CARs and TRUCKs: how engineered T cells become living factories · CARs and TRUCKs: how engineered...

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

hinrich.abken@uk-koeln.de

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