100 309 100 240

ImmTOR

Rapamycin

PLA, PLA-PEG

ImmTOR

Macrophages

Dendritic cells

B cells

6 hr post IV injection of fluorescent

nanoparticles in mice

• ImmTOR is a biodegradable nanoparticle

that encapsulates rapamycin, an mTOR

inhibitor

• Intravenous injection of ImmTOR results

in selective accumulation in the spleen

and liver, where it is endocytosed by

dendritic cells (DC) and macrophages

• ImmTOR is designed to be co-administered with biologic drugs

such as gene therapy vectors to prevent the formation of ADAs

through the induction of immune tolerance and thus enable

sustained therapeutic activity of the biologic

Background

Mitigation of Immunogenicity with Tolerogenic Nanoparticles

Spleen

ImmTOR AAV virion Regulatory

T cell

Naïve T cell

B cell

Helper

T cell

Dendritic cell

Naïve T cell

Induction of tolerogenic

dendritic cells

Immune tolerance mediated by regulatory T cells

Prevention of IgG to AAV

• The co-administration of ImmTOR and AAV is designed to induce the formation of

regulatory T cells that prevent the formation of IgG against AAV and enable

repeated AAV administrations

Abstract

ImmTOR (formerly SVP-Rapamycin or SVP[Rapa])

• ImmTOR co-administration is beneficial for AAV-driven transgene expression in the gene therapy setting

• ImmTOR treatment suppresses the development of anti-capsid AAV IgG, thus enabling repeated vector administration

• Separately, ImmTOR rapidly induces elevated transgene expression immediately after AAV administration. This increase of transgene

levels correlates with higher vector DNA copy number and higher transgene mRNA production and requires admixing ImmTOR to AAV

• The first dose benefit of ImmTOR effect is not directly related to its immunomodulating activity but is related to elevated AAV liver trafficking;

pre-admixing of AAV vector to ImmTOR may counteract partial AAV neutralization in vivo

• Collectively, ImmTOR co-injection with AAV provides a two-pronged benefit, enabling higher initial transgene expression in parallel with anti-

AAV IgG suppression. The latter, in turn, allows for repeat vector administration. Together ImmTOR enables the potential for significant dose-

sparing.

Conclusions

70%*

Initial observation

We have earlier shown that tolerogenic ImmTOR™ nanoparticles

encapsulating rapamycin block adaptive immune responses

against the AAV capsid, thereby enabling repeat administration of

AAV vectors. Here we further demonstrate that ImmTOR also

enhances transgene expression after the first dose of AAV vector

in naïve mice. This beneficial effect of ImmTOR is independent of

its effects on adaptive immunity; it is seen in β2-microglobulin- and

Rag2-deficient mice and cannot be achieved in vivo by free

rapamycin. Admixing ImmTOR and AAV is required for enhanced

transgene expression after the first dose but not for inhibition of

the antibody response to AAV. ImmTOR affects multiple aspects

of AAV biology at first dose, including its trafficking to liver cells.

The requirement for admixing for the first dose benefit on

transgene expression suggests a physical association between

ImmTOR and AAV that may facilitate AAV uptake by hepatocytes

and results in increased vector copy numbers and transgene

mRNA expression. This multi-pronged mechanism of ImmTOR

action makes it an attractive candidate to enhance systemic gene

therapeutic applications. The first dose benefit of adding ImmTOR

to AAV gene therapy is immediate, dose-dependent and not

mouse strain-specific. It can also overcome low levels of pre-

existing antibodies to AAV. The rapid and enhanced transgene

expression may enable faster onset of therapeutic effects

achieved at lower AAV doses and coupled with the inhibition of

antibodies against AAV to enable vector redosing.

ImmTOR treatment leads to immediate benefit for AAV-driven

transgene expression, while also suppressing the formation of

IgG to AAV and thus enabling repeat vector administrations

Co-administration of AAV and ImmTOR leads to increased transgene

expression in immunodeficient mice

Tolerogenic ImmTOR™ Nanoparticles Enhance Vector Transduction, mRNA synthesis

and Transgene Expression after Initial and Repeated Administrations of AAV-based

Gene Therapy Vectors

Petr Ilyinskii, Chris Roy, Alicia Michaud, Gina Rizzo, Stephanie Elkins, Teresa Capela,

Aparajita Chowdhury, Sheldon Leung, Takashi Kei KishimotoSelecta Biosciences, Watertown, MA USA

A. SEAP expression in serum B. AAV IgG development (top OD)

• Groups of C57BL/6 female mice were administered AAV-SEAP with or without ImmTOR.

• Rates of SEAP expression in group treated with ImmTOR to that receiving AAV-SEAP alone

(100% baseline) are shown for all time-points (A, top line). AAV boosts are shown by

arrows.

• Ratios of post-boost (d63-d70) to pre-boost (d46) SEAP levels are shown (A, bottom line)

A

B

First dose benefit of ImmTOR is independent of adaptive

immunity, transgene identity, or mouse strain or gender

B. AAV-SEAP was injected (1×1010 VG) alone or with ImmTOR at 100 µg to β2-

microglobulin KO or wild-type C57BL/6 mice and serum SEAP activity measured.

A single dose of AAV8-Luc (1×1010 VG) was

injected alone or with ImmTOR at 50 µg and the

whole-body luminescence measured 14 or 35 days

later using IVIS (four groups of 5 C57BL/6 males).

ImmTOR co-administration with AAV8 vector via retro-orbital route leads to

elevated transgene expression in male and female BALB/c mice

AAV8-SEAP was injected into retro-orbital plexus at 1×1010 VG alone or with ImmTOR at 100

µg into male or female BALB/c mice and serum SEAP levels measured after initial and

repeated vector administrations (shown by arrow). Relative expression levels for each time-

point and boosting efficacies are shown.

Anc80-SEAP was administered four times at two dose levels. The lower dose virus was injected either alone or admixed with ImmTOR (100 µg on days 0, 70, 167 and 200 µg on day 244).

Serum SEAP (A) and Anc80 IgG (B) were measured on days indicated. Expression levels in groups treated with ImmTOR and with high-dose AAV alone are shown for each time-point as % vs.

expression in the group treated with low-dose virus as 100). Expression levels on day 19 are shown as lines. IgG is shown as top OD. Timing of repeat AAV administrations is shown by arrows.

14 350

1×106

2×106

4×106

4.5×106

5×106

Days after AAV injection

Ph

oto

ns/s

ec (

- b

/gro

un

d)

AAV-Luc (1x1010)

AAV-Luc (1x1010) + ImmTOR, d0

*

ImmTOR elevates AAV8-driven luciferase expression in vivo

7 120

2000

4000

6000

8000

Time after AAV-SEAP prime (d)

RL

U

2m -/- mice

2m -/- mice + ImmTOR

Wt mice, untreated

Wt mice + ImmTOR**

***

***

***

An

ti-A

AV

Ig

G T

op

OD

(4

50-5

70)

AAV-SEAP, 1x1010

VG AAV-SEAP + ImmTOR, 100g0.0

0.4

0.8

1.2

1.6

d11

d18

d7

d46

d32

d63

d70

A

B

A. AAV8-SEAP alone or admixed with ImmTOR was injected (d0, 56) into female wt C57BL/6

and Rag2-KO mice and serum SEAP levels measured. SEAP activity in ImmTOR-treated groups

is shown in bold as % vs. activity in untreated group as 100% with readings from KO mice

italicized. Ratio of SEAP activity after repeat AAV injection vs. that at immediately preceding

time-point is also shown.

19 47 63 89 103 119 1740

10000

20000

30000

40000

50000

Time after AAV injection (d)

RL

U

AAV-SEAP (BALB/c, males)

AAV-SEAP + ImmTOR, 100 g (BALB/c, males)

AAV-SEAP (BALB/c, females)

AAV-SEAP + ImmTOR, 100 g (BALB/c, females)

100 296 100 252 100 273 100 283 100 320 100 356 100 342

1.2 1.3 1.3 1.4 0.8 1.0 1.6 1.8 1.4 1.5

100 228 100 237 100 245 100 216 100 225 100 372 100 393

1.5 1.6 1.8 1.7 1.3 1.2 1.2 1.9 0.8 1.4

d5

6

d112

ImmTOR must be admixed to AAV in order to provide

transgene expression increase, which is a function of higher

vector transduction via enhanced hepatocyte trafficking

Three groups of mice (C57BL/6 females, 20 per

group) were injected with 1×1010 vg of AAV8-

SEAP alone or combined with ImmTOR at 100

µg of rapamycin, either as an admix or

sequentially and then five mice from each group

were analyzed at every time-point for AAV DNA

(A) and SEAP mRNA (B) levels in liver as well as

for serum SEAP activity (C). Intracellular AAV

DNA is quantified in vg/cell and SEAP mRNA in

fold increase (GAPDH-normalized) over naïve

mice. Separately, three groups of mice (C57BL/6

females, ten per group) were injected with 1×1010

vg of AAV8-SEAP alone or combined with

ImmTOR at 100 µg of rapamycin, either as an

admix or sequentially and IgG against AAV was

measured at times indicated (D).

A B C

72h 168h 312h 672h0

2×104

4×104

6×104

8×104

1×105

1.2×105

1.4×105

SE

AP

mR

NA

fo

ld in

cre

as

e (

vs

. n

aÏv

e)

72h 168h 312h 672h0

5000

10000

15000

20000

Avera

ge R

LU

695 138

411 91

336 115 212 99AAV8-SEAP, 1E10 VG

AAV8-SEAP + 100 µg ImmTOR (admix)

AAV8-SEAP + 100 µg ImmTOR (non-admix)

72h 168h 312h 672h0

20

40

6080

100

120

VG

/ C

ell

An

ti-A

AV

Ig

G T

op

OD

(4

50-5

70)

AAV8, 11010

vg AAV8 + ImmTOR, admix AAV8 + ImmTOR, non-admix0.0

0.2

0.4

0.6

0.8

1.0

1.2d7

d12

d20

d47

d33

2.0

2.5

3.0

3.5

4.0

LR

P-1

+F

4/8

0- C

D11b

- (A

647

+, %

)

*

*

Anc80-A647

ImmTOR-A488

Anc80-A647 + ImmTOR-A488

Naive

5 7 5 7 5 7 5 7

0

4

8

12

16

Time after injection (d)

LR

P-1

+F

4/8

0- C

D11b

- (G

FP

+, %

) **

*

Anc80-GFP, 5E+10 vg

ImmTOR-Cy5, 200 g

Anc80-GFP + ImmTOR-Cy5

Naive

Anc80-GFP Anc80-GFP + ImmTOR-Cy50

5

10

15

20

Experimental groups (8 mice/each)

LR

P-1

+F

4/8

0- C

D11b

- (G

FP

+,

% o

f to

tal) GFP+/ImmTOR- GFP+/ImmTOR+

19 61 77 82 151 179 186 200 242 251 257 263

0

20000

40000

60000

80000

Time after Anc80 prime (d)

RL

U (

SE

AP

Exp

ressio

n)

Anc80-SEAP, 1/1/5/10×1010 VG Anc80-SEAP, 1/1/5/10×1010 VG + ImmTOR Anc80-SEAP, 5/5/5/10×1010 VG

d70d167 d244

178 283 169 262 313 252 330 229 349 271

772 211 785 226 753 251

496 223 629 232 682 244 686 212

** * **ns *

** ** ** **

**** **

An

ti-A

nc

80 Ig

G T

op

OD

(4

50

-57

0)

Anc80, 1/1/5/10×1010

vg Anc80, 1/1/5/10×1010

vg; ImmTOR Anc80, 5/5/5/10×1010

vg

0.0

0.4

0.8

1.2

1.6d19

d61

d77

d82

d151

d179

d186

d242

d251

d257

d263

12 19 75 89 1030

10000

20000

30000

40000

Time after AAV8 treatment (d)

RL

U

AAV8-SEAP, 1×1010VG, Rag2-KO

AAV8-SEAP, 1×1010VG + ImmTOR, 100 g; Rag2-KO

AAV8-SEAP, 1×1010VG, wt

AAV8-SEAP, 1×1010VG + ImmTOR, 100 g; wt

140 265 318 126 190 232 209 283 257 233 265 211 207 234 194

1.7 1.8 1.0 1.7 1.6 1.5 0.9 1.3 1.5 1.4 0.9 1.2

d56

** **

ns

ns

****

ns

ns ****

**

ns

ns

**

***

ns

**

****

ImmTOR co-administration with AAV permits dose sparing and multiple repeat dosing

A B

C

D

ImmTOR and AAV admix is necessary for elevated transgene

expression, but not for AAV antibody suppression

AAV and ImmTOR admix leads to increased virion trafficking to hepatocytes

A. A488-labeled ImmTOR and A647-labeled Anc80 were injected alone or admixed, livers taken at 48 hrs,

processed to single-cell suspension, and stained with antibodies to markers indicated. Fractions of A647-positive

hepatocytes are shown. B. Cy5-labeled ImmTOR and GFP-expressing Anc80 were injected alone or admixed, livers

taken at 5 or 7d and hepatocytes analyzed as in A. C. Fractions of GFP-positive populations from day 7 samples in

B are shown, which for of Anc80-eGFP and ImmTOR-Cy5 admix are divided into GFP+Cy5– and GFP+Cy5+.

Emergence of new GFP-positive

cell population if ImmTOR is used

48 hours

Partial AAV neutralization is prevented by pre-admixing with ImmTOR

Passive mouse

immunization with

a panel of human

sera followed by

administration of

AAV-SEAP with

or w/o ImmTOR.

A B