immune monitoring of pnk-007, an allogeneic, off the shelf ......pnk-007 102016 drug substance prior...
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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
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Days in Culture
Typical Growth Culture
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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
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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.
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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
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006-1002 001-1002 007-1001
day 0
day 7
day 1
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001-1003
Cohort 1 (1x106 PNK cells/kg) Cohort 2 (3x106 PNK cells/kg) Cohort 3 (10x106 PNK cells/kg)
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002-1001
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Figure 1. PNK-007 cell persistence in peripheral blood
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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
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