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Approaches to overcome CAR-T cell toxicities: anti-cytokine antibodies and suicide genes
Attilio Bondanza, MD PhDInnovative Immunotherapies Unit
San Raffaele University Hospital and Scientific Institute
A. Bondanza receives research funding from Molmed Spa and is theinventor of CAR-T cell technologies acquired by and/or licensed toMolmed Spa
1) Type of costimulatory endodomain (CD28 > 4-1BB)2) Nature of the targeted antigen, e.g. CD193) Tumor burden (high > low)4) CD4/CD8 composition
The risk of severe cytokine release syndrome (CRS) after CAR-T cell therapies depends on:
A Bondanza Costem Berlin 27.10.2017
Severe neurotoxicties (e.g. brain edema) after CAR-T cell therapies are:
1) More likely in certain tumors than in others (ALL > NHL)2) Due to fludarabine conditioning3) Independent from CRS4) Effectively controlled by tocilizumab
A Bondanza Costem Berlin 27.10.2017
The “perfect” suicide gene to be implemented in CAR-T cell therapies needs to be:
1) Non-immunogenic2) Fast-acting3) Actionable with prodrugs that cross the blood-brain barrier4) Effective and highly penetrant
A Bondanza Costem Berlin 27.10.2017
CARs are synthetic biology receptors made up of mAb-derived targeting motifs and TCR/costimulatory endodomains
mAb scFv CAR
VL
VH VL
linkerspacer (CH2-CH3)
CH2
CL
CH1
CH3ITAM(CD3z)
VH
TCRs CARsHLA-dependent HLA-independentIntracellular Ags Surface AgsProtein Ags Protein, sugar and lipid AgsLow affinity (10-3-10-4) High affinity (10-8-10-10)Killing and proliferation Killing
PI3K,TRAF-2CD28, 4-1BB
A Bondanza Costem Berlin 27.10.2017
CD28 and 4-1BB differently affect the pharmacokinetics of CAR-T cells
Time
Circ
ulat
ing
T ce
lls
CD28 CAR-T1-4
4-1BB CAR-T5-9
5Porter et al, NEJM 20116Porter et al, STM 20117Grupp et al, NEJM 20138Turtle et al, JCI 20159Maude et al, NEJM 2014
1Brentjens et al, STM 20132Kochenderfer et al, JCO 20143Lee et al, Lancet Oncol 20154Wang et al, Blood 2016
A Bondanza Costem Berlin 27.10.2017
Antitumor responses by CD19 CAR-T differ between B-cell tumors but not between costimulatory endodomains
Disease Complete response (CR) rateChronic lymphocytic leukemia (CLL)1,2 30-40%Non-Hodgkin lymphoma (NHL)3,4 50-70%Acute lymphoblastic leukemia (ALL)5-9 80-90%
1Porter et al, NEJM 2011 6Brentjens et al, STM 20132Porter et al, STM 2011 7Maude et al, NEJM 20143Kochenderfer et al, JCO 2014 8Lee et al, Lancet Oncol 20154Wang et al, Blood 2016 9Turtle et al, JCI 20155Grupp et al, NEJM 2013
A Bondanza Costem Berlin 27.10.2017
Cytokine release syndrome (CRS) is caused by by-stander activation of innate immunity
A Bondanza Costem Berlin 27.10.2017
Severe CRS has also been observed with BCMA CAR-T cells
Abbas-Ali et al, Blood 2016
A Bondanza Costem Berlin 27.10.2017
Severe CRS is equally frequent with CAR-T cells having CD28 or 4-1BB costimulatory endodomains
Davila et al, Sci Transl Med 2014
8/30 = 26%
Maude et al, N Eng J Med 2014
7/16 = 43%
A Bondanza Costem Berlin 27.10.2017
Severe CRS is more likely in the case of high tumor burdens regardless of CD4/CD8 composition
Turtlle et al, J Clin Invest 2016
A Bondanza Costem Berlin 27.10.2017
1) Type of costimulatory endodomain (CD28 > 4-1BB)2) Nature of the targeted antigen, e.g. CD193) Tumor burden (high > low) – better, E:T ratio!4) CD4/CD8 composition
The risk of severe CRS after CAR-T cell therapies depends on:
A Bondanza Costem Berlin 27.10.2017
1) More likely in certain tumors than in others (ALL > NHL)2) Due to fludarabine conditioning3) Independent from the cytokine release syndrome4) Effectively controlled by tocilizumab
Severe neurotoxicties (e.g. brain edema) after CAR-T cell therapies are:
A Bondanza Costem Berlin 27.10.2017
Severe neurotoxicities are preceded by cytokine release syndrome and ineffectively controlled by tocilizumab
Turtlle et al, J Clin Invest 2016
15/29 = 51%
A Bondanza Costem Berlin 27.10.2017
Severe neurotoxicities are independent from fludarabineconditioning and apparently less frequent in NHL
Kochenderfer et al, J Clin Oncol 2015
3/15= 20%
1) More likely in certain tumors than in others (ALL > NHL)2) Due to fludarabine conditioning3) Independent from cytokine release syndrome4) Effectively controlled by tocilizumab
Severe neurotoxicties (brain edema) after CAR-T cell therapies are:
A Bondanza Costem Berlin 27.10.2017
1) Non-immunogenic2) Fast-acting3) Used for managing cytokine release syndrome/neurotoxicities4) Effective and highly penetrant
The “perfect” suicide gene to be implemented in CAR-T cell therapies needs to be:
A Bondanza Costem Berlin 27.10.2017
HSCs
T-cell precursors
Thymus
Donor
Non-alloreactive TK cells
Alloreactive TK cells
T cells
TK cells
HSV-TKtransduction
selection expansion
Clonal expansion of alloreactive TK cells
Host-tolerantT cells
Immune Reconstitution
GvIGvT
GvHD Suicide gene machinery activation
Prodrug (GCV)Drug
Allograft Host
Bone Marrow
Oliveira et al., Curr. Opin. Hematol., 2012
Ciceri et al, Lancet Oncology 2009Lupo-Stanghellini et al, ASH 2014
TK is a slow, yet highly penetrant suicide gene capable of reverting GVHD
Nil EGFR CD44v6
NIL
CD19.28
z
EGFR.28z
CD44v6
.28z
0
1
2
3* **
Der
mal
hC
D3
IHC
(sco
re)
H&E
hCD
3 IH
C
NSG mice +Full-thickness human skin +TSCM/CM CAR-T cells (5x10E6)
CD44v6 CAR-T cells are not toxic to human skin engrafted onto NSG mice
A Bondanza Costem Berlin 27.10.2017
CD44v6 CAR-T cells cause selective monocytopenia in NSG mice reconstituted with human HSCs
0 7 14 21 280
200
400
600
Time from infusion (days)
hCD
45+
cells
/mic
roL
CD19 CAR-T cellsCD44v6 CAR-T cells
NSG-SGM3 mice (human SCF, GM-CSF, IL-3)HSCs (cord blood, 50,000/mouse)CD44v6 CAR-T cells (cord blood, 2x10E6/mouse)
Casucci et al, Blood 2013
0 7 14 21 280
125
250
375
500
Time from infusion (days)
hCD
19+
cells
/mcr
oL
0 7 14 21 280
10
20
30
40
50
Time from infusion (days)
hCD
14+
cells
/mic
roL
****** *** ******
A Bondanza Costem Berlin 27.10.2017
TK is a slow, yet highly penetrant suicide gene capable of reverting GVHD
CTRLNW
LNMS
0
2
4
6
8
Live
r wei
ght (
g) ***
0.01 0.1 1 10 1000
20
40
60
80
100
GCV (µM)
Via
bilit
y (%
)
TK-CARTK
LTR LTRHSV-TK mut2 CAR CD44v6∆NSV
A Bondanza Costem Berlin 27.10.2017
1) Non-immunogenic2) Fast-acting3) Actionable with prodrugs that cross the blood-brain barrier4) Effective and highly penetrant
The “perfect” suicide gene to be implemented in CAR-T cell therapies needs to be:
A Bondanza Costem Berlin 27.10.2017
A phase I/IIa clinical trial of anti-CD44v6 CAR-T cells in relapsed/refractory AML and MM will begin in 2018
t
Patient screen& blood draw
Manufacture,QC & release
(2 weeks)
Cryo-preservation
Short & long-term follow up:assessment of safety / efficacy
Lymphodepleting chemotherapy:
Infusion of CAR T cellsd0: 33%, d1: 66%
d-6 to d-2: fludarabine 30mg/mqd-6 to d-5: cyclophosphamide 2,5g/mq
d0 – d180
Lymphocyto-apheresis
(4 weeks beforechemotherapy)
Centers: San Raffaele, Milano (A Bondanza, F Ciceri)Wurzburg University (H Einsele)Ospedale Pediatrico Bambino Gesù, Roma (F Locatelli)Sant Pau Hospital, Barcelona (J Sierra)University Hospital Ostrava (R Hajek)
A Bondanza Costem Berlin 27.10.2017
AcknowledgementsMonica CasucciLaura Falcone
Barbara CamisaMaddalena Noviello
Benedetta Nicolis di Robilant (now at Uni Stanford US)Fabiana Gullotta (now at Uni Basel CH)
Margherita NorelliEdoardo Galli (now at ETH Zurich CH)
Beatrice GrecoMatteo Doglio
Ayurzana Purevdorj (now at Uni Wien AT)Silvia Arcangeli
Marco MontagnaMarta Biondi
Marta MorescoAntonella Antonelli
Ilaria Palamà (Joint Lab at CNR-Nanotec, Lecce IT)Luca Cossa
CollaborationsFabio Ciceri
Chiara BoniniLuigi Naldini
Bernhard GentnerPietro Genovese
Gianpietro Dotti (Uni North Carolina US)Aurore Saudemont (Anthony Nolan, London UK)
Giuseppe Gigli (CNR-Nanotec, Lecce IT)Sara Deola (Sidra Medical Center, Doha QATAR)
Claudio Bordignon (Molmed Spa, Milano IT)Francois Meyer (TxCell SA, Valbonne FR)
Christian Klein (Roche Glycart, Schlieren CH)Daniel Olive (ImmCheck Therapeutics, Marseille FR)