approaches to overcome car-t cell toxicities: anti ... · ospedale pediatrico bambino gesù, roma(f...

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


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


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


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


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


Cytokine release syndrome (CRS) is caused by by-stander activation of innate immunity


Severe CRS has also been observed with BCMA CAR-T cells

Abbas-Ali et al, Blood 2016


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%


Severe CRS is more likely in the case of high tumor burdens regardless of CD4/CD8 composition

Turtlle et al, J Clin Invest 2016


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:


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:


Severe neurotoxicities are preceded by cytokine release syndrome and ineffectively controlled by tocilizumab

Turtlle et al, J Clin Invest 2016

15/29 = 51%


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:


1) Non-immunogenic2) Fast-acting3) Used for managing cytokine release syndrome/neurotoxicities4) Effective and highly penetrant



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


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


hCD

19+

cells

/mcr

oL

0 7 14 21 280

10

20

30

40

50


hCD

14+

cells

/mic

roL

****** *** ******


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


1) Non-immunogenic2) Fast-acting3) Actionable with prodrugs that cross the blood-brain barrier4) Effective and highly penetrant



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)


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)

approaches to overcome car-t cell toxicities: anti ... · ospedale pediatrico bambino gesù, roma(f...

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