01.2018 | THE SCIENTIST 45CREDIT LINE
N
atural killer (NK) cells, known for
their ability to kill tumor cells, are
promising agents for cell-based
cancer immunotherapies. CRISPR-Cas9
gene editing can be used to effectively
modify the genetic makeup of inducible
pluripotent stem cell (iPSC)-derived
NK cells towards this end, but there is
an urgent need to characterize their
potency and efficacy as a preclinical
cancer immunotherapy. IsoPlexis’ single-
cell proteomics system addresses this
challenge by connecting each immune
cell to the many cytokines it secretes,
revealing correlations to in vivo outcome
across a range of disease areas.The Polyfunctional Strength
Index (PSI) delivers correlative
potency data
The IsoPlexis system identifies which cells
are polyfunctional (i.e., those powerful
cells that secrete multiple cytokines) and
quantitates the cytokine concentrations
within each cell. PSI combines these two
single-cell metrics to effectively identify
highly potent immunotherapies.PSI reveals cell potency of gene-
edited, iPSC-derived NK cells
To study the role of cytokine-inducible
SH2-containing protein (CIS) in human
NK cell anti-tumor activity, IsoPlexis’s
systems analyzed the potency of iPSC-
derived NK cells with the CISH gene
deleted using CRISPR-Cas9 technology.
A 10X higher PSI of the gene-edited
NK cells was found relative to control
wildtype NK cells, driven by increased
antitumor cytokine production (e.g.,
Granzyme B, IFN-γ, MIP-1α, Perforin,
TNF-α). Researcher Dr. Dan Kaufman,
PI of this study, said, “Our studies have
used CRISPR-Cas9 gene editing in
human induced pluripotent stem cells to
produce natural killer cells with improved
antitumor activity. Analysis of these cells
using the IsoPlexis system was veryvaluable to characterize the improved
polyfunctional cytokine response that
plays a key role in improved activity of
these iPSC-derived NK cells.”Increase in PSI of gene-edited
NK cells correlates with improved
in vivo response of the NK cell
therapy
To test CISH-/- human NK cells in vivo,
immunodeficient NSG mice were
inoculated with the human leukemia
cell line, MOLM-13, and split into three
groups: untreated mice, mice treated
with control wildtype NK cells, and mice
treated with the CISH-knockout NK cells.
The study revealed substantial tumor
growth in untreated mice and wildtype
NK treated mice, while tumor growth
was nearly absent in mice receiving theCISH-KO NK treatment. This strongly
suggests both a correlation between in
vitro PSI and in vivo mouse antitumor
activity, as well as therapeutic efficacy.Conclusion
IsoPlexis’ single-cell system improves
engineered immune-cell therapy R&D, by
providing metrics such as PSI, indicative
of improved potency of gene-edited cells,
and potentially correlating to in vivo
efficacy of engineered cell therapies.Improving Preclinical Discovery of CRISPR-
Engineered Immune Cell Therapies
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PSI reveals cell potency, post gene edits, which correlates with in vivo response. (A) CISH-/- knock-out
cells showed increased polyfunctional cell subsets (left) and 10x higher PSI (right) compared to the wild
type samples. These results indicate that CIS plays a key role in regulating NK cell activation-induced exhaus-
tion and that Notch activation prevents this exhaustion and enables production of functionally hyperactive
NK cells. (B) CISH-/- iPSC-NK demonstrates better anti-tumor activity in vivo. NSG mice were inoculated
with the human leukemia cell line Molm13; mice were subsequently left untreated or were treated with either
WT-iPSC-NK or KO-iPSC-NK cells. Thirty-five days after inoculation, two of four untreated mice had died and
the other two showed significant tumor load, WT-iPSC-NK mice showed substantial tumor growth, while in
CISH KO-iPSC-NK mice tumor growth was nearly absent, consistent with the 10x higher in vitro PSI of CISH
KO-iPSC-NK cells. Reference: Zhu et al, ASH 2018.