CELLECTIS S.A. Roman GaleCo1, Isabelle Chion-‐So9nel1, Annabelle Gariboldi1 , Agnès Gouble1 and Julianne Smith1
1 Cellec's SA, Paris, France
Poster #2289#1 Introduc9on
-‐ Mul'ple Myeloma (MM) is a B-‐cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow.
-‐ Although currently available therapies can improve pa'ent's overall survival, MM s'll remains incurable in most of the cases.
-‐ The use of autologous chimeric an'gen receptor (CAR)-‐redirected T-‐ cells has allowed to achieve long-‐term durable remissions in pa'ents with B cell leukemia, indica'ng that CAR technology may become a new alterna've in cancer treatment.
-‐ In the present work we have assessed the feasibility of CAR-‐ mediated targe9ng of the CS1 an9gen (SLAMF7), which is highly expressed on tumor cells from most pa9ents with MM.
-‐ Expression of CS1 on normal CD8+ T-‐cells is poten9ally an obstacle for the development of CAR T-‐cells against this protein, since an9gen-‐expressing T cells will be targeted, impac9ng both on the number and the phenotype of the final CAR T-‐cell popula9on.
-‐ Transcrip'on Ac'vator-‐Like Effector Nuclease (TALEN) gene edi'ng technology was used to inac'vate the CS1 gene in T-‐cells, prior to transduc'on with a len'viral vector encoding an an'-‐CS1 CAR. An'-‐
#3 CS1 gene inac9va9on in human T-‐cells
CS1 is expressed on CD8+ T-‐cells, and these cells can be targeted during amplifica'on of CAR T-‐cells
TALEN® targe'ng the coding sequence of the human CS1 gene were designed, and the corresponding mRNAs were transfected in primary T-‐cells using PulseAgile electropora'on technology.
Flow cytometry analysis revealed high levels of CS1 gene inac9va9on in CD8+ T-‐cells.
Control CS1 TALEN
CS1
CD8
#6 an9-‐CS1 CAR ac9vity is enhanced in CS1 KO T-‐cells
Degranula'on ac'vity upon co-‐culture of CAR T-‐cells with CS1 expressing cells (NCI-‐H929 or L363) was measured by detec'ng CD107a expression in CD8+/CAR+ cells a]er 6h of co-‐culture. CS1neg cells were used as a control.
CS1 KO cells are able to degranulate in the presence of the CS1 an9gen at the same levels that non-‐edited cells expressing the same CAR.
Cytotoxic ac'vity against L363 and NCI-‐H929 cells was also measured by flow
#8 TCR/CS1 gene inac9va9on in human T-‐cells
Control TRACTALEN TALEN® targe'ng the CS1 gene
TCR
were co-‐transfected with those targe'ng the human TCR gene. The TCR(-‐) cells were purified and the % of NHEJ at each locus was quan'fied by DeepSeq.
FSC
LOCUS | % NHEJ | INS | DEL | WT | UA |
CS1 | 85.9 ± 5.2 | 2.8 ± 0.7 | 83.2 ± 5.1 | 13.9 ± 5.2 | 0.8 ± 0.2 |
TRAC | 89.4 ± 3.8 | 3.6 ± 0.1 | 76.0 ± 1.6 | 9.3 ± 3.5 | 12.9 ± 1.9 |
#9 UCARTCS1 an9-‐tumor ac9vity in vivo
NOG mice were sublethally irradiated (1,44 Gy) 8 days before injec'on of T-‐ cells. At Day (-‐7) 106 L363-‐Luciferase cells/mice were iv injected.
Mice were then infused with CAR(-‐) T-‐cells or UCARTCS1 cells (CS1/TCR KO, CAR+ T-‐cells). Bioluminiscent signal was assessed at D(-‐1), D7, D14, D21 and D28 post injec'on of T-‐cells.
tumor ac'vity of gene-‐edited CAR T-‐cells was validated in vitro.
#4 Genera9on of an9-‐CS1 CAR
cytometry, upon co-‐culturing the effector and target cells for 4h.
Ac'vity is normalized to consider an equal transduc'on efficiency between
NOG mice
Day -‐7
Day 0 Day 7 Day 14
Day 21 Day 28
-‐ Gene edi'ng technology was also used here to inac'vate the TCR
constant (TRAC) gene, to minimize the poten'al for engineered T-‐ cells to mediate Gra] versus Host Disease (GvHD).
-‐ Finally, we evaluated the in vivo ac'vity of double knockout CAR T-‐ cells (UCARTCS1) by performing experiments in an orthotopic MM mouse model, showing that CS1/TCR disrupted T-‐cells were able to mediate an in vivo an'-‐tumoral ac'vity.
CS1
An scFv targe'ng the extracellular domain of the human CS1 an'gen (SLAMF7) was used to construct a chimeric an'gen receptor containing a 41BB co-‐s'mulatory domain and the CD3 ac'va'on domain.
mock and CS1 KO T-‐cells.
Even if differen9al ac9vi9es are observed among the 9 donors tested, CS1 KO cells show a higher cytotoxic ac9vity when compared to mock transfected cells.
CAR(-‐)
T-‐cells
UCARTCS1
T-‐cells
1x106 L363-‐Luc cells
1x107 UCARTCS1
T cells
Bioluminescence Imaging
Our results show that mul9plex genome edi9ng is possible and can lead to the produc9on of double KO TRAC/CS1 T-‐cells, allowing large scale manufacturing of allogeneic, non alloreac9ve CS1 specific T-‐cells with enhanced an9-‐tumor ac9vity.
#5 CS1 CAR expression in human T-‐cells
#2 Allogeneic approach to target CS1
One of the key barriers for adop9ve transfer of 3rd party CAR T-‐cells can be overcome via TALEN® gene edi9ng technology to disrupt expression of the
T-‐cells were transfected with mRNAs encoding the TALEN® targe'ng the CS1 gene (or mock transfected), and transduced 2 days later with the len'viral vector encoding the an'-‐CS1 CAR.
7 days a]er transduc'on T-‐cells were analyzed for CD8 and CAR expression by flow cytometry.
#7 Genera9on of UCARTCS1 cells
UCARTCS1 cells display an9-‐tumor ac9vity in vivo and increase mice overall survival
TCR, allowing the use of any donors' T-‐cells and minimizing the risk of
GvHD.
This technology can also be used to inac9vate the CS1 gene in the same cell, allowing a more efficient produc9on of allogeneic an9-‐CS1 CAR T-‐cells
T-‐cell ac9va9on
D0
TALEN mRNA
electropora9on
D3
LV-‐CAR
transduc9on
D5
CAR detec9on and ac9vity tests
D12
UCARTCS1 cells can be produced using cells from a healthy donor in a
process designed for GMP compa'bility.
The CS1 KO is performed prior to CAR transduc'on, and the TCR KO cells are purified at the end of the produc'on process.
#10
Conclusions and Perspec9ves
The percentage of CD8+ cells is drama9cally reduced in non-‐edited cells upon an9-‐CS1 CAR expression, indica9ng that CD8+ cells are being targeted during the expansion step. Furthermore, a concomitant enrichment of CAR+ cells is observed in these cells compared to CS1 gene-‐ edited cells.
This communica'on expressly or implicitly contains certain forward-‐looking statements concerning Cellec's SA and its business. Cellec's SA is providing this communica'on as of this date and does not undertake to update any forward-‐ looking statements contained herein as a result of new informa'on, future events or otherwise. This communica'on contains Cellec's' proprietary informa'on. TALEN® and Cellec's® are trademarks owned by the Cellec's Group.
We show here that TALEN®-mediated disrup9on of the CS1
gene in T-‐cells is efficient and improves in vitro ac9vity of T-‐cells harboring an an9-‐CS1 CAR.
We have previously demonstrated that TALEN® mediated inac9va9on of the TCR constant (TRAC) gene can be achieved at high frequencies and eliminate the poten9al for edited T-‐cells to mediate Grao versus Host Disease (GvHD).
Mul9plex genome edi9ng can lead to the produc9on of double KO (TRAC and CS1) T-‐cells, allowing large scale manufacturing of allogeneic, non alloreac9ve CS1 specific T-‐cells that could display enhanced an9-‐tumor ac9vity.
Gene edi9ng technology offers the possibility of developing an off-‐the-‐shelf CAR T-‐cell based frozen product that would be immediately available for administra9on to a large number of MM pa9ents.
Alexandre Juillerat, Cécile Schiffer-Mannioui, Alan Marechal, Jean Marie Filhol, Anne-Sophie Gautron, Julien Valton, Julianne Smith, Laurent Poirot and Philippe Duchateau
Cellectis 8 rue de la Croix Jarry, 75013 Paris, France.
#1 Abstract
#2 Development of a multichain CAR (mcCAR) derived from FceRI
#3 Principle and design of mcCAR with inducibleactivity
#4 Surface detection of FKPB/FRB-CAR
Adoptive immunotherapy using engineered T-cells has emerged as a powerful
A FcRI
mcCAR-e
mcCAR4
A
approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so- called chimeric antigen receptors (CARs). CARs are synthetic receptors that associated an extracellular targeting moiety with one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combines advantages of MHC unrestricted target
chain
chain
chain
B
VH
scFV
VL A
scFV
C D 8
4
1
B B
Fab'2
B
scFv
CD8a TM + Intracel
WT FKBP FRB
FKBP/FRB
Vehicle
FRB/FKBP
recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective strategy to cancer therapy, potential adverse effects such as Cytokines Release Syndrome (CRS)
Rapamycin
scFv
ITAMs CD3ζ
scFv
FRB CD8a TM + Intracel
FKBP CD8a TM + Intracel
FSC
B
Rapamycin AP21967
and/or the risk of on-target off-tumor targeting are still a major concern. To date only Suicide mechanisms that can eradicate the engineered T-Cell "at will" or
4-1BB
scFv
scFv
FRB L
FKBP L
FKBP
FRB
CD8a TM + Intracel
CD8a TM + Intracel
Fold increase [MFI]
Fold increase [MFI]
20 30
1525
20
mRNA CAR transfection approaches have been proposed to addresses this safety
issue.
Here, we describe the development of a small molecule based switch-on technology to control the surface presentation of a chimeric antigen receptor in T-cells. By grafting protein domains that can interact upon addition of a small molecule drug in the hinge domain of the CAR architecture, we are able to turn a T-cell endowed with an engineered CAR from an off-state to an on-state, in term surface presentation as well as for its specific cytolytic properties. This system offers the advantage of a "transient CAR T-cell" for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. This non- lethal control system of CAR engineered T-cells represents an important advance for the safety of this technology.
Anti CD19 Anti ROR1 Anti CD19 Anti ROR1
Functional design of multichain CAR for adoptive immunotherapy.
A: To generate a multi-chain CAR (mcCAR) construct based on FceRI receptor, the extracellular domain of the alpha chain was deleted and replaced by the CD8a stalk and an scFv specific for a given antigen (CD19) to constitute an antigen-recognition domain . Several variants were also designed (type, number and position of the added sequences) which showed show significant and specific degranulation, cytokine secretion and cytotoxicity against Daudi cells (data not shown). We focused on mcCAR4 containing CD3ξ ITAMs and 4-1BB intracellular domain.
B: T cells bearing stably expressed mcCAR4 show potent activity against target cells in vitro.
Alpha chain extracellular organization
Schematic representation of the engineered mcCAR.
A: Principe of inducible conformational changes in the hinge domain. The CAR T-cell performance is intimately linked to an optimal interaction of the scFv to the targeted antigen. We thus conceived a system where controlled variations in the conformation of the hinge that separates the scFv from the cell membrane could be obtained upon addition of a small molecule.
B: Design of the hinge domains that incorporate FRB and/or FKBP domains. To switch the scFv/antigen interaction between on/off states, we inserted either the FRB, the FKBP12, or fusion of the FRB and FKBP12 between the CD8a hinge and the scFv domains. All constructs are based on mcCAR4
10 15
10
5
5
0 0
Surface detection of the engineered CAR in response to small molecules.
Primary T cell with were transfected with mRNAs encoding each chain of the multichain CAR (mcCAR). Upon addition of rapamycin or rapalog, we monitored conformational changes of the extracellular hinge domain by tracking the Fab'2 domain of the CD19-targeting scFv (100 nM, 20 h) (A). While the addition of rapamycin had no effects on the mcCAR, FRB-mcCAR and FKB-mcCAR), it strongly improved (up to 15 fold, B) the surface detection of the FKBP/FRB-mcCAR and FRB/FKBP-mcCAR constructs turning the system from an off to an on state.
#5 Surface expression is dose dependant (rapalog AP21967)
#6 FKPB/FRB-CAR T-cell cytotoxic activity
#7 FKPB/FRB-CAR T-cell in vivo activity against CD123+ tumors
#7 Conclusions
A B
D EC50: 8.2 0.6
AP21967 [10 nM]
100
Recent clinical implementation of adoptive cell transfer of CAR engineered T-cells has proven a powerful and successful approach to
1500
D1/2 EC50: 9.6 1.3 NT
Normalized surface detaction
1000
500
0
D1/4
EC50: 10.1 3.1
0
Tacrolimus [nM]
1
10
33
100
500
Vehicle CD19pos
Targeted cell lysis [%]
80 Rapalog CD19pos Vehicle CD19neg Rapalog CD19neg
60
40
p
cancer immunotherapy. The capability to control T cells endowed permanently with such molecules is a key feature concerning the safety of this technology.
Here, we describe a strategy to create a small molecule based switch technology to control the engineered CAR T-cells. Our approach is based on inducing deliberate conformational changes in the hinge
0 0.1 10
Rapalog (nM)
Characterization of the small molecule switch-on system.
Fab'2
domain that separate the scFv from the cell membrane. Although our
20 approach is applicable to current CAR design (data not shown), we
chose to implement this strategy in a novel CAR architecture that relies
0 on the FceRI receptor scaffold. The particularity of this design resides in
A: To evaluate the AP21967 usable dose range for the switch-on system we
performed a dose response assay . T-cells were transfected with three doses (D, D1/2 and D1/4) of mRNA coding for the engineered CAR and were treated with increasing amount of AP21967 rapalog. The Fab'2 region of the scFv is detected. The results we obtained indicated a maximum signal induction at 100 nM and an EC50 value of approximately 10nM (8.2-10.1 nM) that was independent from the amount of transfected engineered CAR. Remarkably, the EC50s are in range with rapamycin concentrations reported in peripheral blood or tumor tissue of patients, suggesting that the switch-on system may
be sensitive to clinically relevant concentration.
B: Competition experiment between AP21967 (10 nM) and tacrolimus (0 to 500 nM). N=2, error bars denote s.d. The possibility to further modulate the system using alternative small molecule competitors offers additional control of the engineered CAR T-cells. To illustrate the possibility to tune the amount of CAR locked in an on-state at the cell surface, we used the tacrolimus (FK506), a small molecule known to bind to the FKBP12 without enabling to form a complex with the FRB. AP21967 (or rapamycin) and tacrolimus have identical FKBP12 binding core and compete for the same binding site within the FKBP moiety. T-cells transfected with the engineered CAR were incubated with a fixed amount of AP21967 (10 nM) and increasing amount of tacrolimus (0 to 500 nM) and the surface labelling of the scFv was recorded. Addition of increasing amounts of tacrolimus competed with AP21967 for the binding site on FKBP and decreased the surface detection of the CAR
NT mcCAR FKBP/FRB*
Specific cytolytic properties of the engineered CAR T-cells.
The effect of the AP21967 rapalog on the cytolytic capacites of the of the CAR T cells toward model antigen presenting cell was assessed in a flow- based cytotoxicity assay. The CD19pos and a CD19neg target cell viability was measured after coculture with engineered CAR T-cells in presence or absence of AP21967. Effector/target ratios was set to 20:1. NT represents non-transfected T-cells, N=2, error bars denote s.d.
The engineered FKBP/FRB-CAR T-cell presented a significant cell lysis activity only in presence of the AP21967. We showed that the hinge engineering did not impair the specificity feature of the engineered T-cell as no cytotoxicity was observed on CD19neg target cells
G1: T cell-Vehicle G2: T cell + Rapalog G3: Switch On-Vehicle G4: Switch On + Rapalog
In vivo inducible activity cytolytic properties of the engineered CAR T-cells.
The effect of the AP21967 rapalog on the cytolytic capacites of the of the CAR T cells towards model antigen presenting cell was assessed in vivo using human tumor cell line xenograft into immunodeficient NOG mice. Firefly luciferase-expressing MOLM13 cells (0.25 x 106 cells/mouse) were injected intraveinously. 7 days later, mice received a single i.v. infusion of 15 x 106 T cells containing 10 x 106 FKLBP/FRB-CAR+ cells or non-transduced control T cells. Following T cell injection, AP21967 treatment started and consisted of2 daily IP injections at 3 mg/kg/injection for 10 consecutive days. Vehicle was used as control for AP21967. Bioluminescence was analyzed 2 to 3 times a week until sacrifice of the animals.
the possibility to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture.
We first showed that the multichain CAR architecture could redirect T- cells to various antigens. Second, we showed that the hinge engineering approaches allowed to turn a T-cell endowed with an engineered CAR from an off-state to an on-state. By controlling the scFv presentation at the cell surface upon addition of the small molecule, our all-in-one system allowed to further induce the cytolytic properties of the engineered T-cell. Finally, we provide initial in vivo data showing anti- tumor activity of our inducible CAR. This activity is not maintained once the small molecule treatment is interrupted.
Overall, this non-lethal system offers the advantage of a "transient CAR T-cell" for safety while letting open the possibility of multiple specific cytotoxicity cycles using a small molecule drug. Finally, similar strategies that perturb optimal presentation of the antigen targeting moiety of CAR may be easily implemented in order to promote a small molecule off-switch.
This communication expressly or implicitly contains certain forward‐looking statements concerning Cellectis SA and its business. Cellectis SA is providing this communication as of this date and does not undertake to update any forward‐looking statements contained herein as a result of new information, future events or otherwise. This communication contains Cellectis'proprietary
information. TALEN® and Cellectis® are trademarks owned by the Cellectis Group.
Cellectis SA published this content on 20 April 2016 and is solely responsible for the information contained herein.
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