Next generation NKG2D-based CAR-T cells (CYAD-02): Co-expression of a single shRNA targeting MICA and MICB improves cell persistence and anti-tumor efficacy in vivo

Poster 3931

Martina Fontaine 1, Benjamin Demoulin 1, Simon Bornschein 1, Susanna Raitano 1, Steve Lenger 2, Hidevaldo Machado 2, Jonathan D Moore 3, Panagiota A. Sotiropoulou 1, David E. Gilham1

1. Research and Development, Celyad, Mont‐Saint‐Guibert, Belgium; 2. Horizon Discovery, Lafayette, CO; 3. Horizon Discovery, Cambridge, United Kingdom

© Celyad SA 2019

● The rapid approval of two anti-CD19 chimeric antigen receptor (CAR) T-celltherapies and the advanced development of anti-BCMA CAR T-cell therapiesdemonstrate the potential of the approach in B-cell malignancies. However,targets with a similar profile for CAR-T cell therapy in other diseases includingacute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) arelacking.

● The Natural Killer group 2D (NKG2D) receptor binds to 8 stress-induced ligands(NKG2DL): the MHC I chain-related A and B (MICA, MICB) and the UL16 bindingprotein family (ULBP) 1-6 (Figure 1A). These ligands are absent or expressed atvery low levels in normal tissues but found at high frequency across a widerange of tumors, rendering NKG2D a promising tool for cancer immunotherapy.

● CYAD-01 is an autologous CAR-T cell therapy based on the full-length humanNKG2D receptor fused to the intracellular domain of CD3ζ (Figure 1B). CYAD-01 is currently evaluated in relapsed/refractory (r/r) AML/MDS patients inPhase I clinical trials (THINK: poster 3826 and DEPLETHINK: poster 3844),displaying promising results in hematological malignancies.

● Activated T-cells transiently upregulate NKG2DL, leading to recognition by theCAR and thus limiting the in vitro cell expansion. We have previously shown thatrobust cell yields are generated with the addition of a blocking antibody and aPI3K inhibitor during manufacturing (mAb manufacturing process).

● In this work, we investigated the ability of an optimized short hairpin RNA(shRNA) technology to modulate NKG2DL expression on NKG2D CAR-T cells(termed CYAD-02) and to determine whether this confers an increase in anti-tumor activity compared to CYAD-01.

● CYAD-02 cells produced with the OptimAb manufacturing process displayan enriched early memory phenotype (Figure 5A) and increased in vitrocytolytic activity upon culture with PANC-1 cancer cells, compared toCYAD-01 T-cells cultured in standard conditions (mAb manufacturingprocess) (Figure 5B).

● To develop a stress-test animal model titrated for minimal activity ofCYAD-01, mice were injected with an aggressive AML cell line (THP-1),followed by 3 weekly injections of 0.3, 1 or 10 x 106 CYAD-01 cells. Thedoses of 0.3 and 1 x 106 cells per injection showed only a slight trend toincrease mouse survival, while injection of 10 x 106 CYAD-01 cells led toreduction in tumor burden and significantly increased mouse survival(Figure 6A and B). Based on those results, the intermediate dose of 3 x 106

CYAD-01 cells was used in the subsequent experiments.

● Injections of 3 x 106 Mock, CYAD-01 or CYAD-02 T-cells in the abovedescribed stress-test animal model of AML, titrated for minimal activity ofCYAD-01, revealed that CYAD-02 cells exhibit better engraftment(measured by the expression of human CD45 in the peripheral blood byflow cytometry) and elicit a strong anti-tumor response (measured bybioluminescence signal), resulting in a significant increase in mousesurvival compared to CYAD-01 cells (Figure 7A to C).

BACKGROUND RESULTS - 2FIGURES

CONCLUSIONS● The NKG2DL MICA and MICB are upregulated on T-cells upon activation,

limiting the in vitro cell expansion of NKG2D CAR-T cells.

● The shRNA-mediated knockdown of MICA and MICB expression onNKG2D CAR-T cells enhances in vitro expansion.

● CYAD-02 T-cells co-expressing the NKG2D CAR with a shRNA targetingMICA and MICB show an enrichment in early memory phenotype andbetter anti-tumor activity in vitro as compared to CYAD-01manufactured with the mAb process.

● CYAD-02 T-cells display increased in vivo engraftment, persistence andanti-tumor activity, which suggests that CYAD-02 could generate astronger and durable anti-tumor activity in patients as compared toCYAD-01 manufactured with the mAb process.

● The IND application for CYAD-02 has been accepted by the US FDA andthe CYCLE-1 Phase I study (NCT04167696) is scheduled to be initiated inearly 2020.

DISCLAIMER

This poster is published for information only.The views expressed are those of the authors and not necessarily those of the organizations named herein.

F i g u r e 1 : N K G 2 D r e c e p t o r r e c o g n i z e s 8 d i f f e r e n t s t r e s s l i g a n d s e x p r e s s e d i n a l a r g e v a r i e t y o f t u m o r s

Image adapted from Fernández-Messina, et al. Front. Immunol. 2012

A B

F i g u r e 2 : N K G 2 D l i g a n d e x p r e s s i o n i s u p r e g u l a t e d o n T - c e l l s u p o n a c t i v a t i o n a n d l e a d s t o d e c r e a s e d i n v i t r o c e l l e x p a n s i o n i n t h e a b s e n c e o f o p t i m i z e d m a n u f a c t u r i n g

A B

● T-cell activation during manufacturing induces transient up-regulation ofNKG2DL (mainly MICA and MICB), decreasing in vitro expansion (Figure 2A andB). The addition of a NKG2D blocking antibody and a PI3K inhibitor duringmanufacturing enabled the production of robust cell yields, suggesting thatdownregulating MICA and MICB expression on NKG2D CAR-T cells could be ameans to increase in vivo persistence.

● As both MICA and MICB are polymorphic genes and exhibit a high level ofhomology, a clinically relevant means of knocking down their expression isshRNA-mediated targeting of a common MICA and MICB region. Jurkat andHCT-116 cells were transduced with 20 different shRNAs, and MICA and MICBprotein expression was assessed by flow cytometry, using an antibodyrecognizing both MICA and MICB. This screening led to the selection of theshRNA #12 (Figure 3).

● A single retroviral vector encoding both the NKG2D CAR and the candidateshRNA was engineered (CYAD-02 CAR-T cells). The shRNA implementation inthe CAR vector efficiently downregulated both MICA and MICB in primary Tcells (Figure 4A), resulting in increased in vitro cell expansion (Figure 4B).

RESULTS - 1

JurkatMICA/B

HCT116MICA/B

neg. ctl.123456789

1011121314151617181920

40

60

80

100

120

neg. c

tl. 12 14 16 4 3 20 11 19 2 13 5 9 18 15 7 6 1 10 17 8

0

20

40

60

80

100

120

140

160

F i g u r e 3 : R e l a t i v e e x p r e s s i o n o f M I C A a n d M I C B p r o t e i n s i n c a n c e r c e l l l i n e s t r a n s d u c e d t o e x p r e s s s h R N A s t a r g e t i n g b o t h M I C A a n d M I C B

A B

F i g u r e 4 : T a r g e t i n g M I C A a n d M I C B w i t h a s i n g l e s h R N A i n c o r p o r a t e d i n t h e C A R v e c t o r l e a d s t o d e c r e a s e o f M I C A a n d M I C B e x p r e s s i o n o n T - c e l l s a n d e n h a n c e d i n v i t r o e x p a n s i o n

A B

F i g u r e 7 : C Y A D - 0 2 T - c e l l s d i s p l a y e n h a n c e d p e r s i s t e n c e a n d a n t i - t u m o r a c t i v i t y i n a n a g g r e s s i v e A M L m o d e l t i t r a t e d f o r m i n i m a l a c t i v i t y o f C Y A D - 0 1 c e l l s

A

B

C

Mock CYAD-01 CYAD-02

F i g u r e 5 : C Y A D - 0 2 T - c e l l s p r e s e n t a n e n r i c h m e n t i n e a r l i e r m e m o r y p h e n o t y p e a n d e n h a n c e d a n t i - t u m o r a c t i v i t y i n v i t r o

Mock CYAD-01 CYAD-02

CD45RA

CD

62L

0 8 16 24 32 40 48 56 64 72 80 88 96

0

20

40

60

80

100

120

Incubation time (hours)

PAN

C-1

Viab

ility

(%)

Mock

CYAD-02CYAD-01

A B

0 10 20 30 40 50 60106

107

108

109

1010

1011

1012

Time (Days)

Biol

umin

esce

nce

(pho

tons

/ se

c / m

m2 )

F i g u r e 6 : D e v e l o p m e n t o f a s t r e s s - t e s t A M L m o u s e m o d e l t i t r a t e d f o r m i n i m a l a c t i v i t y o f C Y A D - 0 1 T - c e l l s

0 10 20 30 40 50 60106

107

108

109

1010

1011

1012

Time (Days)

Biol

umin

esce

nce

(pho

tons

/ se

c / m

m2 )

0 10 20 30 40 50 60106

107

108

109

1010

1011

1012

Time (Days)

Biol

umin

esce

nce

(pho

tons

/ se

c / m

m2 )

0 10 20 30 40 50 600.001

0.01

0.1

1

10

100

Time (Days)

% h

CD

45+ C

D3+

cells

in th

e bl

ood

0 10 20 30 40 50 600.001

0.01

0.1

1

10

100

Time (Days)

% h

CD

45+ C

D3+

cells

in th

e bl

ood

0 10 20 30 40 50 600.001

0.01

0.1

1

10

100

Time (Days)

% h

CD

45+ C

D3+

cells

in th

e bl

ood

Mock CYAD-01 CYAD-02

0 10 20 30 40 50 600

20

40

60

80

100

Time (Days)

Perc

ent s

urvi

val

Survival (3 donors)

0 10 20 30 40 50 600

20

40

60

80

100

Time (Days)

Perc

ent s

urvi

val

Biol

umin

esce

nce

(pho

tons

/ se

c / m

m2 )

A B

p<0.001

MICA/B shRNA #

MIC

A/B

shR

NA

#

CYAD-01CYAD-02

0

20

40

60

80

100

MICA MICB