CAR NK cell therapy Ulrike Koehl - CDDF
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CAR NK cell therapy
Ulrike Koehl
12.04.2019
Fraunhofer Institute for Cellular Therapeutics and Immunology (IZI),
UKL
© Fraunhofer IZI Institute of Clinical Immunology, University of Leipzig (UL/UKL),
UL
Institute of Cellular Therapeutics, Hannover Medical School (MHH), Germany
Disclosure
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.
© Fraunhofer IZI
Köhl 2.3.2017
The era of Advanced Therapy Medicinal Products
Kymriah® (EMA)
Zalmoxis®
Yescarta® (EMA)
Strimvelis® Yescarta® (FDA)
Alofisel®
Kymriah® (FDA)
Imlygic® Luxturna®
Spherox®
Holoclar®
2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019
USA Europe
Chondrocelect®
(withdrawn in 2017) Glybera® Provenge®
(expired in 2017) (withdrawn in 2015)
MACI®
(expired in 2018) CAR
T cells
Tissue Engineered Product CTL019 trial / Kymriah Europe CAR T Melanoma trial
Somatic Cell Therapy Medicinal Product Press conference NOVARTIS Coop.; Miltenyi Biotec,
Gene Therapy Medicinal Product and Fraunhofer IZI 8/2018 H. Abken (Regensburg),
U. Köhl (MHH)
© Fraunhofer IZI
CAR = chimeric antigen receptor Koehl U … Abken H. HGT 2018 Priesner C … Koehl U. HGT 2016
Aleksandrova K … Koehl U. Transfusion Medicine and Hemotherapy 2019
Clinical CAR T cell studies
Number of Clinical studies
Ongoing CAR T cell studies
Started CAR T cell studies
6/2018
Clinicaltrials.gov
Hartman J, et al. EMBO Mol Med, 8 2017
Paediatric r/r ALL – ELIANA
75 patients
◼ > 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
© Fraunhofer IZI
CAR = chimeric antigen receptor Maude SL et al. New Engl J Med 2018
Overview – CAR NK cells
transduction
➢ Autologous CAR T cells – other limitations:
- manufacturing time consuming, expensive
NK cells
T cells - in some cases failure in manufacturing
- relapse due to contaminating leukemic cells
in the product (Ruella M, Nature Medicine 2018)
Cancer
cell ➢ CAR NK cells for advanced strategies
- Allogeneic donor NK cells as an „off the shelf“
therapy
CAR ➢
- CAR NK cells for improved killing functionality
NK cell - Possibility to overcome tumour immune
escape?
© Fraunhofer IZI CAR = chimeric antigen receptor
Köhl 2.3.2018 NK = natural killer cells
Natural Killer (NK) cells
• CD56+CD3- NK cells comprise 2-18%
Tumour or leukaemic cell
of lymphocytes in the peripheral blood
CD56brightCD16negative (immunregulatory)
CD56dim CD16positive (cytotoxic)
• Major role in killing of tumour cells
best in case of MHC negative targets
MICA BAG6
• Inhibitory and activating receptors:
- KIRs
NKG2D NKp30
- NCRs (NKp30, NKp46, NKp44)
- NKG2D
KILLING
NKp46
NKp44
- NK
KIR
HLA-A,-B
cell
++- IL-2
NK cell -C, -E
Non-HLA
ligands
NCR
KIR (inhibitory and activating)
CD16 NKG2D
KIRs NCRs and NKG2D (activating only)
© Fraunhofer IZI KIR (killer immunglobulin like receptors)
IgV IgC ITAM immunoreceptor tyrosine-based activation motifs) NCR (natural cytotoxicty receptors)
Mechanisms of immune escape in the tumor
microenvironment
Induction of Treg and MDSC Reduced Expression However:
Inhibition of CD8+ and NK Cells of MHC class I Improved
NK cell
attack
Inhibition of DC
Maturation
Disrupted expression
of immune checkpoint
ligands
Induction of Immune
Cell Apoptosis
Chabanon et al (2016) Clin Cancer Res; 22, 4310
Signaling and CARs in primary human NK cells
© Fraunhofer IZI
Oberschmidt O, Kloess S, Koehl U. Front Immunol 2017
results
NK cell immunotherapy NK-DLI = NK donor lymphocyte infusion
Allogeneic NK-DLI post haplo-SCT (Clin Gov No NCT 01386619)
Patients with high risk leukaemia and malignant tumours
Leukapheresis GMP GMP
Donor /
Parents NK cell purification IL-2 expansion NK cell application
(CD3 depl./ CD56 sel.) (1000 U/ml, 10 days) patient
Advantage
➢ No severe adverse events in patients
haplo-SCT
➢ Primary aim >10x10 6 CD56+CD3-/kgBW: 41/49
1st NK cell 2nd NK cell 3rd NK cell
CD34 sel./ application application application
➢ No graft versus
CD3/CD19 depl.host disease if T cells < 25x10 3/kg
➢ IL-2 stimulation → improved NK cell cytotoxicity
Disadvantage
0 (+3) +40 +100 [days post SCT]
➢ Tumor immune escape mechanism (TIEMs)
Kloess et al. Eur J Immunol 2010; Kloess et al. Oncoimmunol 2015
© Fraunhofer IZI
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
solMICA dependent tumor immune escape inhibits NK
results
cells in patients with Neuroblastoma
tumor cell MICA
pImpaired NK cell cytotoxicity in patients results
with head and neck cancer (HNSCC)
n=67 patients with HNSCC
peripheral blood screening NKG2D PE CD45FITC/NKG2DPE NKG2D PE CD45FITC/NKG2DPE
autologous NK cells
healthy control soluble MICA low HNSCC soluble MICA high
20
significant pClinical trials with CAR expressing NK cells
CAR:
NCT03579927 CD19 Lymphoma Cord blood I/II not yet MD Anderson C. CD19-CD28-zeta-
CCCR: Chimeric and leukaemia NK cells recruiting Houston, USA 2A-iCasp9-IL15
Costimulatory (K. Rezvani)
© Fraunhofer
Converting ReceptorIZI NCT03656705 CCCR Non-small Cell Lung NK92 I recruiting Hospital of Xinxiang
Henan, China
Kloess S … Koehl U. Transfusion Medicine and Hemotherapy 2019Redirected “CAR” NK-92 cell line results
R. Esser
anti-GD2 Neuroblastoma Neuroblastoma
UKF-NB-3 (NB) tumor
events
GD2 Expression
fluorescence Esser R et al. J of Cellular and Molecular Medicine 2011
coop. U. Köhl (MHH), W. Wels (FFM), T. Tonn (Dresden)
anti-ErbB2/HER2
events
© Fraunhofer IZI
Schönfeld et al. Molecular Therapy 2014 CAR ExpressionChimeric Antigen Receptor Vector Design
for primary human NK cells A. Schambach
▪ 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
© Fraunhofer IZI
coop.: A. Schambach, MHHCAR expressing NK cells redirected against CD19 results
MOI1
Alpha SIN vector dNK cells CD19-CAR-dNK cells CD19-CAR-dNK cells
vs. BV173 vs. BV173 vs. BV173+CD19mAB
Transduction of mature
primary human dNK cells feasible blocking
Mock alpha
EGFP
FSC-H
EGFP [%]
dNK cells : CD19+ BV173 incubation time [h]; E:T ratio: 5:1
MOI
© Fraunhofer IZI
Suerth J et al. J Mol Med August 2015Secretions of cytokines and pro-apoptotic results
molecules by CAR NK cells
CD56brightCD16dim&neg
CD56dimCD16pos
(immune regulatory)
(cytotoxic)
E/T E/T
1:1 5:1 anti-inflammatory: 1:1 5:1
IL-4
++ 0 + +
IL-10
+ + + +
pro-inflammatory:
++ + IL-6 + +
++ + IL-17A + -
+ + IFNg - -
++ ++ TNFa + +
pro-apoptotic:
+ 0 GrA ++ +
0 0 GrB ++ ++
- - Perforin + +
0 IZI
© Fraunhofer + Granulysin 0 ++
Köhl 27.03. 2017Primary CAR expressing NK cells
redirected against AML cell lines and
patients own leukemic cells
123+ AML
© Fraunhofer IZI Coop.: M. Morgan, A. Schambach, M. Heuser, M. SauerCAR expressing NK cells redirected against CD123+ results
IL-2 activated NK cells CD123-CAR-NK cells CD123-CAR-NK cells
against KG1a against KG1a against KG1a + CD123 mAB
MOI3
CD34 PC7
CD34 PC7
CD34 PC7
blocking
le u k e m ia b la s ts [c e lls /µ l]
300
CD56 APC CD56 APC CD56 APC
250
200
150
100
+
C D123
50
0
0 .1 5 18 24 0 .1 5 18 24 0 .1 5 18 24
© Fraunhofer IZI NK cells : CD123+ KG1a incubation time [h]; E:T ratio: 10:1
Kloess et al. Human Gene Therapy 2017 Coop.: A. Schambach, MHHCAR NK cells against patient´s CD123+AML results
S. Kloess
Cytotoxicity Mock EGFP CD123 CAR/ CD123 CAR/
100
EGFP EGFP + anti-
MOI1
C y to to x ic ity [% ] CD123
75
CAR-NK cells
50 vs.
patient´s AML
25
(E/T: 1:1)
0
5 24 5 24 5 24 5 24
tim e [ h ]
Side effects
CAR-NK cells vs. HLMVEC (E/T: 1:1)
100
75 Mock EGFP CD123CAR/EGFP
C y to to x ic ity [% ]
50
25
20
10
0 Coop.:
5 24 5 24 5 24
M. Heuser,
tim e [ h ]
A. Schambach MHH
© Fraunhofer IZI
Kloess et al. Human Gene Therapy 2017 Kloess et al. Human Gene Therapy 2019CD123CAR expressing NK cells and EGFP+ mock NK cells
aginst CD123 positive KG1a targets
anti-CD123-CAR NK cells EGFP+ anti-CD16-APC, EGFP+ NK cells
CD123+KG1a cell proliferation dye: eFluor®450, anti-CD34-PE
NK:KG1 E:T ratio: 5:1; MOI1
Klöß 27.06.2017Clinical scale – CAR expressing NK cells results
Validation
Upscaling Development
Product-
Quality control
development
Release of product
GMP-compliant protocol
d N K c e ll e x p a n s io n
100 cell expansion
80
60
x - fo ld e x p a n s io n
40
20
1 .5
1 .0 IL -2
fe e d e r c e lls + IL -2
0 .5
fe e d e r c e lls + IL -2 1 /-2
0 .0
0 2 4 6 8 10 12 14
D a y s in c u ltu r e
© Fraunhofer IZI „Off the shelf product“ Advanced Therapy Medicinal Product
SOP=standard operation protocol CMC=chemical manufacturing and control IMPD=investigational medicinal product dossierCAR 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
Gay F et al.
➢ CAR effector cells with Clin Lymph.,
Myeloma and
transient
© Fraunhofer IZI cytokine secretion Leukemia,
review 2017ulrike.koehl@izi.fraunhofer.de Kantons-Spital Basel, CH
www.mh-hannover.de/zelltherapeutika.html J. Passweg M. Stern C. Kalberer
www.izi.fraunhofer.de
Experimental Haematology, MHH, D
Fraunhofer IZI, Leipzig, D
A. Schambach M. Morgan H. Büning T. Moritz
G. Schmiedeknecht S. Ulbert
K. Kebbel F. Horn
J. Lehmann T. Grunwald
Tumorimmunology, Regensburg, D
S. Mitzner U. Demuth H. Abken
S. Fricke S. Klöss
T. Tradler A. Schäfer University Cancer Center Leipzig, D
U. Platzbecker F. Lordick U. Hacker
Clinical Immunology, University Leipzig, D
U. Sack A. Boldt Transfusion Medicine, Leipzig, D
R. Henschler
Inst. Cellular Therapeutics, MHH Hannover, D
R. Esser L. Arseniev
K. Aleksandrova
BSD Hessen/BW und BSD Ost, D
W. Glienke
O. Oberschmidt C. Priesner E. Seifried C. Seidl T. Tonn
J. Leise
Pediatric Hematol/ Oncol., MHH, D
Georg Speyer House Frankfurt, D C. Kratz M Sauer B Maecker-Kolhoff
W. Wels
Transfusion Medicine, MHH, D
Pediatric Hematol/ Oncol., Frankfurt, D R. Blasczyk
.
L. Goudeva B. Eiz-Vesper
T. Klingebiel D. Schwabe P. Bader S. Hünecke
Haematol. /Oncol./ SCT, MHH, D
© Fraunhofer IZI
A. Ganser M. Heuser M. Eder C. Könecke…. and thanks for listening
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