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 engineered pre-defined specificity cancer cancer T cell T cell 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

Fc µ R may be a good candidate for CAR T cell targeting CLL CLL #1 CLL #2 CLL #3 Fc µ R 6B10 FAIM3 CD19 CD19 FAIM3 1E4 HM14 Faitschuk et al., Blood (2016) FcµR

anti-Fc µ R CAR scFv CD28 ζ CD3 + T cells CD19 + B cells CLL cells Faitschuk et al., Blood (2016)

Anti-Fc µ R 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-Fc µ R 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 • 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 immune competent cells mouse (CEA + , R luc + ) (CEA tg) i.v. CAR adoptive CD3 + CTLs transfer (anti-CEA CAR, G luc + ) scFv CD28 ζ

The CEAtg mouse displays the human pattern in CEA expression small intestine CEA tg wt large intestine large intestine 5 CEA 4 3 serum sCEA [ng/ml] small intestine 2 lung CEA 1 0 CEA tg wt stomach

The CEAtg mouse displays the human pattern in CEA expression small intestine CEA tg wt large intestine large intestine transplanted pancreatic carcinoma cells in the CEA tg mouse CEA 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 + ) T cells (CAR + ) tumor (CEA + ) tumor (CEA + ) day +3 day 0 day +7 day +9 day +21 day +23 day +37 day +35

No severe auto-immunity by anti-CEA CAR T cells T cell imaging CAR T cells T cells w/o CAR CAR T cells CEA tg mouse CEA tg mouse wt mouse CEAtg mouse treated with anti-CEA CAR T cells small & large intestine, stomach appendix pancreas stomach lung spleen heart kidney lung CEAtg mouse treated with T cells w/o CAR liver small & large intestine, appendix pancreas stomach lung spleen heart kidney spleen

CAR T cells establish secondary tumor rejection secondary tumor challenge escence (photons) 1500 * CEA+ (right flank) day 25 CEA- (left flank) 1000 mean luminesc 500 day 46 0 day 25 day 46 left flank: CEA - tumour right flank: CEA + tumour

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 tumor cells CEA + CAR 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 tumor cells iIL-12 CEA + CAR 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 CEA + CAR 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 CEA + CAR tumor cells T cells

T cells engineered with CAR inducible IL-12 a LS174T (CEA + ) Colo320 (CEA - ) 2 2 anti-CEA CAR LTR BW431/26scFv IgG CD3ζ IL-12 [ng/ml] anti-CD30 CAR LTR HRS3scFv IgG CD3ζ 1 1 iIL-12 (NFAT) 6 IL-12 IRES Neo r 2 2 γ [ng/ml] 1 1 1 1 IFN- γ innate immune cells 100 100 cytotoxicity [%] 75 75 activate CEA - 50 50 tumor cells 25 25 0.6 1.2 2.5 5 10 0.6 1.2 2.5 5 10 CEA + CAR effector T cells [x 10 3 ] tumor cells anti-CEA CAR anti-CEA CAR + iIL-12 w/o T cells

T cells engineered with CAR inducible IL-12 C15A3 (CEA + ) a b c 2000 2000 2000 T cells w/o T cells CAR T cells CAR + 1500 1500 1500 iIL-12 1000 1000 1000 500 500 500 e [mm 3 ] 0 0 0 0 20 40 60 0 20 40 60 0 20 40 60 tumor volume MC38 (CEA - ) d e f g 2000 2000 2000 2000 T cells CAR T cells CAR + irrad. 293T cells + iIL-12 cIL-12 1500 1500 1500 1500 iIL-12 + irrad. C15A3 T cells CAR 1000 1000 1000 1000 (CEA + ) + irrad. C15A3 (CEA + ) 500 500 500 500 0 0 0 0 0 20 40 60 0 20 40 60 0 20 40 60 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 anti-CEA CAR anti-CD30 CAR w/o + iIL-12 + iIL-12 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 ] anti-CEA CAR anti-CEA CAR anti-CD30 CAR w/o + iIL-12 + iIL-12 5 5 5 5 4 4 4 4 3 3 3 3 2 2 2 2 * 1 1 1 1 0 0 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 days after T cell injection 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 w/o IL-12 iIL-12 CD11b CD11b CD11a CD11a CD80 CD80 CD11b CD11b CD18 CD18 CD86 CD86 IL-12R IL-12R IL-12R IL-12R IL-12R IL-12R IL-12R IL-12R overlay overlay overlay overlay CD11a/b + CD18 + CD11b + CD80 + CD86 + IL-12R + cells IL-12R + cells 25 p < 0.004 25 p < 0.002 20 20 cells per field cells per field 15 15 10 10 5 5 0 0 - iIL-12 w/o iIL-12 w/o

Activated macrophages are involved in killing CEA - tumor cells non-treated mice 1500 myeloablation + IL-12 myeloablation + IL-12 + macrophages [mm 3 ] 1000 w/o myeloablation + IL-12 macrophage depleted mice tumor volume [m w/o myeloablation, w/o IL-12 w/o myeloablation, w/o IL-12 isotype F4/80 500 p < 0.001 isolated macrophages 0 0 5 10 15 20 day

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-TNF α mAb control IgG mAb 400 anti-TNFα 10.000) mAb 300 photons/s/sr (x 10 200 100 control 0 IgG mAb -1 4 9 14 19 day

Other inducible effector molecules? L κ IL-12 L κ scFv hi IL-12 L κ scFv hi IL-12 hi IL-2 L κ scFv Fc hi IL-12 hi IL-2

innate immune cells activate inducible cytokines cytokines CAR CAR redirected T cells 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 r edirected for antigen- u nrestricted c ytokine-initiated k illing innate immune cells activate inducible cytokines cytokines CAR CAR redirected T cells T cells engineered to secrete (combi-)cytokines which activate innate cells to attack those cancer cells which are not recognized by the CAR.

1 st 2 nd 3 rd 4 th generation A scFv spacer CARs CD3 ζ CD28 CD28 CD28 CD3 ζ CD3 ζ 4-1BB/ OX-40 CD3 ζ B iCAR CD45/ PD-1/ CTLA-4 C scFv 1 scFv 2 split CARs activating CD3 ζ CD28 scFv 1 scFv 2 inhibiting CD3 ζ CD45/ PD-1/ CTLA-4 D PD-1 switch receptor CD28