2 1 05

4 1 05

6 1 05

8 1 05

C D 3 T c e l l c o u n t

D a y p o s t P N K - 0 0 7 i n f u s i o n

ce

ll

s/

mL

b

lo

od

s c r e e n 0 7 1 4 2 1 2 8

N D

Background• Natural Killer (NK) cells are innate immune cells which play an important role in host

immune surveillance against pathogenic infection and cell transformation. Multiple studies

adoptively transferring NK cells in clinical settings have demonstrated the potential of NK

cells to induce remissions for hematological indications with a consistent safety profile.

• Celularity has developed a GMP procedure for generating Placenta-derived intermediate

Natural Killer cells (PNK-007) from placental/umbilical cord blood CD34+ cells. This

technology platform enables the scalable commercial production of a uniform allogeneic NK

cell therapy.

• PNK-007 shows cytotoxic activity against various cancer cell lines and is being evaluated

for the treatment of relapsed/refractory AML patients in a Phase I study. Here, we provide

translational data monitoring PNK-007 in vivo persistence and phenotypic characterization

for patients enrolled in the multi-center CCT-PNK-001-AML study in the context of immune

profiling and disease state.

PNK-007 manufacturing process overview

Immune monitoring of PNK-007, an allogeneic, off the shelf NK cell in a

Phase I study of acute myeloid leukemia

William van der Touw1, Lin Kang1, Julie Curtsinger2, Vanessa Voskinarian-Berse1, Bhavani Stout1, Mohamad A. Hussein3, Sarah Cooley2, Jeffery S. Miller2,

Robert Hariri1, Xiaokui Zhang1

1Celularity, Inc, Warren, NJ; 2University of Minnesota, Minneapolis, MN; 3Celgene Corporation, Summit, NJ.

INTRODUCTION

Cryopreserved PNK-007 Drug Substance PNK-007 Product

Cytokine Cocktail-driven

Expansion/Differentiation Process

Cryopreserved

CD34+ Bank

CD34 Donor cell

Screen/Testing

•Recovery Culture

•Washing

• Formulation

•Release Assays

• Shipping 2-8º C

• Administer IV

Freeze for DS Release

•Donor Capacity: 10,000 - 50,000 fold expansion

•Viability: >80% NK viability (7AAD)

•Purity: >85% CD56+/CD3-

•Functionality: >50% K562 cytotoxicity @ 10:1 E:T

•Process Capacity:12 billion cell yield (DS)

•Quality: Systems, testing, residuals, closure

0

1

2

3

4

5

0 7 14 21 28 35

Ex

pa

nsi

on

Fo

ld

Days in Culture

Typical Growth Culture

103

102

101

100

104

Donor Isolation

SCREENING/BASELINE

Day-28 to-7 Day-6 to-2

TREATMENTDay 0 24 Months 24 Months

FOLLOW-UP

Informed consent

and Screening

Conditioning

Regimen

Dose escalation 3 + 3

design at 4 different dose

levels

PNK-007 infusion: cells at

the dose specific to the

current cohort will be

infused Day 0

IL-2 SC every other day for 6

doses starting

Day 0

Follow-up Study

Days 7, 14, 21,

28, 42, 60, 100

Maximum

Tolerated Dose

Analysis Study

Day 28

Follow-up

Months 6, 12, 24

Conditioning regimen: Fludarabine 25 mg/m2 x 5 days, start day -6, Cyclophosphamide 60 mg/kg x 2 days on day -5 and -4 (if <4

months since transplant, omit day -4 dose).

IL-2 to facilitate NK cell survival and expansion: IL-2 at 6 million units SC beginning Day 0, every other day for 6 total doses.

Acetaminophen and diphenhydramine: Acetaminophen 650 mg PO and diphenhydramine 25 mg PO prior to PNK-007 infusion and

4 hours after PNK-007 and IL-2 SC

RESULTS

Conclusions

• Circulating PNK-007 cells persist between 7 to 28 days (mean = 11 days)

following IV infusion at dose level ≥3x106 cells/kg.

• Persistent PNK cells maintain NK lineage marker expression with subsets

upregulating CD57 and KIR. PNK-007 stain for granzyme B, perforin, IFNg,

but not inhibitory checkpoint molecules PD-1, TIM3, and LAG3.

• Serum analysis demonstrated absence of allo-HLA antibodies in all subjects

• Cy/Flu + PNK-007 reduces AML blasts in the blood between days 0 and 7

post infusion, but increasing blast counts was observed by day 28.

• RR AML patients have poor recovery of neutrophils, lymphocytes, and

myeloid-derived suppressor cell expansion associated with AML blasts.

• Treg are a significant proportion of T cells at day 7. T cell expansion occurs

by day 14 followed by contraction by day 21. PD-1 expression can be high

but is variable by patient.

Discussion

Our data demonstrate the feasibility of monitoring allogeneic PNK-007

and immune correlatives in patients for up to 28 days post infusion. Limited

normal hematopoiesis was observed in conditioned RR AML patients and we

observed presence of immune-suppressed subpopulations. The results of this

study will be used to inform the design of future trials in AML.

Subject 007-1002 PBMC (and bone marrow aspirate when available) was characterized for PNK-007 cell persistence

and extended phenotype characterization. A) Persistent PNK-007 cells were identified by gating on HLA-B27-CD56+

cells. PNK-007 102016 drug substance prior to infusion is shown as a staining control. B) Gated PNK-007 cells from

A) were analyzed for expression of NK lineage and immunomodulatory cell surface markers. C) To measure cytokine

responses, total cells were stimulated 4 hours in the presence of PMA+ionomycin +brefeldin A followed by surface

staining and intracellular staining following cell fixation and permeabilization.

CD57Ki67 Pan-KIR TIM3 PD-1 4-1BBLAG3

PNK 102016

FMO control

day 14 PB

HLA-B27

CD94 CD11a NKp30 NKp46CD56 CD16DNAM-1

PNK 102016 screen PB day 14

BM

day 14 PB day 28 PB

CD

56

PNK 102016FMO controlday 14 BMday 14 PBday 28 PB

A

B

C

A) IL-2 and IL-15 were measured in

AML serum samples by Luminex

(Millipore) relative to normal serum. All

patients are plotted versus post PNK-

007 infusion day. Day 7 IL-2 spike

corresponds to the rhIL-2 dosing

window. B) Alloantibodies against HLA-

A/B/C were tested using FlowPRA flow

cytometry test (OneLambda).

Alloantibody binding is shown as FITC

signal geometric mean versus controls.

CONCLUSIONS

#LB-070

002-1001

006-1002

002-1003

001-1002

007-1002

007-1001

002-1004

001-1003

008-1001

A:

B:

C:

D:

E:

F:

G:

H:

I:

D B

F

AH

E

I

D

F

B

A

E,H,I

DE

B

H

I

F

A

Figure 2. PNK-007 cells further mature in vivo

Figure 3. Elevated IL-2/IL-15 and absence of allo-HLA antibodies in serum

A

Figure 4. AML blast and immune reconstitution post PNK-007 infusion Figure 6. Immune checkpoint expression on T cells post PNK-007 infusion

Peripheral blood characterized by hematology analyzer complete blood count and flow cytometry. Left black) AML

blasts percentages were identified by FACS based upon light scatter, aberrant expression of CD34, CD33, CD45, and

CD56. Center blue) Neutrophils were determined by CBC count. Right green) CD3 T cells were identified by FACS

based upon CD45+CD3+ cells among lymphocytes.

Figure 5. PBMC cell profiling post PNK-007 infusion

Figure 6. Blood T cell recovery and subset distribution post

PNK-007 infusion

CD3+CD4-CD8- NKT CD8 CD4+Foxp3+ CD4+ Tconv

A

B

PBMC were characterized by multiparameter flow cytometry (CD45, CD38, CD11b, CD14, CD19, CD33, CD56, CD3,

HLA-DR, CD34) to distinguish immune cell subsets. Cell percentages were normalized to leukocyte cell counts to

determine immune cell subset blood concentration.

A) CD4 and CD8 T cell subsets shown were normalized for blood concentration as done in Figure 5. Over the 4

weeks post PNK-007, the T cell compartment expands from day 7 to 14. The day 14 sample was unavailable for

subjects 002-1001, 007-1001 and 002-1004. T cells contract by day 21, except in 001-1002 and 007-1001 where

CD8 T cells remain elevated. B) The percentage of each T cell subset among CD3+ T cells is summarized

highlighting the higher proportion of Treg at day 7 and sustained proliferation of CD8 T cells in 001-1002 and 007-

1001 beyond day 14.

Sample permitting, T cell subsets were monitored for proliferation (Ki-67) and expression of PD-1 immune checkpoint.

Total CD4 T cells of effector and regulatory subsets were proliferating at day 14 while all CD8 T cell subsets were

proliferating. While T cell PD-1 expression was upregulated from baseline post PNK-007 injection on CD4 and CD8 T

cell subsets, variability among patients was high with 007-1002 expressing negligible PD-1 at all time points.

N/A N/A

N/A N/A

N/A N/A

N/A N/A

CD

4 T

ce

lls

CD

8 T

ce

lls

001-1002 007-1001 007-1002

bas

eli

ne

Da

y 1

4D

ay 2

1b

as

eli

ne

Da

y 1

4D

ay 2

1

PD-1 Ki-67 PD-1 Ki-67 PD-1 Ki-67

FMO control

Naïve CD4

CD4 Teff + EM

CD4 Foxp3+

FMO control

Naïve CD8

CD8 Teff

CD8 CM + EM

Overview of PNK-001-AML clinical protocol

NK cells among patient peripheral blood mononuclear cells (PBMC) were identified based on the CD45+CD3-CD56+

population shown after excluding debris, dead cells, and doublets. The indicated HLA allele specific antibodies

distinguished PNK-007 from patient endogenous NK cells. Plots highlighted in red indicate the presence of PNK-007

persistence detected based upon negative and positive staining controls.

002-1003 008-1001002-1004007-1002

N/A

N/A

006-1002 001-1002 007-1001

day 0

day 7

day 1

4

N/A

001-1003

Cohort 1 (1x106 PNK cells/kg) Cohort 2 (3x106 PNK cells/kg) Cohort 3 (10x106 PNK cells/kg)

N/A

002-1001

N/A

N/A

N/A

Figure 1. PNK-007 cell persistence in peripheral blood

N/AN/A

CD

56

PNK

HLA-A3

PNK

HLA-A3

PNK

HLA-A2

PNK

HLA-B27

PNK

HLA-B27

subject

HLA-B27

PNK

HLA-B27

PNK

HLA-A3

HLA

allele

day 2

1-2

8

0100

200

300

400

500

600

D a y 2 8

D a y 2 1

D a y 1 4

D a y 7

D a y 0

s c r e e n in g

(- ) c o n tr o l

(+ ) c o n tr o l

H L A -A /B /C

P a n e l R e a c t iv e A n t ib o d y T e s t

F IT C G e o m . M e a n

Perforin TNFa IFNgGranzyme B

PNK 102016

unstim control

day 14 PB

PM

A+

Iono

stim

ula

tion

B

1 1 02

1 1 03

1 1 04

1 1 05

1 1 06

1 1 07

A M L b l a s t c o u n t

D a y p o s t P N K - 0 0 7 i n f u s i o n

ce

ll

s/

mL

b

lo

od

s c r e e n 0 7 1 4 2 1 2 8

N D

1 1 02

1 1 03

1 1 04

1 1 05

1 1 06

1 1 07

n e u t r o p h i l c o u n t

D a y p o s t P N K - 0 0 7 i n f u s i o n

ce

ll

s/

mL

b

lo

od

s c r e e n 0 7 1 4 2 1 2 8

N D