off the shelfstrategyfor car-t therapyusingcordblood · tcr mhc i target cell tumor cell + + cik...

Off the shelf strategy for CAR-T therapy using cord blood

Martino Introna

TCR

MHC I

Target cell

Tumorcell

++

CIK cellRestricted

killingNon MHC-restricted

killing

CD3/CD8/CD56 T/EMRA

DNAM1LFA1

CD56NKG2DNKp30

Adapted by Pievani et al, Blood, 2011

Study Design

BM or PB transplant

Taperedimmunosuppressionand chemotherapy

only if needed

Molecular or cytogenetic or hematologic

RELAPSE

1st Standard

DLI(1x106/kg)

2ndStandard

DLI(1x106/kg)

1stCIK

infusion

2ndCIK

infusion

3rdCIK

infusion

Follow-up

Combina-tion

1st CIK cells infusion

2nd CIK cells infusion

3rd CIK cells infusion

1 1x106/Kg 1x106/Kg 5x106/kg

2 1x106/Kg 5x106/kg 5x106/kg

3 1x106/Kg 5x106/kg 10x106/kg

4 5x106/kg 5x106/kg 10x106/kg

3 weeks 3 weeks 3 weeks 3 weeks

Introna M. et al, BBMT, 2017

Study FlowPatients enrolled in the study protocol (N=74)

Patients allocated to CIK treatment (N=60)

Patients completing study protocol (N=43)

1ST DLI

2ND DLI

1ST CIK

2ND CIK

3RD CIK

2 death before starting therapy

4 disease progression and death1 GvHD

3 disease progression and death2 GvHD1 drop out

7 disease progression and death2 GvHD

4 disease progression and death1 insufficient cell supply1 hemolitic anemia2 GvHD

1 excluded for protocol violation



Clinical outcomes according to type of relapse

AML

ALL

Characteristics All (N=73) Pediatrics (N=15) Adults (N=58)

Preparative regimen, N(%)MAC 53 (72.6) 12 (80.0) 41 (70.7)RIC 20 (27.4) 3 (20.0) 17 (29.3)

Stem cell source*, N(%)PB 42 (59.2) 2 (14.3) 40 (70.2)BM 29 (40.8) 12 (85.7) 17 (29.8)

Donor, N(%)SIB 37 (50.7) 5 (33.3) 32 (55.2)MUD 31 (42.4) 8 (53.4) 23 (39.6)HLA aploidentical 5 (6.9) 2 (13.3) 3 (5.2)

Relapse type, N(%)Hematologic 44 (60.3) 6 (40.0) 38 (65.5)Molecular + Cytogenetic 29 (39.7) 9 (60.0) 20 (34.5)

Months from diagnosis to HSCT, median (range) 6 (2-102) 24 (5-62) 5 (2-102)

Months from HSCT to relapse, median (range) 6 (0.5-124) 6 (1-26) 6 (0.5-124)


CIK II STUDY ACKNOWLEDGMENTS

USC Hematology of Bergamo, ItalyDr. F. Lussana, Dr. A. Algarotti, Dr. F. Delaini, Dr. A. Grassi, Dr. C. Micò,Dr. M. L. Ferrari, Dr. E. Todisco, Dr. C. Pavoni, Prof. A. RambaldiDiv. Hematology and TMO, Bolzano, ItalyDr. I. Cavattoni, S. Deola

Center of Cell Therapy “G. Lanzani”, Bergamo, ItalyDr. M. Introna, Dr. J. Golay, Dr. E. Gotti, Dr. R. Valgardsdottir

Department of Pediatrics, University of Milano-Bicocca, Fondazione MBBM, Monza, ItalyProf. E. Biagi, Dr. A. Balduzzi, Dr. A. Rovelli, Dr. S. Napolitano, Dr. G. Sgroi, Dr. E. Marrocco, Prof. A. BiondiDepartment of Hematology, Monza, ItalyDr. M. ParmaApheresis Unit, Monza, ItalyDr. P. Perseghin

Laboratory of Cell and Gene Therapy “S. Verri”, Monza, ItalyDr. G. Gaipa, Dr. D. Belotti, Dr. B. Cabiati

Merker M, et al. BBMT 2019

DLI CIK

MFD 17 31%

3 8%

MUD 25 45%

10 28%

MMFD 13 24%

23 64%

Overt Relapse

10

100

1000

10000

100000

1 2 3 4 5 6 7

TNC

x10

6

X-VIVO X-VIVO+HS

Optimization of method to expand CIK from banked CBUs

Golay et al. Cytotherapy 2018

Adding 1% human serum improves CIK cellexpansion from banked CBUs

0

20

40

60

80

100

120

NKG2DCD27

CD28

CD137

CD154

CD11a

CD49d

CD200R

CD244

CD274

CD272

CD279

CD152

CD223

CD366

% P

ositi

ve c

ells

CD8+ subset CB-CIKPB-CIK

**

**

N=6

0

20

40

60

80

100

CD3+ CD3+CD56+ (CIK) NK

% P

ositi

ve c

ells

Phenotype of CB-CIK in 1%HSAB is similar to that of PB-CIK

0

20

40

60

80

100

CD4+ CD8+

% P

ositi

ve c

ells

***

***CB-CIK N=8PB-CIK N=8CB-CIK N=8

PB-CIK N=8

J Golay, et al. Cytotherapy 2018

0

20

40

60

80

100

CD4 CD8

% P

ositi

ve c

ells

CB-CIKPB-CIK

0

20

40

60

80

100

CD4 CD8

% P

ositi

ve c

ells

CB-CIKPB-CIK

CD

56

CD3

CB-CIK

N=8

PB-CIKA

B

C

*

***

***


Kill

Tumor cell CIK

Bispecific antibody

Target Effector

-20

0

20

40

60

80

100

120

% L

ysis

CB-CIK1 vs REHCB-CIK2 vs REHCB-CIK3 vs BJABPB-CIK1 vs REHPB-CIK2 vs REHPB-CIK3 vs BJAB

- + - + - + BLINA

1:1 3:1 10:1 E:T

0

20

40

60

80

100

CIK-CB PB-CIK

% L

ysis

K562A B

N=3 N=6

***

******

0

20

40

60

80

100

120

- + - + - +

% L

ysis

CB-CIKPB-CIK

N=3

1:1 3:1 10:1

BLINA

*

*

*C

E:T J Golay, et al. Cytotherapy 2018

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Surv

ival

(%)

Time (days)

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Surv

ival

(%)

Time (days)

A BCB-CIK PB-CIK

CTRLBLINACIKCIK+BLINA

******


NOD SCID mice inoculated iv with 106 ALL-2 cells

Start Cell yield at the end of culture Release criteriaTheoretical yield of CIK from whole CBUa

CB-CIK batch number

TNC (x106) put in culture

% CD3+ Days

Fold increaseTNC

TNC x106

CIK x106

% CD3+

%CIK

% NK

%live cells

Other quality controlsb

CIKx106 CIKx106/kg

CB-CIK1 300 46.6 25 22.9 6871 3270 97.9 47.6 2.1 85.3 Pass 11454 163.5CB-CIK2 230 54.2 23 6.4 1472 610 98.3 41.4 1.6 86.3 Pass 1173 16.7CB-CIK3 302 36.5 17 8.5 2574 1387 92.6 53.9 7.0 89.9 Pass 2312 33.0CB-CIK4 732 43.0 20 6.2 4563 1725 91.5 37.8 8.4 95.1 Pass 1725 24.6Median 301 44.8 21.5 7.5 3332 1079 99.3 44.5 4.6 88.1 NA 2018 28.8

Yields and quality of CIK cells expanded from banked CBUs in GMP

aYields were calculated considering the blood volume used for expansion relative to the total CBU volumes available and, in case of CBU1, the actual fraction of TNC put in culture, with respect to total TNCs obtained after washing the CB material on day 0.bGMP quality controls include <7 IU/ml endotoxin, sterility for aerobic and anaerobic microorganisms, absence of mycoplasm


Ficoll/ Wash

Initialexpansion

Medium/large scale expansion in bioreactors

Product wash and medium exchange intofreezing medium

-Gas perm. bags/G-REX/flasks--differentproducts: CIK, T cells

-Sepax: +/-ficoll-Differentcellsources(PB, CB, aphereticmaterial)

-Bioreactor type (Xuri/Quantum/G-REX)-Peristaltic pump (Gatherex)-Growth conditions (cell conc, reperfusion timing, media)-Comparison with flasks-Cell growth measurement methods-T cells, CIK, BET, -Automation

-Sepax-Freezing medium (Cryostor/plasma+ DMSO)-Stability studies-Automation

STEP 1 STEP 3 STEP 4 STEP 5

Options and flow chart for CIK expansion in closed systems (+/-CAR)

-Electropor. In closed system(4D-Nucleofector™ LV Unit)-different cellsources (CB, PB, Aph)

STEP 2

Gene transduction

Initial expansion Medium/large scale expansion in bioreactors

XURI BIOREACTOR

Optimization of expansion steps

Flask

G-REX

Standard method in flasks

G-REX

CIK Expansion in XURI vs FLASK ( 2 validations after some optimization)

A. Time course B. Mean fold increase of TNC at the end of culture with respect to start

N=2 N=2

9-10 days 10-14 days

Or

Add large amount of medium at start

Incubate 10-12 days in standard incubator

Faster growth due to better gas exchange

Gas-permeablemembrane at the bottom

G-Rex vessels

Version with tubings to connect to Sepax/ gatherex to load and collect cells in closedsystem

0

50

100

150

200

250

0 9-11 21-22

Fold

incr

ease

Days

FlasksFlasks ->G-Rex

N=3

flasks

G-Rex or flasks

0

50

100

150

200

250

0 9-11 21

Fold

incr

ease

Days

0.25x106/cm2

0.5x106/cm21x106/cm2

N=3

flasks

CIK Expansion in G-Rex vs FLASK ( set up)

Different cell densities in G.Rex

9-10 days

10-12 daysor

0

20

40

60

80

100

120

0 10

Fold

incr

ease

Days

0.25x106/cm20.5x106/cm21.0x106/cm2

N=2

0

100

200

300

400

500

0 10-11 21To

tal c

ell n

umbe

r (x1

06)

Days

G-RexFlasksN=3

Only 1 G-REX (10-14 days) Only Flasks

CIK Expansion in G-Rex vs FLASKs (optimization)

or

Set up G-Rex Comparison flasks G-REX

PBMC Harvest

CIK-PB in flasks or G-Rex show equivalent cytotoxicityagainst K562

N=3

N=3

*

*

*

NK CIK

Overall phenotype at end of culture is similar for CIK in G-Rex vs flasks, but lower % CD3+CD56+

0 10 20 30 40 50 60 70 80 days

ALL-2 iv survival

CIKBlina

Exp 1734Efficacy of

CIK-G alone andcombined withBlina in ALL2

model

0 10 20 30 40 50 60 700

50

100

CIK-G + blina vs vehicle p=0,0016** Mantel CoxCIK-G vs vehicle p=0,0016** Mantel CoxCIK-G vs CIK-G +Blina p=0,038* Mantel Cox

CIK-G +Blina

vehicleCIK-G

days

Surv

ival

(%)

****

CIK-G are effective in vivo in mice (+/-blinatumomab) in Ph+ ALL model

CIK from CB or PB (latter expanded in G-REX) do not induce GvHD in NSG mice

(Sarah Tettamanti, Fondazione MBBM, Monza, Italy)

0

20

40

60

80

100

120

0 50 100 150 200

Surv

ival

(%)

Days

PBMCCIK-PB in G-RexCIK-CB

Theoretical yields of CIK in G-REX based on initial validations

50x106

PBMC

3x109 TNC

1x109 CIK1 liter and about 10 days

(1 G-REX 100M)

500x106

CB MNC

30x109 TNC

10x109 CIK10 liter and about 10 days

(4 G-REX 500CS)

PB small scale

CB large scale

HLA A HLA B HLA DR11 2 8 18 11 172 24 35 51 11 142 30 13 44 7 11

24 29 18 44 7 112 3 7 35 11 153 33 35 65 1 13

11 30 18 35 11 171 2 51 57 7 132 11 35 50 1 71 23 44 49 7 112 24 35 44 4 131 32 35 51 11 133 11 35 44 4 143 26 38 51 11 132 24 51 62 4 132 24 44 51 8 162 24 44 49 11 132 24 18 58 16 17

24 26 7 35 1 153 24 8 35 16 171 2 52 63 13 152 2 44 51 1 15

25 68 18 51 11 153 32 18 44 7 11

11 24 51 52 11 1526 68 35 44 11 132 24 38 61 11 133 24 13 35 7 111 2 37 57 10 111 2 18 3901 1 16

The 30 CBU with the most frequent

haplotypes in Italy

IBMDRItalian Bone Marrow Donors Registry

In collaboration with Nicoletta Sacchi and Anna Maria Gallina, IBMDR, E.O. Ospedali Galliera, Genova

Minimal HLA matching present in different allo-HSCT settings

Allogenic transplant with CBUHLA A HLA B HLA C HLA DRB1*

x x x xy y y y

Haplo-identical transplantHLA A HLA B HLA C HLA DRB1

x x x xy y y y

Haplo-matching CB-CIK (3/6)HLA A HLA B HLA C HLA DRB1

x x x xy y y y

* High resolution


HLA matching

Patient HLAHLA A HLA B HLA DR1

11 24 18 39 15 16

Banked CB CIK unit HLAHLA A HLA B HLA DR1

02 24 18 58 16 17

At least 3/6 matching


simulation

80 consecutive ASST-PG23 Hematology transplantedpatients

5 batches from each cord blood unit => 150 batchesbanked from the 30 CB selected units

60/80 patients with positive matching75%

Sponsor: Fondazione Tettamanti

Modification and expansion of mononuclear precursors by the SB system

Magnani CF. et al, Oncotarget, 2017

Ø 15-20 patientsØ Da 1 x 106 a 15x106 /kg

dose escalation

Sponsor:Fondazione Tettamanti Strutture:Pediatric patientsASST-Monza, Ospedale San Gerardo – Monza, IT Adult patientsASST-Bergamo, Papa Giovanni XXIII – Bergamo, IT

NCT03389035

Phase I/Iia trial with allogeneicCIK cells transduced by SB CAR CD19 in adult and pediatric ALL patients relapsed after HSCT


CD19.CAR redirect CIK-cell activity against CD19+ cells.


Specific cytokine production of CD19.CAR CIK cells.


In vivo anti-tumor activity of CD19.CAR

CIK

Gene non-viral transfection manufacturing

Viral vectorsEfficient /standardized but time-

consuming, expensive, non random integration

TRANSPOSONSMobile DNA elements naturally occurring in

the genome

Easy to purify, non-immunogenic, random pattern of integration

Cytokine Induced Killer (CIK) as effector cells

Cytokine Induced Killer cells (CIK)

T cells with NK-like cytotoxicity,

enriched in CD3+CD56+

Clinical experience with allogeneic CIK: feasible, safe and well tolerated with reduced risk of GVHD, included in 5

patients with CIK from haploidenticaldonors

(Introna et al. BBMT 2017, EudraCTN°2008-003185-26)

USS Centro di Terapia Cellulare «G. Lanzani», ASST Papa Giovanni XXIII, Bergamo

J. Golay RAQC. CapelliO. PedriniE. GottiR. ValgardsdottirF. CorrentiI. CattaneoS. ZaninelliS. TettamantiC. CuofanoM. Introna QP

USC Hematology, ASST Papa Giovanni XXIII, Bergamo

PI: Prof. A. RambaldiG. GrittiF. Lussana

Hematology,ASST Monza Ospedale S Gerardo

Prof A. BiondiE. BiagiCF. MagnaniN. TurazziS. Tettamanti

Laboratorio di Terapia Genica e Cellulare“Stefano Verri”, ASST Monza Ospedale S Gerardo

G. GaipaB. CabiatiD. Belotti

off the shelfstrategyfor car-t therapyusingcordblood · tcr mhc i target cell tumor cell + + cik...

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