PowerPoint Presentation · Title: PowerPoint Presentation Author: Attilio Bondanza Created Date:...

Should haplo-SCT be performed

with T-cell depletionNO

Fabio CiceriSan Raffaele Scientific Institute

Milano, [email protected]

Two mainplatforms for haplo

Platforms for haplo• CD34+ selection

– Pathogen specific lymphocytes– TK-transduced lymphocytes– IL-10 anergyzed lymphocytes– Photo-allodepleted lymphocytes– CD8 neg. selected lymphocytes– Tregs pos.selected

• CD3/CD19 negative selection

• Unmanipulated BM– Endoxan at day + 3– Basiliximab, Csa,

• Unmanipulated PBSC– Rapamycin

Immunesuppressive tx

Graft manipulation

1- T-cell depleted grafts

are ideal platformsfor manipulated T-cell repletion

MolMed – TK008 IPR/21.A, 2010 - CONFIDENTIAL

HSV-TK approach: engineering of donor T cells (1)

5

Pts with TK cells engraftment Pts with NO TK cells engraftment

TK007 phase II trial: only patients who achieve TK cell engraftment progress to long term IR

MolMed – TK008 IPR/21.A, 2010 - CONFIDENTIAL 7

Phase I/II-TK007 Results)

Infective Adverse Events

Immune-reconstituted pts NOT-Immune-reconstituted pts

Source adopted from: Ciceri F.,Bonini C. et al. Lancet Oncol. 2009 May;10(5):489-500

Months after HCT Months after HCT

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Vβ24

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TK007: T cell repertoire normalizes in 6 months

0%

20%

40%

60%

80%

100%

At IR 6 m afterTK cellsinfusions

1 yearafter TK

cellsinfusions

Controls

Dis

trib

ution o

f TC

R V

b f

amili

es

MonoclonalOligoclonalPoliclonal

TK007: TKcells induce a rapid recovery of immune responses to EBV and CMV

TK007: Immune reconstitution obtained with TK cells add-back is protective against CMV

Peaks of CMV antigenemia Days of antiviral therapy

11

TK008 phase III trial

R (3:1)

Haplo-HSCT + TK

Haplo-HSCT

Key inclusion criteria:

• AML-ALL at high-risk in first CR

• AML-ALL in ≥ second CR

• secondary AML in CR

• absence of HLA-matched family or unrelated donor

Stratification

• complete response (1st v > 1st), very-high-risk vs high-risk

• country

Primary endpoint:

• Leukemia-free survival

Secondary aims:

• NRM, overall survival, immune-reconstitution, engraftment, aGvHD, cGvHD, relapse, disease-free survival, infectious, safety, quality of fife, pharmacoeconomics

• 1-year NRM standard haplo:60%

• 1-year NRM ph II study TK: 37%

• Relapse rate standard haplo 25%

• Relapse rate ph II study TK 18%

• Power = 80%; HR= 0.55

• LFS 30% in standard haplo

• LFS 52% expected in TK008 exp arm

• 91 events (death + leukemia relapse)

• N=170 patients

N=127

N=43

New developments in T-cell engineering

12

What are the optimal T cell subsets for effective and persistent cancer immunotherapy?

Naïve

CD45RA+CD62L+

CD127+

Central Memory

CD45RA-CD62L+

CD127+

Terminal Effector

CD45RA+CD62L-

Effector Memory

CD45RA-CD62L-

Death sensitivity

Effector functions

Proliferative potential

Lymphnode homing

How to generate gene modified central memory T cells?

Schluns et al., 2003

Memory

Cell-sized paramagnetic beads coated with anti-CD3/anti-CD28 antibodies (Invitrogen)

Preserved alloreactivity with IL-7 & IL-15

Kaneko et al, Blood 2009

CD3/CD28-beads + IL-7 & IL-15 NOD.scid HLA-mismatched skin

0,1

1

10

100

0 2 4 6 8 10 12 14 16

time from infusion (wk)

log

leuk

emic

chi

mer

ism

(log

%)

9 wk delay

HA-1-specific TK+ lymphocytes generated with B+IL7/IL15 delay human leukemic outgrowth in vivo

PBSCMV line fromPBLHA-1 line fromPBLHA-1 line fromTK+ cells

P<0.01

Bondanza et al., Blood 2011

Exploiting TCM lymphocytes for an effective T cell based gene therapy

TCM

TK-TCM in haplo for advanced leukemia

RV

CAR GENE TRANSFERto generate effective tumor-specific lymphocytesable to expand and persist in cancer patients

LV LV/ZFN

Bondanza et al, Blood 2006Kaneko and Mastaglio et al, Blood 2009Bondanza et al., Blood 2011

T cell receptor (TCR) gene editing

Targeting critical antigens for oncogenic transformation to avoid tumor escape with high-affinity TCR

S-S

Vα Vβ

Cα Cβ

S-S

Vα Vβ

Cα CβS-S

WT TCR Cys-modifiedTCR

Vα 21 - Vβ 21HLA-A2 restricted

• 1st disease targeted: Acute myeloid leukemia

• 1st Antigen targeted: WT1 – Overexpressed by several tumors

(Van Driessche, Leukemia 2005)

– Involved in oncogenic transformation– WT1 is immunogenic in humans

(Gao, Blood 2000; Xue Blood 2005)

– WT1-specific high avidity optimized TCR availables

Kuball et al, Blood 2007

Full Editing of T-Cell Specificity

baCD3/CD28 stimulation

242 9

ZF

N-T

RA

C

TRAC-ZFNs

8

CD

3

FL1

α-chain NEG

100 101 102 103 104100

101

102

103

104

55.6 0

044.4

Sorting CD3-

FL1

CD

3

α-chain edited

α-ch

ain

LV

WT1-α chain

WpreRREGA TCR α

0 102 103 104 105

0

102

103

104

105

0

078.9

21.1

100

baCD3/CD28 stimulation

2 11 18

FL1

CD

3

α-β NEG

ZF

N-T

RB

C

TRBC-ZFNs

0 102 103 104 105

0

102

103

104

105 76.8 0

023.2

Sorting CD3-

WT1-β chain

WpreRREGA TCR β

β-ch

ain

LV

Full TCR-editedLymphocytes

Vβ21

CD

3

0 102 103 104 105

0

102

103

104

105 0.83 23.1

0.9675.1 Pol

yclo

nal

Stim

ulat

ion

Vβ21

CD

3

PolyclonalStimulation

0 102 103 104 105

0

102

103

104

105

0.83

90.21.46

7.52

α/β edited T-cells recognize and lyse WT1pos primary leukemias

TCR-transferred TCR-α/β-edited

E/T Ratio E/T Ratio

% L

ysis

% L

ysis

AML1 (HLA-A2+)

AML 2 (HLA-A2+)

AML 3 (HLA-A2-)

α/β edited T-cells do not display off-target reactivity and do not mediate GvHD in NSG mice

PBMCPBS

**

***

TCR-transferredTCR-α/β-edited

TCR

-α/β

-edi

ted

PBM

CTC

R-tr

ansf

erre

d

PBS

NSG mice

PBMC

PBS

TCR-α/β-edited

TCR-transferred

1 4 7 1 4 7 1 4 7 1 4 7Days after infusion

Infused cells At sacrifice

Vβ2

1 R

FI

% V

β21 cells / hC

D45 cells

**

allogeneic SCT in advanced leukemia

23

T-cell depleted haplo is ineffective in advanced leukemia

210

1,0

0,8

0,6

0,4

0,2

0,0

Ciceri et al, Blood 2008

The European Group for Blood and Marrow Transplantation

Cy/TBI vs. BuCy in resistant AMLCy/TBI vs. BuCy in resistant AML: LFS

years

23 ± 2%

23 ± 2%

Cy/TBI n=388

BuCy n=395

P : 0.41

A Nagler et al, in preparation

Unmanipulated graft in haplo-SCT

Macrolytic antibiotic that inhibits the mammalian target of rapamycin (mTOR)

– Inhibition of cell cycle entry in stimulated T cells

– Direct toxic effect on dendritic cells

– Anti-angiogenetic activity

– Clinical activity on refractory AML

– Induction of Foxp3 expression and maintenance of Tregs

Hypothesis: in vivo Tregs expansion under rapamycin

Treosulfan-based conditioning and Rapamycin-ATG-F-based

GvHD prophylaxis prior tounmanipulated allogeneic haematopoietic stem cell

transplantation from a mismatched donor in patients with advanced hematological

malignancies

TrRaMM

Eudract 2007-5477-54

TrRaMM: Conditioning

-6 -5 -4 -3 -2 -1 0 +1 +2

Treosulfan 14 g/m2 for 3 days

Rapamicyn +MMF/Myf

Fludara 30 mg/m2 for 5 days

PBSC:4-10x106 CD34

No in vitro manipulation!

ATG-Fresenius 10 mg/kg for 3 days

Rituximab 500 mg

TrRaMM: Inclusion criteria

• Advanced disease

• Absence of HLA-matched donor

• Age > 1 and ≤ 70 years

• Karnofsky Index ≥ 80%

• Sorror HCT-CI ≤ 4

• Written informed consent

• March 2007- April 2010• 79 patients (43 ♂, 36 ♀):

– 45: 6/12 matched– 18: 7/12 matched– 16: 8 or 9/12 matched

• Previous allogeneic transplant (19 pts):– 14 MRD– 2 MUD– 2 MMRD – 1 MRD and MMRD

• Median age: 47 years (range 14-69) • Median HCT-CI : 2 (range 0-4)

TrRaMM: Patients characteristics

AML CR1 5CR2 12

> CR2/refractory 32

ALL CR2 2> CR2/refractory 8

MDS 3

MPN 3

LPD HD 7NHL 3

Biphenotypic AL 1

GS/CLL/CMML 3

Total 79

TrRaMM: Disease status at transplant

TrRaMM: Results

• Median Follow-up 391 days (238-1187)

• 29 patients alive 22 in CR, 27 out of IS

• 50 patients dead 20 for NRM, 30 for PD

TrRaMM: engraftment & chimerism

69/75 patients: FULL DONOR at d+30

Immune reconstitution

Pattern of immune reconstitution

In vivo Tregs expansion and function

TrRaMM: acute and chronic GvHD

TrRaMM: disease relapse

TrRaMM: mortality and survival

The European Group for Blood and Marrow Transplantation

alternative donors options

Lack of HLA-id donor

Unrelatedmismatched

Cord-blood

Family Haploidentical

Intention-to-treat in allo SCT

p = ns

AML early phaseintention-to-treat: outcome

Experimental treatments better performed in clinical trials

HSR. Exp. Hematol.Elena PROVASIZulma MAGNANIAttilio BONDANZALuca VAGOMonica CASUCCI Shin KANEKOSara MASTAGLIOVeronica VALTOLINASerena Kimi PERNAMaddalena NOVIELLOAlessandra FORCINABarbara CAMISANicoletta CIERIGiacomo OLIVEIRALaura FALCONE

HSR TIGET Pietro GENOVESEAngelo LOMBARDOOscar MUNIZ PELLOMario AMENDOLALucia SERGI SERGILuigi NALDINI

FHCRC, SeattleP.D. GREENBERGJ. KUBALL

SANGAMO BIOSCIENCESM. HOLMESP. GREGORY

HSR BMT UNITM.T. LUPO-STANGHELLINIJacopo PECCATORIMassimo BERNARDIConsuelo CORTI

HSR Dept. PathologyMaurilio PONZONIFrancesca SANVITOClaudio DOGLIONI

HSR Mol. and Functional Immunogenetics UnitKatharina FLEISCHHAUER

HADASSAH H, JerusalemShimon SLAVINShoshana MORECKIAliza ACKERSTAIN

ISTITUTO CLINICO HUMANITAS, MilanoLuca CASTAGNAArmando SANTORO

HAMMERSMITH, LondonJane APPERLEYEduardo OLAVARRIA

HANNOVER MEDICAL SCHOOL, HannoverEvy WEISSINGERArnold GANSERMichael STADLER

G.PAPANICOLAOU HOSPITAL , Thessaloniki Evangelia YANNAKI Athanasios FASSAS, Achilles ANAGNOSTOPOULOS

MolMed SpAClaudio BORDIGNONAnd all the staff!!!

ACKONWLEDGEMENTS

PowerPoint Presentation · Title: PowerPoint Presentation Author: Attilio Bondanza Created Date:...

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