Pooling signaling and costimulatory domains in a flexible CARpool design

Lorraine Springuel*, Jennifer Bolsée*, Amélie Velghe, Sophie Agaugué and David E. Gilham

Research & Development , Celyad SA, Mont-Saint-Guibert, Belgium

*Denotes co-first authors

#333

B A C K G R O U N D

CARs are modular receptors that consist of a target bindingmoiety fused to structural domains including an extracellularspacer, a transmembrane region and intracellular signalingdomains. These signaling regions typically comprise atandem alignment of co-stimulatory (e.g. CD28, CD137) andactivating (CD3ζ) domains that upon target binding initiateactivation of T cell effector functions. This linearconfiguration displays a rigid spatial orientation and ratio ofco-stimulation to activation domains. To address this, wehave developed a novel mix-and-match approach (CARpool)where the costimulatory signal is provided in trans onaccessory proteins that associate with the antigen bindingchain via transmembrane-mediated interactions, potentiallydriving the ability to tailor T cell responses upon CARactivation.

R E S U L T S

CARpool design

By exploiting the ability of NK activating receptors toassemble as multi-subunit complexes via interactionsbetween membrane-embedded opposite charges, severalCD3ζ-containing CAR chains were designed using thetransmembrane (TM) and cytoplasmic (CYP) domains ofNKG2D or NKp44, able to associate with DAP10 and DAP12respectively [1-3]. NKG2D TM was replaced by a polyleucinesequence with the positively charged residue at position 11or 12, allowing its interaction with DAP10. Each CAR includeda B7H6 specific scFv. The CAR- and accessory protein-encoding sequences were co-expressed with a selectionmarker using 2A self-cleaving sites. These constructs werecompared to a classical second-generation CAR constructemploying CD28 as costimulatory domain [4] (Figure 1).

Cell surface expression varied between CARpool designs

CAR and selection marker expressions were assessed byflow cytometry (Figure 2). NKG2D-based CARpools showedreduced expression levels compared to the reference CAR.NKp44-based CARpool constructs containing the CD8αhinge showed expression levels largely superior to therespective CD28 hinge-containing CARs. Addition of CD28costimulatory domain to DAP12 significantly decreased CARcell surface expression

R E S U L T S

Cellular phenotype was not altered by CARpool expression

CARpool T cells exhibited a trend to express less CD25activation marker compared to the reference CAR (Figure 3).Analysis of the differentiation status based on theexpression of CD62L and CD45RA delineating 4 functionalsubsets (Naïve, Central memory, Effector memory andEffector T cells) revealed that our cells were mainlycomposed of memory cells. No major difference in activationand memory phenotype between CARpool and referenceCAR T cells could be observed.

CARpool T cells showed potent in vitro anti-tumor activity

All CARpool T cells showed potent cytotoxicity against thecervix carcinoma cell line Hela (Figure 4). NKG2D-basedCARpool T cells (R=11 and R=12) showed similar cytolyticactivities. NKp44-based CARpool T cells incorporating theCD28 hinge were more potent compared to those with theCD8α hinge and were equivalent to the reference CAR.Supplementation of DAP12 with CD28 costimulatory domaindid not improve cell functionality.

To interrogate whether target cell engagement by thedistinct CARpool T cells drives distinct cytokine releaseprofiles, supernatants of coculture with Hela cells wereanalyzed using a multiplex assay (Figure 5). T cells bearingthe NKp44 CARpool with the CD28 hinge co-expressingDAP12 showed the highest levels of secretion for all types ofcytokines equivalent to the reference CAR T cells. Hereagain, fusion of DAP12 to CD28 cytoplasmic domain did notalter the pattern and levels of cytokine secretion.

F I G U R E S & T A B L E S

C O N C L U S I O N S

These studies provide proof-of-concept for novelmodulatory CAR complexes with improved flexibilitycompared to a classical CAR design. Currently ongoingwork and future directions include:

• In vivo evaluation of CARpool anti-tumor activity• Incorporation of different scFv targeting other

antigens (e.g. CD19)• Interchange of costimulatory domains

REFRENCES:

[1] Garrity D et al. PNAS 2005;102:7641-7646 [3] Lanier L Immunol Reviews 2009;227:150-160

[2] Feng J et al. PLOS Biology 2006;4:0768-0779 [4] Wu M et al. Gene Therapy 2015;22:675-684.

F I G U R E 1 : C A R p o o l d e s i g n s

F I G U R E 2 : E x p r e s s i o n o f C A R p o o l o n h u m a n T c e l l s

F I G U R E 4 : A n t i - t u m o r a c t i v i t y o f C A R p o o l T c e l l s

CA

R

Selection marker

CA

R

Selection marker

0

25

50

75

100

125

F I G U R E 3 : P h e n o t y p e o f C A R p o o l T c e l l s

F I G U R E 5 : C y t o k i n e s e c r e t i o n o f C A R p o o l T c e l l s

Refere

nce

NKG2D R=11

NKG2D R=12

CD28H DAP12

CD8H D

AP12

CD28H DAP12-CD28

CD8H D

AP12-CD28

0

5000

10000

15000

20000

25000IFN-

(pg/

mL)

Refere

nce

NKG2D R=11

NKG2D R=12

CD28H DAP12

CD8H D

AP12

CD28H DAP12-CD28

CD8H D

AP12-CD28

0

200

400

600

800

1000IL-4

(pg/

ml)

Refere

nce

NKG2D R=11

NKG2D R=12

CD28H DAP12

CD8H D

AP12

CD28H DAP12-CD28

CD8H D

AP12-CD28

0

1000

2000

3000

4000

5000IL-17A

(pg/

ml)

0

20

40

60

80

100

% o

f po

siti

ve c

ells