Download NK cells Expansion and Activation for Cancer Immunotherapy

NK cells Expansion and Activation for Cancer Immunotherapy
Arkadi Yakirevitch*, Naama Zabari, Jacob Schachter, Michal J. Besser
Ella Institute for Melanoma Research and Department of Otorhinolaryngology-H&NS*
Sheba Medical Center, Tel-Hashomer, Israel
Background
Background
¾Natural
¾Natural Killer
Killer (NK)
(NK) cells
cells are
are large
large granular
granular lymphocytes
lymphocytes of
of the
the innate
innate immune
immune system,
system, comprising
comprising ~
~ 15%
15% of
of all
all circulating
circulating lymphocytes
lymphocytes
¾In
¾In contrast
contrast to
to BB and
and TT cells,
cells, they
they do
do not
not rearrange
rearrange the
the TT cell
cell receptor
receptor or
or the
the immunoglobulin
immunoglobulin genes
genes
¾NK
¾NK cell
cell killing
killing does
does not
not require
require that
that the
the tumor
tumor cells
cells express
express intact
intact self-MHC
self-MHC antigens.
antigens. In
In addition,
addition, NK
NK cells
cells are
are able
able to
to be
be catalytic
catalytic without
without
prior
prior sensitization.
sensitization. Further
Further activation
activation of
of NK
NK cells
cells by
by cytokines
cytokines can
can increase
increase killing
killing activity
activity and
and broaden
broaden the
the spectrum
spectrum of
of malignant
malignant cells
cells that
that
are
are killed.
killed. Activated
Activated NK
NK cells
cells kill
kill target
target cells
cells through
through various
various mechanisms,
mechanisms, predominantly
predominantly through
through the
the release
release of
of cytolytic
cytolytic enzymes
enzymes
¾Donor
¾Donor NK
NK cell
cell infusion
infusion can
can provide
provide potent
potent antitumour,
antitumour, antiviral
antiviral and
and engraftment-facilitating
engraftment-facilitating activity,
activity, without
without causing
causing GVHD
GVHD
¾In
¾In order
order to
to avoid
avoid GVHD
GVHD in
in clinical
clinical application,
application, alloreactive
alloreactive NK
NK cells
cells need
need to
to be
be expanded
expanded in
in extensive
extensive TT cell-depleted
cell-depleted setting
setting
Materials
Materials and
and methods
methods
Study
Study Purpose
Purpose
To
To define
define the
the best
best algorithm
algorithm of
of the
the isolation,
isolation,
activation
activation and
and expansion
expansion of
of the
the NK
NK cells
cells for
for clinical
clinical
¾Cell
¾Cell source:
source: human
human peripheral
peripheral blood
blood mononuclear
mononuclear cells
cells
¾Isolation:
¾Isolation: CD3
CD3 depletion
depletion using
using magnetic
magnetic microbeads
microbeads cell
cell sorting
sorting
use
use under
under good
good manufacturing
manufacturing practice
practice conditions
conditions
¾Purity
¾Purity estimation:
estimation: flow
flow cytometry
cytometry analysis
analysis
Exp.
No.
Checked parameters
1
Materials and Methods
Results
Various conditions of
incubation
Medium types: X-VIVO, CellGro,
AIM-V, DMEM
IL-2 concentration 62.5-1000 IU/ml
PHA concentration: 1-10 μg/ml
NK: feeder cell proportion:
1:1, 1:10, 1:10 (2 rounds)
OKT-3 +/Incubation of fresh and
Fresh and frozen NK and feeder
frozen NK and feeder cells
cells of the same donor
Donor-to-donor variations in Fresh NK cells of two donors
incubation results
T cells depletion
CD3 depletion of PBMCs on 2X LS
columns--> 1XLD.
Number of feeder rounds
1 or 2 rounds (the 2nd on the 7th
day)
during expansion
2
3
4
5
Effect of feeder
preincubation with OKT3
Preincubation with OKT3
Preincubation with OKT3 ->
washing
Direct adding of OKT3 to culture
PBMC separation on 2 followed columns has a
benefit in context of T cell contamination/NK purity
Second round of feeders results in high purity
Growth Curves, Experiment 5
250
400
6
200
NK Number, X10
NK Fold Expansion
No donor dependence of expansion rates
Preincubation with OKT3 has a positive effect on
both purity and expansion of the NK population
Experiment 5, Day 19
150
100
50
0
20%
High purity of NK cells with use of CellGro
Low purity of NK cells with use of AIM-V
Unsatisfying expansion rates with use of IL-2 or
PHA alone
Optimal IL-2 concentration 500 IU/ml
Optimal PHA concentration 5 μg/ml
2nd round of feeder seems of benefit
Better expansion rates of the fresh NK cells
40%
60%
80%
100%
Purity
300
200
100
0
0
Day
13
19
Medium
Feeders
OKT3
AIM-V
X10x10
direct addition
X-VIVO
X10x10
direct addition
X-VIVO
x10x10
feeder preincubation with OKT3
X-VIVO
x10x10
feeder preincubation with OKT3 -> wash