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CARs and TRUCKs: how engineered T cells become living factories - - PowerPoint PPT Presentation
CARs and TRUCKs: how engineered T cells become living factories - - PowerPoint PPT Presentation
CARs and TRUCKs: how engineered T cells become living factories Hinrich Abken Centre for Molecular Medicine Cologne University of Cologne and Dept I for Internal Medicine University Hospital of Cologne hinrich.abken@uk-koeln.de T cells with
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- 1. Targeting leukemia/lymphoma by CAR T cells
is clinically successful
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54 CAR T cell trials targeting CD19 (May 2016)
Holzinger, Barden, Abken, Cancer Immunol Immunother 2016
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CD19 CAR T cell therapy of B cell leukemia is successfull, however, associated with relapse of leukemic cells which lost the targeted antigen
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CD19 CAR T cell therapy is specific but not selective for B leukemic cells
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CLL #3
6B10 FAIM3
CLL #2 CLL #1
FcµR may be a good candidate for CAR T cell targeting CLL
FcµR CD19
FAIM3 1E4 HM14 FcµR
CD19
Faitschuk et al., Blood (2016)
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CLL cells CD19+ B cells CD3+ T cells ζ scFv
CD28
anti-FcµR CAR
Faitschuk et al., Blood (2016)
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CLL cells healthy B cells allogeneic
Anti-FcµR CAR T cells eliminate CD19+ CLL cells but not CD19+ B cells
autologous
Faitschuk et al., Blood (2016)
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Anti-FcµR CAR T cells prolong disease free survival in a mouse model as do anti-CD19 CAR T cells
Faitschuk et al., Blood (2016)
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- 1. „Tumor associated antigens“ are not exclusively
expressed by tumor cells.
- 2. Tumors are extremely heterogenous
with respect to targetable surface antigens
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- 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
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CAR T cells to target pancreatic cancer cells in the tolerant, immune competent mouse
pancreatic cancer cells
(CEA+, R luc+)
immune competent mouse (CEA tg)
- rthotopic
installation
CD3+ CTLs (anti-CEA CAR, G luc+)
CAR
i.v. adoptive transfer ζ scFv
CD28
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large intestine wt CEA tg
CEA small intestine large intestine
4 5
The CEAtg mouse displays the human pattern in CEA expression
small intestine
CEA lung stomach
1 2 3
CEA tg wt
serum sCEA [ng/ml]
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large intestine wt CEA tg
CEA small intestine large intestine
transplanted pancreatic carcinoma cells 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
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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
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small & large intestine, appendix kidney lung pancreas stomach spleen heart
CEAtg mouse treated with anti-CEA CAR T cells
T cell imaging
CAR T cells CEA tg mouse CAR T cells wt mouse stomach lung T cells w/o CAR CEA tg mouse
No severe auto-immunity by anti-CEA CAR T cells
small & large intestine, appendix kidney lung pancreas stomach spleen heart
CEAtg mouse treated with T cells w/o CAR spleen liver
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CAR T cells establish secondary tumor rejection
CEA+ (right flank) CEA- (left flank)
secondary tumor challenge day 25
1000 1500
escence (photons)
*
left flank: CEA- tumour right flank: CEA+ tumour day 46
500
mean luminesc
day 25 day 46
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- 1. „Tumor associated antigens“ are not exclusively
expressed by tumor cells.
- 2. Tumors are extremely heterogenous
with respect to targetable surface antigens
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innate immune cells
CEA- tumor cells
activate
How to activate innate immune cells in the targeted tumor lesion for an anti-tumor attack?
CEA+ tumor cells
T cells
CAR
tumor cells
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innate immune cells
CEA- tumor cells
activate
How to activate innate immune cells in the targeted tumor lesion for an anti-tumor attack?
CEA+ tumor cells
T cells
CAR
tumor cells
iIL-12
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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
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anti-CEA CAR CD3ζ LTR BW431/26scFv IgG iIL-12 Neor (NFAT)6 IL-12 IRES anti-CD30 CAR CD3ζ LTR HRS3scFv IgG
T cells engineered with CAR inducible IL-12
CEA+ tumor cells
T cells innate immune cells
CAR
CEA- tumor cells
activate
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a
LS174T (CEA+) Colo320 (CEA-) γ [ng/ml] IL-12 [ng/ml] 1 2 1 2 1 2 1 2
anti-CEA CAR CD3ζ LTR BW431/26scFv IgG iIL-12 Neor (NFAT)6 IL-12 IRES anti-CD30 CAR CD3ζ LTR HRS3scFv IgG
T cells engineered with CAR inducible IL-12
anti-CEA CAR w/o anti-CEA CAR + iIL-12 effector T cells [x 103] IFN-γ cytotoxicity [%] 1 1 25 50 75 100 25 50 75 100
0.6 1.2 2.5 5 10 0.6 1.2 2.5 5 10
CEA+ tumor cells
T cells innate immune cells
CAR
CEA- tumor cells
activate
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500 1000 1500 2000 20 40 60 500 1000 1500 2000 20 40 60 500 1000 1500 2000 20 40 60 T cells w/o T cells CAR T cells CAR + iIL-12
C15A3 (CEA+) e [mm3]
a b c
T cells engineered with CAR inducible IL-12
500 1000 1500 2000 20 40 60 500 1000 1500 2000 20 40 60 500 1000 1500 2000 20 40 60
days after tumor inoculation
T cells CAR + iIL-12
- irrad. 293T cells
cIL-12 T cells CAR + iIL-12 +
- irrad. C15A3
(CEA+) 500 1000 1500 2000 20 40 60 T cells CAR + irrad. C15A3 (CEA+)
MC38 (CEA-) tumor volume
d e f g
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CEA+ tumor cells
(CB luc)
CEA- tumor cells
(R luc)
w/o anti-CEA CAR + iIL-12 anti-CEA CAR anti-CD30 CAR + iIL-12
T cells engineered with CAR inducible IL-12 mediate control of CEA- cancer cells in CEA+ tumors
right flank: MC38 cells (CEA-) + C15A3 cells (CEA+) left flank: MC38 cells (CEA-)
tumor volume [cm3]
anti-CD30 CAR + iIL-12
1 2 3 4 5 5 10 15 20 1 2 3 4 5 5 10 15 20 1 2 3 4 5 5 10 15 20 1 2 3 4 5 5 10 15 20
w/o anti-CEA CAR + iIL-12 anti-CEA CAR
days after T cell injection
* time after adoptive T cell transfer [days]
19
19 19 19
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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-12R IL-12R
- verlay
- verlay
p < 0.004 cells per field
CD11a/b+ CD18+ IL-12R+ cells
iIL-12 w/o 5 10 15 20 25
- IL-12R
- verlay
IL-12R
- verlay
iIL-12 w/o p < 0.002
CD11b+ CD80+ CD86+ IL-12R+ cells
cells per field 5 10 15 20 25
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Activated macrophages are involved in killing CEA- tumor cells
non-treated mice macrophage depleted mice
1000 1500
[mm3] w/o myeloablation, w/o IL-12 myeloablation + IL-12 myeloablation + IL-12 + macrophages w/o myeloablation + IL-12
isolated macrophages isotype F4/80
500 5 10 15 20
day tumor volume [m p < 0.001 w/o myeloablation, w/o IL-12
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IL-12R F4/80 TNF-α
- verlay
CAR iIL-12 T cell treated tumor
Activated tissue macrophages in tumors produce TNF-α
CAR T cell treated tumor
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day -1 +3 +10 +17 anti-TNFα mAb 300 400 500
anti-TNFα mAb control IgG mAb
10.000)
Activated tissue macrophages kill CEA- tumor cells through TNF-α
control IgG mAb 100 200
- 1
4 9 14 19
day photons/s/sr (x 10
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Lκ IL-12 Lκ scFv IL-12 hi
Other inducible effector molecules?
IL-2 Lκ scFv IL-12 hi hi Fc IL-2 Lκ scFv IL-12 hi hi
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innate immune cells
activate
inducible cytokines
T cells
CAR
cytokines CAR redirected T cells engineered to secrete (combi-)cytokines which activate innate cells to attack those cancer cells which are not recognized by the CAR.
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innate immune cells
activate
inducible cytokines
TRUCKs:
T cells redirected for antigen-unrestricted cytokine-initiated killing
T cells
CAR
cytokines CAR redirected T cells engineered to secrete (combi-)cytokines which activate innate cells to attack those cancer cells which are not recognized by the CAR.
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1st 2nd 3rd
CD3ζ scFv spacer
A
generation
B
iCAR
4-1BB/ OX-40 CD45/ PD-1/ CTLA-4
CARs
CD28 CD3ζ CD28 CD3ζ CD28 CD3ζ
4th
C
CD28 PD-1
D
split CARs
activating inhibiting
switch receptor
CD3ζ CD28 scFv 1 scFv 2 CD3ζ CD45/ PD-1/ CTLA-4 scFv 1 scFv 2
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CAR drivers
Markus Chmielewski Danuta Chrobok Elena Faitschuk Carola Jahnke Petra Hofmann Astrid Holzinger Andreas Hombach Birgit Hops Dorottya Horvath Johannes Kühle
Collaborators
- H. Büning, MH Hannover
- T. Blankenstein, MDC, Berlin
- R. Debets, Erasmus, Rotterdam
- Z. Eshhar, Weizmann, Rehovot
- B. Giebel, P. Horn, UK Essen
- R. Handgretinger, UK Tübingen
- R. Kiessling, Karolinska, Stockholm
- C. Renner, U Zurich
- B. Seliger, Halle
- W. Uckert, MDC, Berlin
Johannes Kühle Jennifer Makalowski Alexandra Martyniszyn Anja Meier Victória Nagy Gunter Rappl Tobias Riet Nicole Riet
- W. Uckert, MDC, Berlin
- G. Vereb, U Debrecen