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 engineered pre-defined specificity T cell cancer cell The aim: To give patient s immune cells specificity for targeting autologous cancer cells.
1. Targeting leukemia/lymphoma by CAR T cells is 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 successfull, 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
FcµR may be a good candidate for CAR T cell targeting CLL FcµR CLL #1 CLL #2 CLL #3 6B10 CD19 FAIM3 FAIM3 1E4 HM14 Faitschuk et al., Blood (2016) FcµR
anti-fcµr CAR scfv CD28 ζ CLL cells CD19 + B cells CD3 + T cells Faitschuk et al., Blood (2016)
Anti-FcµR CAR T cells eliminate CD19+ CLL cells but not CD19+ B cells CLL cells healthy B cells allogeneic autologous Faitschuk et al., Blood (2016)
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)
1. Tumor associated antigens are not exclusively expressed by tumor cells. 2. Tumors are extremely heterogenous with respect to targetable surface antigens
CAR T cells for treating adenocarcinoma CEA is a validated target T cells engineered with anti-cea CAR 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 cells in the tolerant, immune competent mouse orthotopic installation pancreatic cancer cells (CEA +, R luc + ) immune competent mouse (CEA tg) CD3 + CTLs (anti-cea CAR, G luc + ) CAR i.v. adoptive transfer scfv CD28 ζ
The CEAtg mouse displays the human pattern in CEA expression small intestine CEA tg wt large intestine 5 large intestine CEA 4 3 small intestine CEA serum scea [ng/ml] 2 1 lung 0 CEA tg wt stomach
The CEAtg mouse displays the human pattern in CEA expression small intestine CEA tg wt large intestine large intestine CEA transplanted pancreatic carcinoma cells in the CEA tg mouse CEA + tumor w/o tumor small intestine H&E lung CEA CEA stomach
Imaging tumor and CAR engineered T cells tumor imaging T cell imaging T cells (CAR + ) tumor (CEA + ) T cells (CAR + ) tumor (CEA + ) day 0 day +3 day +9 day +7 day +23 day +21 day +37 day +35
No severe auto-immunity by anti-cea CAR T cells T cell imaging CEAtg mouse treated with anti-cea CAR T cells CAR T cells CEA tg mouse T cells w/o CAR CEA tg mouse CAR T cells wt mouse small & large intestine, appendix stomach pancreas stomach lung spleen heart kidney lung CEAtg mouse treated with T cells w/o CAR small & large intestine, appendix pancreas stomach lung liver spleen heart kidney spleen
CAR T cells establish secondary tumor rejection secondary tumor challenge left flank: CEA - tumour right flank: CEA + tumour day 25 day 46 mean lumines scence (photons) 1500 1000 500 0 * day 25 day 46 CEA+ (right flank) CEA-(left flank)
1. Tumor associated antigens are not exclusively expressed by tumor cells. 2. Tumors are extremely heterogenous with respect to targetable surface antigens
How to activate innate immune cells in the targeted tumor lesion for an anti-tumor attack? innate immune cells activate CEA - tumor cells CAR CEA + tumor cells T cells
How to activate innate immune cells in the targeted tumor lesion for an anti-tumor attack? innate immune cells activate CEA - tumor cells iil-12 CAR CEA + tumor cells T cells
Why IL12? recruits innate and adaptive effector cells activates T cells, NK cells, CD11b + myeloid derived cells promotes T H 1 cell polarization and reverses T H 2 polarization improves MHC class I presentation increases IP-10, MIG chemokine secretion alters extracellular matrix (MMPs, VEGF,endothelial cell adhesion molecules ) decreases angiogenesis innate immune cells activate CEA - tumor cells CAR CEA + tumor cells T cells
T cells engineered with CAR inducible IL-12 anti-cea CAR LTR BW431/26scFv IgG CD3ζ anti-cd30 CAR LTR HRS3scFv IgG CD3ζ iil-12 (NFAT) 6 IL-12 IRES Neo r innate immune cells activate CEA - tumor cells CAR CEA + tumor cells T cells
T cells engineered with CAR inducible IL-12 anti-cea CAR anti-cd30 CAR iil-12 LTR BW431/26scFv IgG LTR HRS3scFv IgG (NFAT) 6 IL-12 IRES CD3ζ CD3ζ Neo r a LS174T (CEA + ) Colo320 (CEA - ) 2 2 IFN-γ γ [ng/ml] IL-12 [ng/ml] 1 1 2 2 1 1 innate immune cells activate CEA - tumor cells cytotoxicity [%] 100 75 50 25 100 75 50 25 CAR CEA + tumor cells 0.6 1.2 2.5 5 10 0.6 1.2 2.5 5 10 effector T cells [x 10 3 ] anti-cea CAR anti-cea CAR + iil-12 T cells w/o
C15A3 (CEA + ) T cells engineered with CAR inducible IL-12 2000 1500 a b c 2000 2000 T cells w/o T cells CAR T cells CAR + 1500 1500 iil-12 1000 1000 1000 tumor volume [mm 3 ] 500 0 0 20 40 60 MC38 (CEA - ) 2000 1500 1000 500 500 0 0 20 40 60 500 0 0 20 40 60 d e f g T cells CAR + iil-12 2000 1500 1000 500 T cells CAR + iil-12 + irrad. C15A3 (CEA + ) 2000 1500 1000 500 T cells CAR + irrad. C15A3 (CEA + ) 2000 1500 1000 500 irrad. 293T cells cil-12 0 0 20 40 60 0 0 20 40 60 0 0 20 40 60 0 0 20 40 60 days after tumor inoculation
T cells engineered with CAR inducible IL-12 mediate control of CEA - cancer cells in CEA + tumors anti-cea CAR + iil-12 anti-cea CAR anti-cd30 CAR + iil-12 w/o CEA + tumor cells (CB luc) CEA - tumor cells (R luc) 0 19 0 19 0 19 0 19 time after adoptive T cell transfer [days] tumor volume [cm 3 ] 5 4 3 2 1 0 anti-cea CAR + iil-12 0 5 10 15 20 * 5 4 3 2 1 0 anti-cea CAR 0 5 10 15 20 5 4 3 2 1 0 anti-cd30 CAR + iil-12 0 5 10 15 20 days after T cell injection 5 4 3 2 1 0 w/o 0 5 10 15 20 left flank: MC38 cells (CEA - ) right flank: MC38 cells (CEA - ) + C15A3 cells (CEA + )
Activated macrophages in tumor lesions treated with CAR iil-12 T cells iil-12 w/o IL-12 iil-12 w/o IL-12 CD11a CD11a CD11b CD11b CD11b CD11b CD80 CD80 CD18 CD18 CD86 CD86 IL-12R IL-12R IL-12R IL-12R overlay overlay overlay overlay CD11a/b + CD18 + IL-12R + cells CD11b + CD80 + CD86 + IL-12R + cells 25 p < 0.004 25 p < 0.002 cells per field 20 15 10 5 0 iil-12 - w/o cells per field 20 15 10 5 0 iil-12 w/o
Activated macrophages are involved in killing CEA - tumor cells non-treated mice 1500 myeloablation + IL-12 myeloablation + IL-12 + macrophages tumor volume [mm 3 ] macrophage depleted mice 1000 isotype F4/80 isolated macrophages 500 0 0 5 10 15 20 day w/o myeloablation + IL-12 w/o myeloablation, w/o IL-12 p < 0.001
Activated tissue macrophages in tumors produce TNF-α IL-12R F4/80 TNF-α overlay CAR iil-12 T cell treated tumor CAR T cell treated tumor
Activated tissue macrophages kill CEA - tumor cells through TNF-α day -1 +3 +10 +17 500 anti-tnfα 400 mab 300 control IgG mab photons/s/sr (x 10.000) 200 100 0 anti-tnfα mab control IgG mab -1 4 9 14 19 day
Other inducible effector molecules? Lκ IL-12 Lκ scfv IL-12 hi Lκ scfv hi IL-12 hi IL-2 Lκ scfv hi IL-12 hi Fc IL-2
innate immune cells activate inducible cytokines CAR T cells 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.
TRUCKs: T cells redirected for antigen-unrestricted cytokine-initiated killing innate immune cells activate inducible cytokines CAR T cells 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.
A 1 st 2 nd 3 rd 4 th generation scfv spacer CD3ζ CD28 CD3ζ CD28 4-1BB/ OX-40 CD28 CD3ζ CARs CD3ζ B C CD45/ PD-1/ CTLA-4 scfv 1 scfv 2 icar split CARs CD3ζ CD28 activating scfv 1 scfv 2 inhibiting CD3ζ CD45/ PD-1/ CTLA-4 D PD-1 CD28 switch receptor
CAR drivers Markus Chmielewski Danuta Chrobok Elena Faitschuk Carola Jahnke Petra Hofmann Astrid Holzinger Andreas Hombach Birgit Hops Dorottya Horvath Johannes Kühle Jennifer Makalowski Alexandra Martyniszyn Anja Meier Victória Nagy Gunter Rappl Tobias Riet Nicole Riet 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 G. Vereb, U Debrecen Grant sponsors Deutsche Forschungsgemeinschaft Deutsche Krebshilfe Wilhelm Sander Stiftung Deutsche José Carreras Leukämie Stiftung Else Kröner-Fresenius Stiftung German-Israeli Foundation BMBF