Effectors: The End of the
Helen E. Heslop,
and Maksim Mamonkin
Genome editing has emerged as a powerful
tool to enhance the potency of effector cell
therapies and potentially improve their
Stadtmauer et al.
at the University
of Pennsylvania recently reported prelimi-
nary results of a ﬁrst-in-man clinical study
using CRISPR/Cas9-edited therapeutic
T cells in patients with advanced malig-
nancies. In this study, peripheral T cells
were obtained from patients with multiple
myeloma or sarcoma. To allow their engi-
neered T cells to be more resistant to tu-
mor-mediated immune suppression, the
cells were then activated and treated with
a mixture of CRISPR/Cas9 ribonucleopro-
teins (RNPs) to disrupt the PDCD1 gene
that encodes inhibitory receptor PD-1 as
well as TRAC and TRBC genes. TRAC and
TRBC encode constant regions of endoge-
nous TCR aand bchains, respectively. By
eliminating endogenous T cell receptor
(TCR) expression, the authors aimed to in-
crease the expression of the transgenic NY-
ESO-1/LAGE-1-speciﬁc TCR they subse-
quently incorporated by lentiviral transduc-
tion. The resulting NY-ESO-1 TCR-trans-
genic CRISPR triple-edited cells, or “NYCE
cells,”were manufactured for four patients
enrolled in the study, of whom three were
infused. Even though the number of patients
treated with NYCE T cells remains insufﬁ-
cient to draw deﬁnitive conclusions about
the safety of their approach or the biological
role of the mutations introduced, the study
marks an important milestone in the devel-
opment and clinical application of gene-edi-
ted effector cell therapy.
Using recombinant Cas9 complexed with
four distinct single guide RNAs (sgRNAs),
the authors disrupted the TRAC, TRBC,
and PDCD1 genes with efﬁciencies ranging
from 15% (for the TRBC gene) to 45% (for
the TRAC gene). The average lentiviral
NY-ESO1 TCR transduction efﬁciency was
3.1%. Highlighting the inhibitory effect of
endogenous TCR and/or PD-1 expression
on the cytotoxicity of TCR-transgenic
T cells, disruption of these genes modestly
increased in vitro killing of tumor by
NYCE T cells. The selected sgRNAs had
minimal off-target activity, as evaluated us-
ing the iGUIDE method, and the identiﬁed
non-speciﬁc sites were not mapped to known
tumor suppressor genes or oncogenes in
T cells. Of note, chromosomal translocations
that arose from multiple simultaneous dou-
ble-strand breaks in CRISPR-edited T cells
were also detected in these patients. How-
ever, these genome alterations were unlikely
to confer survival or proliferative advantage
to the modiﬁed T cells, as the frequency of
both NY-ESO-1 TCR-positive and -negative
T cells carrying edited genes or transloca-
tions gradually decreased after infusion.
Notably, PD-1-deﬁcient T cells did not
expand or persist better than T cells with un-
altered PD-1 expression, consistent with
previous ﬁndings that loss of PD-1 may
reduce the ability of T cells to establish a
long-lived memory population.
Upon infusion, NYCE T cells expanded and
tracked to tumor sites, stabilizing disease in
two of three patients treated on the protocol.
Although NYCE T cells remained detectable
in circulation for up to 9 months after infu-
sion, at least in one patient with myeloma,
the authors documented downregulation
of NY-ESO-1 and LAGE-1 antigens recog-
nized by the transgenic TCR. Evidence of
antigen escape, which has also been
frequently observed in studies using CAR
T cells for B cell malignancies,
contributed to the limited antitumor activity
of NYCE T cells and may warrant the use of
T cells simultaneously targeting multiple
Despite the presence of pre-existing anti-
bodies to the Cas9 protein from Strepto-
a component of the normal
commensal ﬂora in humans, no reactivation
of Cas9-speciﬁc humoral immunity was
observed after administration of NYCE
T cells. The authors attributed this lack
of response to the minimal presence of
residual Cas9 protein in T cells at the time
of cryopreservation and the transient im-
mune suppression of patients induced by
the lymphodepleting treatment and prior
lymphotoxic therapies. While the authors
did not measure activation of endogenous
Cas9-speciﬁc T cells,
these responses, even
if present, did not blunt the expansion of
gene-edited T cells in vivo. Overall, these
results indicate genome editing of thera-
peutic T cells ex vivo using CRISPR/Cas9-
sgRNA RNP complexes results in minimal
additional immunogenicity, largely due to a
very transient persistence of electroporated
Cas9 protein in rapidly dividing T cells prior
Numerous proof-of-concept reports have
utilized genome-editing tools in T cells to
minimize the risk of graft-versus-host dis-
T cell fratricide,
among other applica-
tions. Further, commercial and academic
entities have conducted several clinical
studies using gene-edited T cells, including
a landmark report in 2014 by Tebas et al.
utilizing zinc-ﬁnger nucleases to create
CCR5-edited T cells resistant to HIV infec-
tion. The latest study by Stadtmauer et al.
is the ﬁrst to report the manufacturing,
safety, and clinical activity of CRISPR/
Cas9-edited effector T cells.
Center for Cell and Gene Therapy, Baylor College of
Medicine, Houston Methodist Hospital and Texas
Children’s Hospital, Houston, TX, USA;
Program in Translational Biology and Molecular
Medicine, Baylor College of Medicine, Houston, TX,
Correspondence: Feiyan Mo, Center for Cell and
Gene Therapy, Baylor College of Medicine, Houston
Methodist Hospital and Texas Children’s Hospital,
Houston, TX, USA.
Molecular Therapy Vol. 28 No 4 April 2020 ª2020 The American Society of Gene and Cell Therapy. 995
Recent technological advances could further
improve the efﬁciency and precision of
multiplexed genome editing and minimize
its side effects. Examples include employing
high-ﬁdelity versions of Cas9 with reduced
utilizing base editing
in lieu of non-homologous end-joining
(NHEJ) repair to minimize CRISPR-induced
double-strand breaks and the resulting
ing in desired constructs to replace endoge-
nous genes via homology-directed recombi-
With the ﬁrst clinical
testing of CRISPR/Cas9-edited therapeutic
T cells, this pioneering study of NYCE
T cells is perhaps “the end of the beginning”
of the era of gene-edited immune effectors.
F.M., H.E.H., and M.M. wrote the
H.E.H. is a founder with equity in Allovir
and Marker Therapeutics; has served on
advisory boards for Gilead, Novartis, Tessa
Therapeutics, Kiadis, and PACT Pharma;
and receives research support from Tessa
Therapeutics and Kuur Therapeutics.
The National Cancer Institute
(P50CA126752), the SU2C/AACR (604817)
Meg Vosburg T cell Lymphoma Dream
Team, and the Leukemia and Lymphoma So-
ciety supported the authors.
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