In vivo persistence and proliferation of FC21-NK cells in 33 patients across trials in three clinical settings

Jolie R Schafer1, Soumen Khatua2, Vanessa Valim3, Stefan O Ciurea2, Lucia Silla3, 4, Kinnari Sorathia5, Amanda Campbell5, Robin J. Nakkula5, Prashant Trikha5, Aarohi Thakkar5, Dean A Lee51Kiadis Pharma, Amsterdam, Netherlands2MD Anderson Cancer Center, Texas, United States3Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil4Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil5Nationwide Children’s Hospital, Ohio, United States

Immunotherapy - ClinicalAbstract number S284

DisclosuresKiadis Pharma/CytoSen TherapeuticsEmployment: Jolie SchaferStock/Stock Options: Dean Lee, Stefan CiureaConsulting/Scientific Advisory Board: Dean LeePatents/Licensing Fees: Dean Lee, Prashant Trikha, Aarohi Thakkar

4-1BBL

mbIL-21

NK cell

T cell

B cell

Mono

K562

Donor Blood Collection

Thaw and Infuse

Legend

CD3 Depletion

Buffy CoatFraction

Infuse FreshProduct

Cryopreservation

RecursiveStimulation

Clinical production of FC21-NK cells on K562 feeder cells expressing mbIL21 and 4-1BBL (FC21)

Denman C, PLoSONE 2012, 7:e30264FC21

FC21-NK

FC21 platform allows for 108 fold expansion ofhyperfunctional FC21-NK cells with therapeutic potential

0 7 14 21 28 35 42

100

101

102

103

104

105

106

107

108

109

mbIL15

mbIL21

***

Days

Fo

ld E

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on

Fre

sh

mbI

L15

mbI

L21

Fre

sh

mbI

L15

mbI

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1

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100

1000

10000

IFNg TNF IFNg TNF

Su

pe

rna

tan

t [p

g/m

l]K562mbIL4 K562mbIL21

-20

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pe

rce

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f orig

inal

telo

me

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ngth

Robust in vitroproliferation

Capacity for in vivoproliferation

Enhanced function(cytokine production)

Denman C, PLoSONE 2012, 7:e30264Ray A, PediatrBloodCancer 2019, 66:e27783

RS4

11

MOL

T-4K5

62Rh

30

SJ-G

BM2

NB-e

bcl

Rh41

TC-7

1

COG-

LL-3

17

CHLA

-136

CHLA

-10

CHLA

-9

Ram

os RD

RH-1

8

CHLA

-258

NB-1

643

Kasu

mi-1

CHLA

-266

CCRF

-CEM

Karp

as-2

99

CHLA

-90

Nalm

-6

BT-1

2-20

-10

0

10

20

30

40

50

60

70

80

90

100

Cell Lines

% S

pe

cifi

c ly

sis

Donor 1Donor 2Donor 3Donor 4

Enhanced function(cytotoxicity)

K562mbIL15 K562mbIL21

CyTOF demonstrates bright phenotype of FC21-NK cells

Expansion Day 0

Expansion Day 7

CD56 NKp46 NKp44 NKp30 DNAM1 NKG2D

Aquino A, under review

Expansion Day 14

Completed clinical trials with FC21-NK cells (n=59)‒ Allogeneic

• NCT01904136: 25 patients receiving haploSCT with PTCy for myeloid leukemia, up to 3 infusions each

• 25 patients receiving haplo NK cells after chemotherapy for relapsed/refractory AML, up to 6 infusions each

• NCT02809092: 13 in Brazil • NCT01787474: 12 in US

‒ Autologous• NCT02271711: 9 patients with intraventricular infusions for

relapsed/refractory brain tumor, up to 27 infusions eachKhatua S, Neuro-Onc 2020, in press

Ciurea SO, Blood 2017, 130:1857Silla L, in preparation

Ciurea SO, in preparation

Russo A, Blood 2018, 131:247

HaploSCT + PTCy: NK cell recovery is low and slow, but associated with protection from relapse

T cells dominate over NK cells at all timepoints

Absolute counts of NK and T cells in PB Absolute counts of circulating NK cells and CI of relapse

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7

Treatment Day

Stem cell processing

-7-8 8 9

PBSC or marrow collection

Tacro/MMF beginning day 5GCSF beginning day 7

28

Ad

mit/

Hyd

ratio

n

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7

Fludarabine 40 mg/m2

Stem Cell Collection/Infusion

NK Cell Collection/Infusion

Treatment Day

Stem cell processing

-7-8

Melphalan 140 mg/m2

Peripheral bloodcollection

NK cell expansion x 2 weeks on aAPC

NK cells, escalating dose

8 9

PBSC or marrow collection

Cyclophosphamide 50 mg/kg

Tacro/MMF beginning day 5GCSF beginning day 7

28

Ad

mit/

Hyd

ratio

n

NK cell expansion x 2 weeks on FC21

Level 1: 104/kg (de-escalation)Level 2: 105/kgLevel 3: 106/kgLevel 4: 107/kgLevel 5: 3x107/kgLevel 6: 108/kg

HaploSCT + PTCy: Adjunctive FC21-NK cells used to augment NK cell recovery

Ciurea SO, Blood 2017, 130:1857NCT01904136

0266

0708

NK c

ell p

rodu

ct0

20

40

60

80

100

Per

cen

t S

pec

ific

Lys

is(1

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rat

io)

D30 peripheralblood NK cells

p= 0.03880.2268p=

0266

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NK cell product

D30

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D180

0

5000

10000

15000

20000

Ce

lls/u

L

WBC

2009-0266

2012-0708

D30

D90

D180

1

10

100

1000

Cel

ls/u

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CD3+CD4+

D30

D90

D180

10

100

1000

10000

Ce

lls/u

L

CD3+

D30

D90

D180

1

10

100

1000

10000

Cel

ls/u

L

CD3+CD8+

p = 0.00015

D30

D90

D180

0

200

400

600

800

Ce

lls/u

L

CD56+CD3-

D30

D90

D180

1

10

100

1000

Cel

ls/u

L

CD25

CD10

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NgTN

Fa0

20

40

60

80

100

Cytokine

% P

osi

tive

0266

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p= 0.0028

p= 0.0231

Ciurea SO, Blood 2017, 130:1857

Haplo FC21-NK cells enhance NK reconstitution after PTCy

CD16

+

CD62

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CD24

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CD27

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CD35

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20

40

60

80

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Phenotypic Marker

% P

osi

tive

02660708

Activating Inhibitory

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

2009-0266 / 0266: haplo+PTCy2012-0708 / 0708: haplo+PTCy+FC21-NK

Immune reconstitution in HaploSCT patients receiving FC21-NK

CD3

CD56

35-parameter CyTOF, 8-parameter ViSNE clustering

Healthy Donor FC21-NK Product Patient

T cells FC21- NK “super bright”

“Std” NK

Ciurea SO, in preparation

FC21-NK “super bright”

EHA AbstractEP1487

“Std” NK cells

Phenotypic identification of infused haplo FC21-NK cells

Patient #302(Day 14)

Patient #74(Day 14)

Healthy donor

FC21-NK product

CD3 CD56 NKp46 NKG2D CD57 Perforin Ki67

Haplo FC21-NK cells promote NK-dominant reconstitution

Heal

thy

dono

rs

Patie

nts

Prod

ucts

0

20

40

60

80

100

Per

cen

t su

per

bri

gh

t N

K

% NK cells

% CD56br (of NK)

Russo A, Blood 2018, 131:247Ciurea SO, in preparation

0 14 28 420

20

40

60

80

100

Day

Per

cen

t o

f C

D45

+

T

Total NK

0 14 28 420

20

40

60

80

100

Day

Per

cen

t o

f C

D45

+

Superbright NK

All samples1

10

100

1000

10000

NK

:T r

atio

% NK cells

% CD56br (of NK)

HaploSCT + PTCyHaploSCT + PTCy + FC21-NK

FC21-NK cells proliferate in vivo

FC21-NK cellsStd NK cells

T cellsSt

d NK

Supe

rbrig

ht N

K T0

20

40

60

80

100

% K

i67+

of

sub

po

pu

lati

on

Ciurea SO, in preparation

FC21-NK have increased Ki67 expression (proliferation marker) compared to standard (Std) NK cells and T cells

Std

NK

FC-2

1 N

K

T-ce

lls

(Representative patients at day 14) (All patients, all timepoints)

Haploidentical

0 1 2 3 4 5 6 7

Fludarabine 30mg/m2/dayAra-C 2g/m2/day (4 hours after Flu)NK cell expansion on

aAPC x 14d

G-CSF 5ug/kg/dayDonor Blood obtained prior to beginning FLAG

Cryopreservation

8 9 10 11

NK cell therapy3 days/week x 6 doses

FLAG chemotherapy5 days (4 if age ≥ 60 y)

Rest period of 2-14 days

NK cell infusion

R/R AML: Haplo FC21-NK post FLAG

FC21-NK cell infusion

NCT01787474; NCT02809092

NK cell expansion x 2 weeks on FC21

A B

Haplo FC21-NK cells expand and persist in vivo (Brazil)

NK cells T cells0.001

0.01

0.1

1

10

x10-

3 u

L Day 0- pre-infusion 1

Day 0- post-infusion 1

nsp= 0.04

Day 7- pre-infusion 4

1 2 3 4 5 60.001

0.01

0.1

1

10

Infusion

x 10

-3 u

L

Day 28

T cells

NK cells

Silla L, EHA Poster EP585Manuscript in preparation

NK cells peak at day 7 NK cells show 100-fold accumulation

Flow cytometry analyses of HLA chimerism between patient and donor

Both Patient A and B received the lowest FC21-NK dose (106 cells/kg)

Haplo FC21-NK cells persist for 5 weeks in vivo (US) Patient A Patient B

HLA-A2 HLA-A2 HLA-Bw6

Ciurea SO, in preparation

FC21-NK

FC21-NK

FC21-NK

1 2 3 4

Peripheral bloodfrom patient

NK Infusion

(Treatment Week)

NK cell expansion on aAPC x 14d Cryopreservation

Cell Therapy

Patient enrollment:Draw peripheral blood

3ml/kg (150 max)

4-week Treatment Cycle (x 3)

Dose Escalation

Level 1: 106/m2 IT per infusion TIW

Level 2: 3 x 107/m2 IT per infusion QW

Level 3: 3 x 108/m2 IT per infusion QW

1 2 3 4 1 2 3 4

Cycle 1 Cycle 2 Cycle 3

REST REST REST

Autologous FC21-NK cells for brain tumors (intraventricular infusion)

Khatua S, Neuro-Onc 2020, in pressAutologous FC21-NK cell infusion

NK cell expansion x 2 weeks on FC21

NCT02271711

Autologous FC21-NK cells increase and persist in CSF over 8-week treatment period

Khatua S, Neuro-Onc 2020, in press

Conclusions‒ FC21-NK cells display unique bright phenotype and hyperfunctional

activity with potent therapeutic potential.‒ FC21-NK cell persistence was evaluated in 33 of 59 patients in three

clinical settings• Allogeneic FC21-NK in HaploSCT: functional persistence for 3 weeks,

persistent phenotype and NK cell dominance to at least day 49• Allogeneic FC21-NK after chemo: 100-fold accumulation at high doses,

30% chimerism and at least 5-week persistence at low doses• Autologous FC21-NK: 10-fold accumulation across 8 weeks of

treatment cycles

Lee Lab, Collaborators, and SupportLeeLab

- Prashant Trikha- Meisam Naeimi- Marcelo Pereira

- Ani (Aarohi) Thakkar- Robin Nakkula- Maggie Lamb- Brian Tullius

- Kinnari Sorathia- Amanda Campbell

CollaboratorsUFRGS

- Lucia Silla- Vanessa ValimMD Anderson

- Stefan Ciurea- Soumen Khatua

Former LeeLab- Cecele Denman- Prasad Phatarpekar

- Jolie Schafer- Ariany Aquino-Lopez- Anitha Somanchi- Nitin Chakravarti- Jennifer Foltz- Jena Edwards- Ezgi Elmas

Financial Support- Germain and DiMarco

Families- St. Baldrick’s Foundation- Leukemia & Lymphoma

Society- Hyundai Hope on Wheels- Cancer-Free Kids- Nat’l Pediatric Cancer

Foundation- Pelotonia Foundation