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Jeremiah L. Oyer1, Thomas Dieffenthaller1, Namita Varudkar1, Raquel Arthuzo1, Griffith D. Parks1, Robert Y. Igarashi2, Robert H.E. Friesen2, Alicja J. Copik1
2020 ASGCT Annual Meeting – Abstract #765
1Burnett School of Biomedical Sciences, University of Central Florida, Orlando FL, 2Kiadis Pharma, Amsterdam, Netherlands
Email: [email protected]: [email protected]
We previously developed platforms with membrane bound IL-21 (mbIL21) for high scale expansion of hyper-functional NK cells CSTX002 (FC21) is a K562 cell line modified to express mbIL21 and 4-1bbl and used in co-culture as feeder
cells to stimulate NK cell expansion PM21 particles are plasma membrane particles prepared from FC21 cells PM21 particles allows feeder-cell-free NK cell expansion, now used in GMP production of therapeutic K-NK
cells
Both platforms yield ~1,000x NK cell expansion in ~14 days
• High cytotoxicity• High expression of activating receptors• High production of IFNγ
4-1BBL
mbIL214-1BBL
Expansion with FC21 feeder cells1
Expansion with PM21 particles2
mbIL21
FC21 feeder cells
PM21particles
NK cell
Expanded NK cells
1PLoS One 2012 7(1), e302642Cytotherapy 2016 18(5), 653-63
Hypothesis: Stimulation with Fc (fraction crystallizable domain of IgG1) on surface of feeder cells (FC21) or PM21 particles leads to improved expanded NK cells
CD16
Surface of feeder cells or particles
NK cell
Fc stimulated NK cells:• Better expansion rate or
selection• Higher anti-tumor potency• More favorable phenotype
Construction of FC21.Fc cells
mbIL214-1BBL
CSTX002 feeder cells
(FC21)
Fc
Lentiviral transduction
Fc domain
CSTX002.Fc feeder cells
(FC21.Fc)
PM21.Fcparticles Fc domain
Flow cytometry verification of Fc expression on FC21.Fc
FC21feeder cellsNK cell Expanded
FC21-NKcells
NK cell Expanded FC21.Fc-NKcells
Fc domain
FC21.Fc feeder cells
NK cell expansion is improved beyond day 12with FC21.Fc compared to FC21
Comparative testing of NK cell expansion with two different donors
PM21.Fc-NK cells have higher expression of CD16 Other activating receptors (NKG2D, NKp46, NKp30, NKp44) highly expressed
*NK cells gated on CD56+CD16+CD3-
NK cells from 2 unrelated donors were expanded for 14 days with PM21 and PM21.Fc particles and inspected for expression of key activating receptors.
NK Alone
NK+Cetux
NK+A549
NK+Cetux+A5490
50
100
150
[TN
F] (
pg
/mL
)
PM21 NKPM21.Fc-NK
*
*
NK cells expanded with PM21.Fc particles produce higher amounts of TNFαupon antibody and tumor target engagement
IFNγ and TNFα secretion was measured after incubation with Cetuximab (1 µg/mL), A549 lung cancer cells, or A549+Cetuximab (1 µg/mL). NK cells expanded with PM21.Fc secreted significantly higher amount of TNFα in response to A549 and A549+Cetuximab. IFNγ and TNFα secretion was measured using BD Bead Capture Assay.
NK cells expanded with PM21.Fc have higher cytotoxicity and higher antibody dependent cell cytotoxicity (ADCC) at low E:T ratios
Cytotoxicity against lung cancer (A549) cells in a 2-D culture were inspected on the Incucyte platform with and without Cetuximab for both PM21-NK and PM21.Fc-NK cells. Plots A and B show amount of red-labelled A549 tumor cells over time after addition of NK cells without (A) and with 0.1 µg/mL Cetuximab (B). Cytotoxicity after 24 hours with and without Cetuximab (0.1 µg/mL) (C). NK:A549 ratio = 0.3:1
A B C
FC21-N
K
FC21.F
c-NK
0
20
40
60
80
100
0 10 200.0
0.5
1.0
Hours Elapsed
FC21-NK cellsFC21.Fc-NK cells
ADCC killing of lung cancer (A549) tumor spheroids in combination with Cetuximab is enhanced for NK cells expanded with FC21.Fc vs FC21
Cytotoxicity of NK cells with Cetuximab (3 µg/mL) against red-labeled A549 3D spheroids using the IncuCyte instrument.
✱
FC21.Fc-NK cells appear to proliferate better compared to control in the presence of ovarian cancer SKOV3 tumor spheroids
noTr
astu
zum
abw
ithTr
astu
zum
ab
FC21-NKs FC21.Fc-NKs
NK cells were recovered 3 days after co-culture with tumor spheroids andcounted by flow cytometry.
Addition of Fc domain to FC21 further enhances NK cell expansion
NK cells cultured with PM21.Fc particles had increased CD16 expression after 10-14 days in culture compared to PM21 control
Upon re-engagement of CD16 using a humanized antibody against EGFR (Cetuximab) on EGFR+ A549 target cells, NK cells cultured with FC21.Fc or PM21.Fc exhibited higher cytotoxicity and higher TNFα secretion than control FC21- or PM21-expanded NK cells
These results support the potential use of PM21.Fc-NK cells in combination with ADCC competent antibodies as a cancer therapeutic
Jeremiah Oyer: i.p. licensing to and equity in Kiadis Pharma Robert Igarashi: equity in and employee of Kiadis Pharma Robert Friesen: equity in and employee of Kiadis Pharma Alicja Copik: i.p. licensing to and equity in Kiadis Pharma
