=

DEVELOPMENT OF A NEXT GENERATION ALLOGENEIC CAR-T CELL PLATFORM WITHOUT GENE EDITING

Sotiropoulou PA1, Michaux A1*, Raitano S1*, Bornschein S1, Bolsée J1, Lenger S2, Machado H2, Moore JD3, Gilham DE1.

I N T R O D U C T I O N

M E T H O D S

R E S U L T SF I G U R E S

C O N C L U S I O N S

These experiments demonstrate that theexpression of shRNA in a standard retroviralvector provides a one-step solution to producecells suitable for Allogeneic CAR T cell therapy.Our testing continues, involving theexamination of the anti-tumor potency of suchshRNA-Allo CAR-T cells. Importantly, thisapproach provides a one vector solution thatoffers an alternative to strategies such as geneediting to eliminate the TCR, while alsorequiring no major alteration to current CAR-Tcell manufacturing, which should enable therapid implementation of the approach intoclinical testing.

F I G U R E 3 : C h a r a c t e r i z a t i o n o f T c e l l s e x p r e s s i n g s h R N A t a r g e t i n g C D 3 ζ

F I G U R E 2 : S c r e e n i n g o f t h e s e l e c t e d s h R N A - C D 3 E a n d s h R N A - C D 3ζ i n p r i m a r y T c e l l s u s i n g r e t r o v i r a l v e c t o r s

F I G U R E 1 : I d e n t i f i c a t i o n o f s h R N A s a g a i n s t C D 3 E a n d C D 3ζ c h a i n s t o k n o c k -d o w n t h e T C R / C D 3 c o m p l e x i n J u r k a t T c e l l l i n e u s i n g l e n t i v i r a l v e c t o r s

AFFILIATIONS: 1 Research & Development, Celyad SA, Mont-Saint-Guibert, Belgium; 2 Horizon Discovery, Lafayette, USA; 3 Horizon Discovery, Cambridge, United Kingdom

Autologous CAR-T cell therapy is now aproven breakthrough technology in thetreatment of B cell malignancies and holdspromise in the therapy of all types of cancer.However, autologous CAR-T cell therapy doeshave significant challenges relating to logisticsand product consistency. Allogeneic CAR-Ttherapeutic products could allow treatment ofmultiple patients with cells from the samehealthy donor, increasing productconsistency, likely reducing manufacturingcosts and avoiding the time delay required togenerate an autologous T cell product.Allogeneic CAR-T cell therapy is dependentupon eliminating the activity of theendogenous T Cell Receptor (TCR) within theengineered T cell, thereby preventing theinduction of a potentially life-threatening graftversus host disease (GvHD).In this work, we have explored RNAinterference to modulate the TCR and toassess the potential of shRNA as a platformtechnology for allogeneic CAR T cell therapy.

To disrupt the functionality of the TCRcomplex, we tested several shRNAs for theirability to reduce TCR expression by targetingthe CD3 elements of TCR. The best shRNAcandidates were initially identified in Jurkatcells and then validated in primary human Tcells. Subsequently, a side by side comparisonwas performed to assess the control ofalloreactivity of T cells expressing theselected shRNA. The shRNA-CD3ζ candidate2 (shRNA-CD3ζ-2) was selected as theoptimal shRNA to generate an allogeneic CART cell platform. Finally, the selected shRNAwas compared in vitro and in vivo with a geneediting technology (CRISPR-Cas9) targetingas well the CD3ζ subunit. Specifically, PBMCswere activated via anti-CD3 stimulation,depleted for CD20-positive cells andtransduced with retroviral vectors encodingfor a truncated form of CD19 (tCD19), fortCD19 and shRNA-CD3ζ, or nucleofected withCRISPR-CD3ζ. Transduced cells wereselected based on CD19 expression and thenseeded for 4 days of expansion. At harvest, toeliminate eventual remaining TCR-positivecells, the tCD19/shRNA-CD3ζ, and CRISPR-CD3ζ arms underwent TCR depletion usingmagnetic beads.

Selection of the best shRNA candidate forthe generation of a non-gene editedallogeneic CAR T cell platformMultiple shRNAs targeting CD3E or CD3ζ werescreened in Jurkat T cell line using lentiviralvectors (Figure 1). The shRNA-CD3E-2 andshRNA-CD3ζ-2 were selected based on theirability to downregulate the respective mRNAexpression and the surface levels of TCR.These shRNAs were subsequently tested inprimary T cells (Figure 2). The shRNA-CD3ζwas selected based on the higher inhibition ofTCR function upon in vitro mitogenicstimulation.

Characterization of T cells expressingshRNA-CD3ζThe shRNA-CD3ζ was incorporated in aretroviral vector encoding a control CAR(tCD19) and T cells expressing or not theshRNA-CD3ζ were generated using an 8-dayprocess. As shown in Figure 3, viability, foldincrease, CD4/CD8 ratio and the memoryphenotype were not affected by the shRNA.

Comparison of targeting CD3ζ using shRNAsand CRISPR-Cas9Comparison of T cells generated using shRNAor CRISPR-Cas9 targeting CD3ζ showed thatT cells generated with the two methodsexhibited identical CD3 and TCRdownregulation. Upon mitogenic stimulus invitro, T cells did not upregulate activationmarkers and did not produce IFNγ (Figure 4).

In vivo experiments with T cells adoptivelytransferred to NSG mice (Figure 5) showedthat shRNA targeting CD3ζ protected animalsfrom GvHD. Importantly, T cell persistencewas significantly higher compared to T cellsgenetically engineered with CRISPR-Cas9targeting CD3ζ. While the underlyingmechanism of this persistence is underinvestigation, the current hypothesis is thatthe maintenance of very low TCR expressionby shRNA-CD3ζ bearing T cells drives T cellpersistence without mediating GvHD.

F I G U R E 5 : T c e l l s e x p r e s s i n g s h R N A t a r g e t i n g C D 3 ζ d o n o t g e n e r a t e G v H D w h i l e t h e y m a i n t a i n s i g n i f i c a n t l y h i g h e r p e r s i s t e n c e c o m p a r e d t o a l l o g e n e i c T c e l l s g e n e r a t e d w i t h C R I S P R - C a s 9 t a r g e t i n g C D 3 ζ

shRNA-C

D3E

-1

shRNA-C

D3E

-2

shRNA-C

D3E

-3

shRNA-N

eg

0.0

0.5

1.0

1.5

CD3E RNA

Fo

ld c

han

ge o

ver

sh

RN

A-N

eg

shRNA-C

D3

-1

shRNA-C

D3

-2

shRNA-C

D3

-3

shRNA-N

eg

0.0

0.5

1.0

1.5

CD3ζ RNA

Fo

ld c

han

ge o

ver

sh

RN

A-N

eg

TCRα/β

CTR (tCD

19)

shRNA CD

3

shRNA CD

30.0

0.5

1.0

1.5

CD

3

mR

NA

fo

ld c

ha

ng

e o

ve

r

CT

R (t

CD

19

)

CTR (tCD

19)

shRNA CD

3

shRNA CD

30.0

0.5

1.0

1.5

CD

3

mR

NA

fo

ld c

ha

ng

e o

ve

r

CT

R (t

CD

19

)

0 50 100 150 2000.0

0.1

0.2

0.3

0.4

OKT3 (ng/ml)

IFN

(ng

/ml)

CTR (tCD19)shRNA CD3shRNA CD3

CD3E

CD4 CD8

CTRL-tCD19

shRNA-CD3ζ

CRISPR-CD3ζ

Isotype

0 2 20 100 200

-500

0

500

1000

1500

2000

CD69

OKT3 (ng/ml)MF

I C

D69 o

f th

e

tota

l via

ble

cells CTRL tCD19

shRNA-CD3

CRISPR-CD3

0 2 20 100 200-2000

0

2000

4000

6000

8000

10000

CD25

OKT3 (ng/ml)

MF

I C

D25 o

f th

e

tota

l via

ble

cells

CTRL tCD19

shRNA-CD3

CRISPR-CD3

CTR

L tCD19

shRNA-C

D3

CRIS

PR-C

D3

0

50

100

150

IFNγ release

% IF

N-γ

rele

ase r

ela

tive

to C

on

tro

l tC

D19 ****

CD4

CD8

0

5

10

15

2060

80

100

120

% TCRα/β protein

%T

CRα

/β o

f

CD

4+/C

D8

+ c

ells

**** ****CTRL tCD19

shRNA-CD3

CRISPR-CD3

Control 1

(Vehicle)

Control 2

(Mock)shRNA-CD3ζ

20*106 cells/mouse

CRISPR-CD3ζ

0 5 10 15 20 25 30 35 40 45 500

50

100

Time (Days)

Pe

rce

nt

surv

ival

shRNA CD3CRISPR CD3

CTR (tCD19)

CTR (tCD

19)

shRNACD3

0

10

20

30

40

Fo

ld in

cre

ase

(da

y 4

-8)

Donor 1Donor 2

Donor 3

Donor 4

Donor 5

CTR (tCD

19)

shRNA CD

30

1

2

3

4

CD

4/C

D8

ra

tio

Donor 1

Donor 2

Donor 3Donor 4

Donor 5

CD4+ CD8+

CTR shRNA

CD3CTR shRNA

CD3

CD45RA

CD

62

L

CD45RACD

62

L

Effector

Naïve

Central memory

Effector memory

Diffe

ren

tiation

Donor 1

Donor 2

Donor 3

Donor 4

Donor 5

0

50

100

% o

f th

e t

ota

l via

ble

po

pu

lati

on

Donor 1

Donor 2

Donor 3

Donor 4

Donor 5

0

50

100

% o

f th

e t

ota

l via

ble

po

pu

lati

on

Donor 1

Donor 2

Donor 3

Donor 4

Donor 5

0

50

100

% o

f th

e t

ota

l via

ble

po

pu

lati

on

Donor 1

Donor 2

Donor 3

Donor 4

Donor 5

0

50

100

% o

f th

e t

ota

l via

ble

po

pu

lati

on

CTR (tCD

19)

shRNACD3

020406080

95

100

105

Via

bili

ty (%

)

F I G U R E 4 : C o m p a r i s o n o f t a r g e t i n g C D 3 ζ w i t h s h R N A v e r s u s C R I S P R - C a s 9 i n p r i m a r y T c e l l s

5’LTR 3’LTRy tCD19

Day 0 Day 2 Day 4 Day 8

Activation

Transduction

CD19 purification TCR depletion& harvest

Expansion

7 14 21 28 34 540

10

20

30

40

Time (Days)

Fre

qu

en

cy o

f h

um

an

T c

ells

(%)

shRNA CD3ζ CRISPR CD3ζ

**

********

****

-1 4 8 13 18 22 27 32 36 41 46 5016

18

20

22

24

26

28

Days

We

igh

t (g

)CTR (tCD19)

shRNA CD3CRISPR CD3ζ

< 50% of

animals alive