© Fraunhofer IZI

CAR NK cell therapy

12.04.2019

Fraunhofer Institute for Cellular Therapeutics and Immunology (IZI),

Institute of Clinical Immunology, University of Leipzig (UL/UKL),

Institute of Cellular Therapeutics, Hannover Medical School (MHH), Germany

UKL

UL

Ulrike Koehl

© Fraunhofer IZI

In relation to this presentation, I declare that there are no conflicts of interest.*

➢ “CD20CAR-TIME“ is a joint research project partly funded by the German ministry

of education and research (ref. 01EK1507A-C) within the research programme

“Innovations in Personalised Medicine“.

➢

➢ CTL019 European study trial Kymriah®

➢ Consulting: AstraZeneca, Affimed, Glycostem

* A conflict of interest is any situation in which a speaker or immediate family members have interests, and those may cause a conflict with the current presentation.

Conflicts of interest do not preclude the delivery of the talk, but should be explicitly declared. These may include financial interests (e.g. owning stocks of a related

company, having received honoraria, consultancy fees), research interests (research support by grants or otherwise), organisational interests and gifts.

Disclosure

Köhl 2.3.2017

© Fraunhofer IZI

2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

Imlygic®

Holoclar®

Glybera®(expired in 2017)

Yescarta® (FDA)

Kymriah® (FDA)

Spherox®

Chondrocelect®(withdrawn in 2017) Provenge®

(withdrawn in 2015)

MACI®(expired in 2018)

Zalmoxis®

Strimvelis®

Kymriah® (EMA)

Yescarta® (EMA)

Alofisel®

Luxturna®

Tissue Engineered Product

Somatic Cell Therapy Medicinal Product

Gene Therapy Medicinal Product

The era of Advanced Therapy Medicinal Products

CTL019 trial / Kymriah Europe

Press conference NOVARTIS

and Fraunhofer IZI 8/2018

USA Europe

CAR

T cells

CAR T Melanoma trial

Coop.; Miltenyi Biotec,

H. Abken (Regensburg),

U. Köhl (MHH)

2019

Koehl U … Abken H. HGT 2018 Priesner C … Koehl U. HGT 2016

Aleksandrova K … Koehl U. Transfusion Medicine and Hemotherapy 2019

CAR = chimeric antigen receptor

© Fraunhofer IZI

Hartman J, et al. EMBO Mol Med, 8 2017

Clinical CAR T cell studies

Ongoing CAR T cell studies

Started CAR T cell studies

Nu

mb

er

of

Clin

ica

l stu

die

s

6/2018

Clinicaltrials.gov

Maude SL et al. New Engl J Med 2018

75 patients

Paediatric r/r ALL – ELIANA

◼ > 450 clinical trials (2/2019)

◼ 10% of the studies in Europe, only

◼ To date, 2 products on the market

◼ To date, successful results in

hematological disorders

(most experience in CD19+ diseases)

◼ But very limited efficacy in solid tumors

CAR = chimeric antigen receptor

© Fraunhofer IZI

Köhl 2.3.2018

Overview – CAR NK cells

➢ Autologous CAR T cells – other limitations:

- manufacturing time consuming, expensive

- in some cases failure in manufacturing

- relapse due to contaminating leukemic cells

in the product (Ruella M, Nature Medicine 2018)

transduction

➢ CAR NK cells for advanced strategies

- Allogeneic donor NK cells as an „off the shelf“

therapy ➢

- CAR NK cells for improved killing functionality

- Possibility to overcome tumour immune

escape?

CAR = chimeric antigen receptor

NK = natural killer cells

Cancer

cell

NK cell

NK cells

T cells

CAR

© Fraunhofer IZI

ITAM immunoreceptor tyrosine-based activation motifs)IgCIgV

CD16

NKp46

NKp30

NKp44

NKG2D

NK cell

MICA BAG6

Tumour or leukaemic cell

KIRs

• CD56+CD3- NK cells comprise 2-18%

of lymphocytes in the peripheral bloodCD56brightCD16negative (immunregulatory)

CD56dim CD16positive (cytotoxic)

• Major role in killing of tumour cells

best in case of MHC negative targets

• Inhibitory and activating receptors:

- KIRs

- NCRs (NKp30, NKp46, NKp44)

- NKG2D

KIR (inhibitory and activating)

NCRs and NKG2D (activating only)

KIR (killer immunglobulin like receptors)

NCR (natural cytotoxicty receptors)

HLA-A,-B

-C, -E

NK

cell

KIR

+-

-Non-HLA

ligands+

IL-2NCR

NKG2D

KILLING

Natural Killer (NK) cells

Mechanisms of immune escape in the tumormicroenvironment

Chabanon et al (2016) Clin Cancer Res; 22, 4310

Induction of ImmuneCell Apoptosis

Induction of Treg and MDSCInhibition of CD8+ and NK Cells

Inhibition of DC Maturation

Reduced Expressionof MHC class I

Disrupted expressionof immune checkpoint

ligands

However:

Improved

NK cell

attack

© Fraunhofer IZI

Signaling and CARs in primary human NK cells

Oberschmidt O, Kloess S, Koehl U. Front Immunol 2017

© Fraunhofer IZI

Donor /

Parents

Allogeneic NK-DLI post haplo-SCT (Clin Gov No NCT 01386619)

Patients with high risk leukaemia and malignant tumours

NK cell purification

(CD3 depl./ CD56 sel.)

IL-2 expansion

(1000 U/ml, 10 days)

Leukapheresis GMP

NK cell application

patient

GMP

[days post SCT]

haplo-SCT

CD34 sel./

CD3/CD19 depl.

0

2nd NK cell

application

+40 +100

3rd NK cell

application

NK cell immunotherapy

1st NK cell

application

(+3)

Koehl U et al. Blood Cell Mol Dis 2004; Koehl U et al. Klin Päd 2005; Koehl U et al. Front Oncol 2013, Stern M et al. BMT 2013

NK-DLI = NK donor lymphocyte infusion

Advantage

➢ No severe adverse events in patients

➢ Primary aim >10x106 CD56+CD3-/kgBW: 41/49

➢ No graft versus host disease if T cells < 25x103/kg

➢ IL-2 stimulation → improved NK cell cytotoxicity

Disadvantage

➢ Tumor immune escape mechanism (TIEMs)

Kloess et al. Eur J Immunol 2010; Kloess et al. Oncoimmunol 2015

results

© Fraunhofer IZI

tumor cell MICA

Lyse MMP

cleavage

solMICA

+

NK

NKpatientNK

NKG2D

Kloess S … Koehl U. Eur J Immunol 2010 Kloess S … Koehl U, Oncoimmunol 2016

solMICA dependent tumor immune escape inhibits NK

cells in patients with Neuroblastoma

NK

NK NK

NKNK

NK

NKlysis

IL-2 activated NK cells improve NKG2D mediated

cytotoxicity via scavanging of solMICA in plasma

prior NK cell post NK cell control

0

500

1000

1500

2000

3000

4000

so

lMIC

A [

pg

/ml]

p<0.0001

application application

0 1000 2000 3000 40000

20

40

60

80

100

solMICA [pg/ml]

NK

:NB

Killin

g [

%]

p<0.0002

prior to +0.1h +1h +4h +24h +4weeks 0

1000

2000

3000

4000 NK cell application

solM

ICA

[pg/m

l]

prior to +0.1h +1h +4h +24h +4weeks0

20

40

60

80

100NK cell application

cyto

toxic

ity [%

]

NK

:NB

ratio 1

0:1

results

© Fraunhofer IZI

Impaired NK cell cytotoxicity in patients

with head and neck cancer (HNSCC)

autologous NK cells

regulatory NK cells cytotoxic NK cells

HNSCC HP0

10

20

HNSCC HP0

50

100

[%]

significant p<0.001

0 400 8000

50

100

cyto

toxic

ty[%

] significant p<0.001

sol MICA [pg/ml]

head neck healthy head neck healthy

cancer control cancer control

NKG2D PE CD45FITC/NKG2DPE

healthy control soluble MICA low HNSCC soluble MICA high

n=67 patients with HNSCC

peripheral blood screening

results

NKG2D PE CD45FITC/NKG2DPE

Dysregulation of autologous NK cells in patients and inhibition of

autologous and allogeneic NK cells via soluble plasma MICA/TGFβ

Kloess S et al. Oncoimmunology 2016

To overcome those hurdles: CAR NK cells ?

© Fraunhofer IZI

NCT03579927 CD19 Lymphoma Cord blood I/II not yet MD Anderson C.

and leukaemia NK cells recruiting Houston, USA

NCT03656705 CCCR Non-small Cell Lung NK92 I recruiting Hospital of Xinxiang

Henan, China

CCCR: Chimeric

CostimulatoryConverting Receptor

Clinical trials with CAR expressing NK cells

CAR:

CD19-CD28-zeta-

2A-iCasp9-IL15

(K. Rezvani)

Kloess S … Koehl U. Transfusion Medicine and Hemotherapy 2019

© Fraunhofer IZI

Redirected “CAR” NK-92 cell line

UKF-NB-3

Neuroblastoma

(NB) tumor

anti-ErbB2/HER2

GD2 Expression

anti-GD2

CAR Expression

even

tseven

ts

fluorescence Esser R et al. J of Cellular and Molecular Medicine 2011

coop. U. Köhl (MHH), W. Wels (FFM), T. Tonn (Dresden)

Schönfeld et al. Molecular Therapy 2014

results

R. Esser

Neuroblastoma

© Fraunhofer IZI

Chimeric Antigen Receptor Vector Design

for primary human NK cells

▪ Endodomain: FMC63 → CD19

▪ Endodomain: CD28 + 4-1BB(CD137) +CD3

▪ Codon-optimization: removal of cryptic splice sites, polyadenlyation

signals and other inhibitory sequences

→ CD19 binding leads to signal transduction

→ Enhanced cytotoxicity

coop.: A. Schambach, MHH

A. Schambach

© Fraunhofer IZI

CAR expressing NK cells redirected against CD19

dNK cells

vs. BV173

MOI1

dNK cells : CD19+ BV173 incubation time [h]; E:T ratio: 5:1

CD19-CAR-dNK cells

vs. BV173

CD19-CAR-dNK cells

vs. BV173+CD19mAB

blocking

Suerth J et al. J Mol Med August 2015

Alpha SIN vector

FSC-H

EG

FP

Mock alpha

Transduction of mature

primary human dNK cells feasible

EG

FP

[%

]

MOI

results

© Fraunhofer IZI

Köhl 27.03. 2017

Secretions of cytokines and pro-apoptotic

molecules by CAR NK cells

anti-inflammatory:

IL-4

IL-10

pro-inflammatory:

IL-6

IL-17A

IFNg

TNFa

pro-apoptotic:

GrA

GrB

Perforin

Granulysin

CD56brightCD16dim&neg

(immune regulatory)CD56dimCD16pos

(cytotoxic)

E/T

1:1 5:1

++ 0

+ +

++ +

++ +

+ +

++ ++

+ 0

0 0

- -

0 +

E/T

1:1 5:1

+ +

+ +

+ +

+ -

- -

+ +

++ +

++ ++

+ +

0 ++

results

© Fraunhofer IZI

Primary CAR expressing NK cells

redirected against AML cell lines and

patients own leukemic cells

123+ AML

Coop.: M. Morgan, A. Schambach, M. Heuser, M. Sauer

© Fraunhofer IZI

IL-2 activated NK cells

against KG1a

MOI3

NK cells : CD123+ KG1a incubation time [h]; E:T ratio: 10:1

CD123-CAR-NK cells

against KG1a

CD123-CAR-NK cells

against KG1a + CD123 mAB

blocking

0 .1 5 1 8 2 4 0 .1 5 1 8 2 4 0 .1 5 1 8 2 4

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

CD

12

3+ l

eu

ke

mia

bla

sts

[c

ell

s/µ

l]

CD56 APC CD56 APC CD56 APC

CD

34 P

C7

CD

34 P

C7

CD

34 P

C7

Coop.: A. Schambach, MHH

CAR expressing NK cells redirected against CD123+

Kloess et al. Human Gene Therapy 2017

results

© Fraunhofer IZI

CAR NK cells against patient´s CD123+AML

5 2 4 5 2 4 5 2 4 5 2 40

2 5

5 0

7 5

1 0 0

tim e [h ]

Cy

toto

xic

ity

[%

]

Mock EGFP CD123 CAR/ CD123 CAR/

EGFP EGFP + anti-

CD123

CAR-NK cells

vs.

patient´s AML

(E/T: 1:1)

MOI1

Coop.:

M. Heuser,

A. Schambach MHH

5 2 4 5 2 4 5 2 40

1 0

2 0

2 5

5 0

7 5

1 0 0

tim e [h ]

Cy

toto

xic

ity

[%

]

CAR-NK cells vs. HLMVEC (E/T: 1:1)

Mock EGFP CD123CAR/EGFP

Side effects

Cytotoxicity

Kloess et al. Human Gene Therapy 2017

S. Kloess

results

Kloess et al. Human Gene Therapy 2019

CD123CAR expressing NK cells and EGFP+ mock NK cells

aginst CD123 positive KG1a targets

Klöß 27.06.2017

CD123+KG1a cell proliferation dye: eFluor®450, anti-CD34-PE

anti-CD123-CAR NK cells EGFP+

NK:KG1 E:T ratio: 5:1; MOI1

anti-CD16-APC, EGFP+ NK cells

© Fraunhofer IZI

Clinical scale – CAR expressing NK cells

Product-

development

Development

Quality control

Release of product

Upscaling

Validation

„Off the shelf product“ Advanced Therapy Medicinal Product

GMP-compliant protocol

SOP=standard operation protocol CMC=chemical manufacturing and control IMPD=investigational medicinal product dossier

0 2 4 6 8 1 0 1 2 1 4

0 .0

0 .5

1 .0

1 .5

2 0

4 0

6 0

8 0

1 0 0

d N K c e ll e x p a n s io n

D a y s in c u ltu re

x-f

old

ex

pa

ns

ion

IL -2

fe e d e r c e lls + IL -2

fe e d e r c e lls + IL -2 1 /-2

cell expansion

results

© Fraunhofer IZI

CAR expressing effector cells conclusion

CAR T cells:

➢ Successful clinical CAR T cells studies (~ 450 documented world wide)

➢ Manufacturing failure of autologous CAR T cells needs complementary concepts

Primary human CAR NK cells:

➢ Patients can receive allogeneic haploidentical or „third party NK cells“ without

severe side effects → good candidates for „off the shelf CAR products“

➢ CAR NK cells (alpha retroviral SIN vectors) reached a nearly complete

elimination of CD19+ and CD123+ leukemic cells after 48 h

Improvement in future studies:

➢ CAR expressing cells and checkpoint

inhibitors → combination

➢ CAR effector cells with

transient cytokine secretion

Gay F et al.

Clin Lymph.,

Myeloma and

Leukemia,

review 2017

© Fraunhofer IZI

Inst. Cellular Therapeutics, MHH Hannover, D

R. Esser

W. Glienke

O. Oberschmidt

L. Arseniev

K. Aleksandrova

C. Priesner

J. Leise

ulrike.koehl@izi.fraunhofer.de

www.mh-hannover.de/zelltherapeutika.html

www.izi.fraunhofer.de

Tumorimmunology, Regensburg, D

H. Abken

Kantons-Spital Basel, CH

J. Passweg M. Stern C. Kalberer

A. Schambach M. Morgan H. Büning T. Moritz

Experimental Haematology, MHH, D

Haematol. /Oncol./ SCT, MHH, D

E. Seifried C. Seidl T. Tonn

BSD Hessen/BW und BSD Ost, D

A. Ganser M. Heuser M. Eder C. Könecke

Fraunhofer IZI, Leipzig, D

G. Schmiedeknecht

K. Kebbel

J. Lehmann

S. Mitzner

S. Fricke

T. Tradler

S. Ulbert

F. Horn

T. Grunwald

U. Demuth

S. Klöss

A. Schäfer

Clinical Immunology, University Leipzig, D

U. Sack A. Boldt

W. Wels

C. Kratz M Sauer B Maecker-Kolhoff

Pediatric Hematol/ Oncol., MHH, D

T. Klingebiel D. Schwabe P. Bader S. Hünecke

Pediatric Hematol/ Oncol., Frankfurt, D R. Blasczyk L. Goudeva B. Eiz-Vesper

Transfusion Medicine, MHH, D.

Georg Speyer House Frankfurt, D

Transfusion Medicine, Leipzig, D

R. Henschler

University Cancer Center Leipzig, D

U. Platzbecker F. Lordick U. Hacker

…. and thanks for listening