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CARs and TRUCKs:how engineered T cells become living factories
Hinrich Abken
Centre for Molecular Medicine CologneUniversity of Cologne
andDept I for Internal Medicine
University Hospital of Cologne
T cellcancer
T cells with engineered pre-defined specificity
T cellcancercell
The aim:
To give patient´s immune cells specificity
for targeting autologous cancer cells.
1. Targeting leukemia/lymphoma by CAR T cellsis clinically successful
54 CAR T cell trials targeting CD19 (May 2016)
Holzinger, Barden, Abken, Cancer Immunol Immunother 2016
CD19 CAR T cell therapy of B cell leukemia is succe ssfull,
however,
associated with relapse of leukemic cells
which lost the targeted antigen
CD19 CAR T cell therapy is specific
but not selective for B leukemic cells
CLL #3
6B10
FAIM3
CLL #2CLL #1
FcµR may be a good candidatefor CAR T cell targeting CLL
FcµR
CD19
FAIM3 1E4
HM14
FcµR
CD19
Faitschuk et al., Blood (2016)
CLL cells CD19+ B cells CD3+ T cells
ζ
scFv
CD28
anti-Fc µR CAR
Faitschuk et al., Blood (2016)
CLL cells healthy B cells
allogeneic
Anti-Fc µR CAR T cells eliminateCD19+ CLL cells but not CD19+ B cells
autologous
Faitschuk et al., Blood (2016)
Anti-Fc µR CAR T cells prolong disease free survival in a mo use modelas do anti-CD19 CAR T cells
Faitschuk et al., Blood (2016)
1. „Tumor associated antigens“ are not exclusivelyexpressed by tumor cells.
2. Tumors are extremely heterogenouswith respect to targetable surface antigens
• CEA is a validated target
• T cells engineered with anti-CEA CAR
• soluble (serum) CEA does not block anti -CEA CAR
CAR T cells for treating adenocarcinoma
• soluble (serum) CEA does not block anti -CEA CAR mediated T cell activation
• the same TAA is expressed by healthy tissues
CAR T cells to target pancreatic cancer cellsin the tolerant, immune competent mouse
pancreatic cancercells
(CEA+, R luc+)
immune competentmouse
(CEA tg)
orthotopicinstallation
CD3+ CTLs(anti-CEA CAR,
G luc+)
CAR i.v.adoptivetransfer
ζ
scFv
CD28
large intestine
wtCEA tg
CEA
small intestine
large intestine
4
5
The CEAtg mouse displays the human pattern in CEA expression
small intestine
CEA
lung
stomach
0
1
2
3
CEA tg wt
seru
m s
CE
A [n
g/m
l]
large intestine
wtCEA tg
CEA
small intestine
large intestine
transplanted pancreatic carcinomacells in the CEA tg mouse
CEA+ tumor w/o tumor
The CEAtg mouse displays the human pattern in CEA expression
small intestine
CEA
lung
stomach
H&E
CEA
T cells (CAR+) tumor (CEA+)
day 0
tumor imaging
Imaging tumor and CAR engineered T cells
day +3
T cells (CAR+) tumor (CEA+)
T cell imaging
day +9
day +23
day +37
day +7
day +21
day +35
small & large intestine,appendix
kidney
lungpancreas stomach
spleen heart
CEAtg mouse treated with anti-CEA CAR T cells
T cell imaging CAR T cells CEA tg mouse
CAR T cellswt mouse
stomach
lung
T cells w/o CARCEA tg mouse
No severe auto-immunity by anti-CEA CAR T cells
small & large intestine,appendix
kidney
lungpancreas stomach
spleen heart
CEAtg mouse treated with T cells w/o CAR
spleen
liver
CAR T cells establish secondary tumor rejection
CEA+ (right flank)
CEA- (left flank)
secondary tumor challenge
day 25
1000
1500
mea
n lu
min
esce
nce
(pho
tons
)
*
left flank: CEA- tumour
right flank: CEA+ tumour
day 46
0
500
mea
n lu
min
esce
nce
(pho
tons
)
day 25 day 46
1. „Tumor associated antigens“ are not exclusivelyexpressed by tumor cells.
2. Tumors are extremely heterogenouswith respect to targetable surface antigens
innate immune cells
CEA-
tumor cells
activate
How to activate innate immune cellsin the targeted tumor lesionfor an anti-tumor attack?
CEA+
tumor cells
T cells
CAR
tumor cells
innate immune cells
CEA-
tumor cells
activate
How to activate innate immune cellsin the targeted tumor lesionfor an anti-tumor attack?
CEA+
tumor cells
T cells
CAR
tumor cells
iIL-12
Why IL12?
• recruits innate and adaptive effector cells
• activates T cells, NK cells, CD11b+ myeloid derived cells
• promotes TH1 cell polarization and reverses TH2 polarization
• improves MHC class I presentation
• increases IP-10, MIG chemokine secretion
• alters extracellular matrix (MMPs , VEGF ,endothelial cell adhesion molecules )
• decreases angiogenesis
CEA+
tumor cells
T cells
innate immune cells
CAR
CEA-
tumor cells
activate
anti-CEA CAR CD3ζLTR BW431/26scFv IgG
iIL-12 Neor(NFAT)6 IL-12 IRES
anti-CD30 CAR CD3ζLTR HRS3scFv IgG
T cells engineered with CAR inducible IL-12
CEA+
tumor cells
T cells
innate immune cells
CAR
CEA-
tumor cells
activate
a LS174T (CEA+) Colo320 (CEA -)
γ[n
g/m
l]IL
-12
[ng/
ml]
1
2
1
2
1
2
1
2
anti-CEA CAR CD3ζLTR BW431/26scFv IgG
iIL-12 Neor(NFAT)6 IL-12 IRES
anti-CD30 CAR CD3ζLTR HRS3scFv IgG
T cells engineered with CAR inducible IL-12
anti-CEA CAR
w/o
anti-CEA CAR + iIL-12
effector T cells [x 10 3]IF
N- γ
cyto
toxi
city
[%]
1 1
25
50
75
100
25
50
75
100
0.6 1.2 2.5 5 100.6 1.2 2.5 5 10CEA+
tumor cells
T cells
innate immune cells
CAR
CEA-
tumor cells
activate
0
500
1000
1500
2000
0 20 40 60
0
500
1000
1500
2000
0 20 40 60
0
500
1000
1500
2000
0 20 40 60
T cells w/o T cells CAR T cells CAR+
iIL-12
C15A3 (CEA+)
tum
or v
olum
e [m
m3 ]
a b c
T cells engineered with CAR inducible IL-12
0
500
1000
1500
2000
0 20 40 60
0
500
1000
1500
2000
0 20 40 60
0
500
1000
1500
2000
0 20 40 60
days after tumor inoculation
T cells CAR+
iIL-12
irrad. 293T cellscIL-12
T cells CAR + iIL-12
+irrad. C15A3
(CEA+)
0
500
1000
1500
2000
0 20 40 60
T cells CAR+
irrad. C15A3 (CEA+)
MC38 (CEA-)
tum
or v
olum
e [m
m
d e f g
CEA+
tumor cells(CB luc)
CEA-
tumor cells(R luc)
w/oanti-CEA CAR + iIL-12
anti-CEA CAR anti-CD30 CAR+ iIL-12
T cells engineered with CAR inducible IL-12mediate control of CEA - cancer cells in CEA + tumors
right flank: MC38 cells (CEA-) + C15A3 cells (CEA+)
left flank: MC38 cells (CEA-)
tum
or v
olum
e [c
m3 ] anti-CD30 CAR
+ iIL-12
0
1
2
3
4
5
0 5 10 15 20
0
1
2
3
4
5
0 5 10 15 20
0
1
2
3
4
5
0 5 10 15 20
0
1
2
3
4
5
0 5 10 15 20
w/oanti-CEA CAR+ iIL-12
anti-CEA CAR
days after T cell injection
*
time after adoptive T cell transfer [days]
0 190 19 0 19 0 19
CD11b
CD11a
IL-12R
CD18
CD11b
CD11a
IL-12R
CD18
iIL-12 w/o IL-12
CD86
IL-12R
CD80
CD11b
iIL-12
CD11b
CD86
IL-12R
CD80
w/o IL-12
Activated macrophages in tumor lesions treated with CAR iIL-12 T cells
IL-12RIL-12R
overlay overlay
p < 0.004
cells
per
fiel
d
CD11a/b+ CD18+
IL-12R+ cells
iIL-12 w/o
0
5
10
15
20
25
-
IL-12R
overlay
IL-12R
overlay
iIL-12 w/o
p < 0.002
CD11b+ CD80+ CD86+
IL-12R+ cells
cells
per
fiel
d
0
5
10
15
20
25
Activated macrophagesare involved in killing CEA - tumor cells
non-treated mice
macrophage depleted mice 1000
1500
tum
or v
olum
e [m
m3 ]
w/o myeloablation, w/o IL-12
myeloablation + IL-12
myeloablation + IL-12 + macrophages
w/o myeloablation + IL-12
isolated macrophages
isotype
F4/80
0
500
0 5 10 15 20
day
tum
or v
olum
e [m
mp < 0.001
w/o myeloablation, w/o IL-12
IL-12R F4/80 TNF-α overlay
CAR iIL-12 T celltreated tumor
Activated tissue macrophagesin tumors produce TNF- α
CAR T celltreated tumor
day -1 +3 +10 +17
anti-TNFα
mAb 300
400
500anti-TNFα mAb
control IgG mAb
phot
ons/
s/sr
(x
10.0
00)
Activated tissue macrophageskill CEA - tumor cells through TNF- α
control
IgG mAb0
100
200
-1 4 9 14 19day
phot
ons/
s/sr
(x
10.0
00)
Lκ IL-12
Lκ scFv IL-12hi
Other inducible effector molecules?
IL-2Lκ scFv IL-12hi hi
Fc IL-2Lκ scFv IL-12hi hi
innate immune cells
activate
induciblecytokines
T cells
CAR
cytokines
CAR redirected T cellsengineered to secrete (combi-)cytokineswhich activate innate cellsto attack those cancer cellswhich are not recognized by the CAR.
innate immune cells
activate
induciblecytokines
TRUCKs:
T cells redirected for antigen-unrestricted cytokine-initiated killing
T cells
CAR
cytokines
CAR redirected T cellsengineered to secrete (combi-)cytokineswhich activate innate cellsto attack those cancer cellswhich are not recognized by the CAR.
1st 2nd 3rd
CD3ζ
scFv
spacer
A generation
BiCAR
4-1BB/ OX-40
CD45/ PD-1/ CTLA-4
CARsCD28
CD3ζ
CD28
CD3ζCD28
CD3ζ
4th
C
CD28
PD-1D
split CARs
activating
inhibiting
switch receptor
CD3ζ CD28
scFv 1 scFv 2
CD3ζ CD45/ PD-1/ CTLA-4
scFv 1 scFv 2
CAR drivers
Markus ChmielewskiDanuta ChrobokElena FaitschukCarola JahnkePetra HofmannAstrid HolzingerAndreas HombachBirgit HopsDorottya HorvathJohannes Kühle
Collaborators
H. Büning, MH HannoverT. Blankenstein, MDC, BerlinR. Debets, Erasmus, RotterdamZ. Eshhar, Weizmann, RehovotB. Giebel, P. Horn, UK EssenR. Handgretinger, UK TübingenR. Kiessling, Karolinska, StockholmC. Renner, U ZurichB. Seliger, HalleW. Uckert, MDC, BerlinJohannes Kühle
Jennifer MakalowskiAlexandra MartyniszynAnja MeierVictória Nagy Gunter RapplTobias RietNicole Riet
W. Uckert, MDC, BerlinG. Vereb, U Debrecen
Grant sponsors
Deutsche ForschungsgemeinschaftDeutsche KrebshilfeWilhelm Sander StiftungDeutsche José Carreras Leukämie StiftungElse Kröner-Fresenius StiftungGerman-Israeli FoundationBMBF