RESEARCH ARTICLES

Cite as: E. A. Stadtmauer et al., Science

10.1126/science.aba7365 (2020).

CRISPR-engineered T cells in patients with

refractory cancer
Edward A. Stadtmauer,1,2*† Joseph A. Fraietta,2,3,4,5* Megan M. Davis,5,6 Adam D. Cohen,1,2 Kristy L. Weber,2,7
Eric Lancaster,8 Patricia A. Mangan,1 Irina Kulikovskaya,5 Minnal Gupta,5 Fang Chen,5 Lifeng Tian,5
Vanessa E. Gonzalez,5 Jun Xu,5 In-young Jung,4,5 J. Joseph Melenhorst,3,5,6 Gabriela Plesa,5 Joanne Shea,5
Tina Matlawski,5 Amanda Cervini,5 Avery L. Gaymon,5 Stephanie Desjardins,5 Anne Lamontagne,5
January Salas-Mckee,5 Andrew Fesnak,5,6 Donald L. Siegel,5,6 Bruce L. Levine,5,6 Julie K. Jadlowsky,5
Regina M. Young,5 Anne Chew,5 Wei-Ting Hwang,9 Elizabeth O. Hexner,1,2 Beatriz M. Carreno,3,5,6
Christopher L. Nobles,4 Frederic D. Bushman,4 Kevin R. Parker,10 Yanyan Qi,11 Ansuman T. Satpathy,10,11
Howard Y. Chang,10,12 Yangbing Zhao,5,6 Simon F. Lacey,5,6* Carl H. June2,3,5,6*†

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1
Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 2Abramson Cancer Center,
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 3Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA, USA. 4Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 5Center for Cellular
Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 6Department of Pathology and Laboratory Medicine, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA, USA. 7Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia,
PA, USA. 8Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 9Department of Biostatistics, Epidemiology and
Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 10Center for Personal Dynamic Regulomes, Stanford University School of
Medicine, Stanford, CA, USA. 11Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. 12Howard Hughes Medical Institute, Stanford University
School of Medicine, Stanford, CA, USA.
*These authors contributed equally to this work. †Corresponding author. Email: [email protected] (E.A.S.); [email protected] (C.H.J.)

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight
cancer. We report a first-in-human phase I clinical trial to test the safety and feasibility of multiplex
CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the
endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC) were deleted in T cells to reduce
TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1).
Removal of a third gene encoding PD-1 (PDCD1), was performed to improve anti-tumor immunity. Adoptive
transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic
loci. Though chromosomal translocations were detected, the frequency decreased over time. Modified
T cells persisted for up to 9 months suggesting that immunogenicity is minimal under these conditions and
demonstrating the feasibility of CRISPR gene-editing for cancer immunotherapy.

Gene editing offers the potential to correct DNA mutations, to HIV infection (3, 4). The incorporation of multiple guide
and may offer promise to treat or eliminate countless human sequences in CRISPR–Cas9 permits, in principle, multiplex
genetic diseases. The goal of gene editing is to change the genome engineering at several sites within a mammalian ge-
DNA of cells with single base pair precision. The principle nome (5–9). The ability of CRISPR to facilitate efficient mul-
was first demonstrated in mammalian cells when it was tiplex genome editing has greatly expanded the scope of
shown that expression of a rare cutting endonuclease to cre- possible targeted genetic manipulations, enabling new possi-
ate double-strand DNA breaks resulted in repair by homolo- bilities such as simultaneous deletion or insertion of multiple
gous and non-homologous recombination (1). A variety of DNA sequences in a single round of mutagenesis. The pro-
engineered nucleases were then developed to increase effi- spect of using CRISPR-engineering to treat a host of diseases
ciency and enable potential therapeutic applications, includ- such as inherited blood disorders and blindness is moving
ing zinc finger nucleases, homing endonucleases, closer to reality.
transcription activator-like effector nucleases and CRISPR– Recent advances in CRISPR-Cas9 technology have also
Cas9 (clustered regularly interspaced short palindromic re- permitted efficient DNA modifications in human T cells,
peats associated with Cas9 endonuclease) (2). The first pilot which holds great promise for enhancing the efficacy of can-
human trials using genome editing were conducted in pa- cer therapy. T lymphocytes are specialized immune cells that
tients with HIV/AIDS and targeted the white blood cell pro- are largely at the core of the modern day cancer immunother-
tein CCR5, with the goal of mutating the gene by non- apy revolution. The T cell receptor (TCR) complex is located
homologous recombination and thereby inducing resistance on the surface of T cells, and is central for initiating

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successful anti-tumor responses by recognizing foreign anti- Results
gens/peptides bound to MHC-molecules. One of the most Clinical protocol
promising areas of cancer immunotherapy involves adoptive The phase I human trial (ClinicalTrials.gov NCT03399448)
cell therapy, where the patient’s own T cells are genetically was designed to assess the safety and feasibility of infusing
engineered to express a synthetic (transgenic) TCR that can autologous NY-ESO-1 TCR engineered T cells in patients after
specifically detect and kill tumor cells. Recent studies have CRISPR-Cas9 editing of the TRAC, TRBC and PDCD1 loci.
shown safety and promising efficacy of such adoptive T cell During the manufacturing process, cells were taken out of the
transfer approaches using transgenic TCRs specific for the cancer patient, engineered and then infused back into the in-
immunogenic NY-ESO-1 tumor antigen in patients with mye- dividual. The genetically-engineered T cell product was
loma, melanoma and sarcoma (10–12). One limitation of this termed “NYCE” (NY-ESO-1 transduced CRISPR 3X edited
approach is that the transgenic TCR has been shown to mis- cells) and is referred to as NYCE hereafter. During clinical
pair and/or compete for expression with the alpha and beta development of the protocol, we elected to use a TCR rather
chains of the endogenous TCR (13–15). Mispairing of the ther- than a CAR because the incidence of cytokine release syn-
apeutic TCR alpha and beta chains with endogenous alpha drome is generally less prevalent using TCRs (11). In princi-
and beta chains reduces therapeutic TCR cell surface expres- ple, this allowed a more discriminating assessment of

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sion and potentially generates self-reactive TCRs. whether gene editing with Cas9 was potentially immuno-
A further shortcoming of adoptively transferred T cells genic or toxic when compared with the baseline low level of
has been the induction of T cell dysfunction or exhaustion adverse events observed in our previous clinical trial target-
leading to reduced efficacy (16). PD-1–deficient allogeneic ing NY-ESO-1 with transgenic TCRs (11). The autologous
mouse T cells with transgenic TCRs showed enhanced re- T cells were engineered by lentiviral transduction to express
sponses to alloantigens, indicating that the PD-1 protein on an HLA-A2*0201-restricted TCR-specific for the
T cells plays a negative regulatory role in antigen responses SLLMWITQC peptide in NY-ESO-1 and LAGE-1. The manu-
that are likely to be cell intrinsic (17). The adoptive transfer facturing process, vector design and clinical protocol for
of PD-1 deficient T cells in mice with chronic lymphocytic NYCE T cells are described in the materials and methods sec-
choriomeningitis virus infection initially leads to enhanced tion and are depicted schematically (figs. S1 and S2). Of the
cytotoxicity and later to enhanced accumulation of terminally six patients that were initially enrolled, four patients had
differentiated T cells (18). Antibody blockade of PD-1, or dis- suc-cessfully engineered T cells that were subjected to
ruption/knockdown of the gene encoding PD-1 (i.e., PDCD1), detailed release criteria testing as specified in the FDA
improved chimeric antigen receptor (CAR) or TCR T cell-me- accepted In-vestigational New Drug (IND) application
diated killing of tumor cells in vitro and enhanced clearance (table S1). See fig. S3 for the consort diagram. Of the four
of PD-L1+ tumor xenografts in vivo (19–23). In preclinical patients with cell products available, one patient assigned
studies, we and others found that CRISPR–Cas9-mediated with unique patient number (UPN) 27 experienced rapid
disruption of PDCD1 in human T cells transduced with a CAR clinical progression and was no longer eligible for infusion
increased anti-tumor efficacy in tumor xenografts (24–26). due to the inability to meet protocol mandated safety criteria
Adoptive transfer of transgenic TCR T cells specific for the (see supplementary mate-rials). Of the three patients that
cancer antigen NY-ESO-1, in combination with a monoclonal were infused with CRISPR-Cas9 engineered T cells, two
antibody targeting PD-1, enhanced antitumor efficacy in mice patients had refractory advanced myeloma and one patient
(27). We therefore designed a first-in-human, phase I human had a refractory metastatic sar-coma not responding to
clinical trial to test the safety and feasibility of multiplex multiple prior therapies (Table 1). The patients were given
CRISPR-Cas9 genome editing for a synthetic biology cancer lymphodepleting chemotherapy with cyclophosphamide and
immunotherapy application. We chose to target endogenous fludarabine on days -5 to -3 (i.e., prior to administration with
TRAC, TRBC and PDCD1 on T cells to increase the safety and CRISPR-Cas9 engineered T cells) and a single infusion of 1
efficacy profile of NY-ESO-1 TCR-expressing engineered cells. × 108 manufactured CRISPR-Cas9 engi-neered T cells per
In principle, this strategy allowed us to increase exogenous kg on day 0 of the protocol (fig. S2). No cytokines were
TCR expression and reduce the potential for mixed heterodi- administered to the patients.
mer formation (i.e., by deleting the alpha and beta TCR do-
main genes TRAC and TRBC respectively), and to limit the Characteristics of infused CRISPR-Cas9-
development of T cell exhaustion which can be triggered by engineered T cell products
the checkpoint ligands PD-L1 and PD-L2 (i.e., by deleting The T cell product was manufactured by electroporation of
PDCD1). ribonucleoprotein complexes (RNP) comprising recombinant
Cas9 loaded with equimolar mixtures of sgRNA for
TRAC, TRBC and PDCD1 followed by lentiviral
transduction of the transgenic TCR (Fig. 1A). All
products were expanded to
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>1 × 1010 T cells by the time of harvest (Fig. 1B). The trans- steady state level of engineered cells was lowest in patient
genic TCR could be detected by flow cytometric staining for UPN35, who also had the lowest transduction efficiency
Vβ8.1 or dextramer staining, ranging from 2 – 7% of T cells in (table S2). The persistence of the transduced cells is remark-
the final product (Fig. 1C). The frequency of editing as deter- ably stable from three to nine months after infusion, varying
mined by digital PCR varied according to the sgRNA and was from 5 to 50 cells per μl of blood (Fig. 3B). Using a subject-
approximately 45% for TRAC, 15% for TRBC and 20% for specific piecewise linear model, the decay half-life in days of
PDCD1 (Fig. 1D). Final product transduction efficiency, the transduced cells was 20.3, 121.8 and 293.5 for UPN07,
CD4:CD8 ratio, and dosing are shown in table S2. UPN35, and UPN39. The average decay half-life was 83.9 days
The potency of the final engineered T cells were assessed (15-153, 95% CI) for the three subjects as estimated by a piece-
by co-culture with HLA-A2+ tumor cells engineered to express wise linear mixed effects model that assumes cells decay lin-
NY-ESO-1 (Fig. 2A). The engineered T cells had potent anti- early from day 14 post-expansion and random effects to allow
gen-specific cytotoxicity over a wide range of effector to tar- varying level of expansion (or peak values) across subjects.
get cell ratios. Interestingly, the cells treated with CRISPR- The stable engraftment of our engineered T cells is remarka-
Cas9 were more cytotoxic than control cells transduced with bly different from previously reported trials with NY-ESO-1
the TCR but electroporated without CRISPR-Cas9 (i.e., cells engineered T cells, where the half-life of the cells in blood was

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that retained endogenous TCR). This is consistent with pre- ~1 week (11, 32, 33). Biopsy specimens of bone marrow in the
vious findings in mouse T cells, when a transgenic TCR was myeloma patients and tumor in the sarcoma patient demon-
inserted into the endogenous locus, ablating expression of the strated trafficking of the engineered T cells to the tumor in
endogenous TCR (15). Further studies will be required to de- all three patients at levels approaching those in the blood
termine if PD-1 knockout contributes to the increased po- compartment (Fig. 3A).
tency afforded by knockout of the endogenous TCR. To determine the engraftment frequency of the CRISPR-
We developed a sensitive immunoassay for detection of Cas9 gene-edited cells, we initially used chip-based digital
Streptococcus pyogenes Cas9 protein and quantified Cas9 PCR. With this assay, engraftment of cells with editing at the
early in the manufacturing process, showing declining levels TRAC and PDCD1 locus was evident in all three patients (Fig.
that were <0.75 fg/cell in the harvested final product (Fig. 3C). There was sustained persistence of TRAC and PDCD1 ed-
2C). Using a competitive fluorescence ELISA screen, we its in patient UPN39 and UPN07 at frequencies of 5 to 10% of
found that healthy donors have humoral reactivity to Cas9 in circulating peripheral blood mononuclear cells (PBMC),
serum (Fig. 2D) and T cells (Fig. 2E), confirming previous re- while TRBC edited cells were lowest in frequency and only
ports (28–30). Interestingly, we found that the three patients transiently detected. The low-level engraftment of TRBC ed-
tested at a variety of time points after infusion of the engi- ited cells is likely related to the observation that this locus
neered T cells did not develop humoral responses to Cas9. had the lowest level of editing efficiency in our preclinical
The lack of immunization to Cas9 is consistent with the ex- studies (25), and in the harvested products (Fig. 1D).
tended persistence of the infused cells (Fig. 3) and could be a
consequence of the low content of Cas9 in the infused prod- Analysis of the fidelity of CRISPR-Cas9 genome editing
uct and/or to the immunodeficiency in the patients due to On-and off-target editing efficiency was assessed in the NYCE
their extensive previous treatment histories (Table 1). cells at the end of product manufacturing. Details of the anal-
ysis for UPN07 are shown as an example in Fig. 4, with de-
Engraftment and persistence of infused CRISPR-Cas9- tailed analysis of the other three manufactured products
engineered T cells in cancer patients shown in the table S3. The average on-target CRISPR-Cas9
Three patients with advanced, refractory cancer were given editing efficiency for all engineered T cell products for each
infusions of the CRISPR-Cas9 engineered T cells. The infu- target is shown in Table 3. We used iGUIDE (34), a modifica-
sions were well-tolerated with no serious adverse events tion of the GUIDE-seq method (35), to analyze the Cas9-me-
(Table 2); importantly there were no cases of cytokine release diated cleavage specificity. A complication of assays to assess
syndrome, which is a potentially life threatening systemic in- repair by non-homologous end joining (NHEJ) is that DNA
flammatory response that has been associated with cancer double-strand breaks are formed spontaneously during cell
immunotherapies (31). All three patients were infused with division at high rates in the absence of added nucleases (36),
1 × 108 cells/kg, and due to the considerable variation in TCR which can increase the background in assays of off-target
transduction efficiencies (table S2), the absolute number of cleavage. The distribution of on-target and off-target cleavage
infused engineered T cells ranged from 6.0 × 107 to 7.1 × 108 is expected to vary for the three sgRNAs that were used in the
cells. Despite the variation in engineered cells, there were manufacturing process (fig. S1A). Of the three sgRNA, there
high peak levels and sustained persistence of the engineered were more off-target mutations identified for TRBC than for
cells in the blood of all three patients (Fig. 3A). The peak and the other loci (Fig. 4C and figs. S4 and S5). The sgRNA for

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PDCD1 was the most specific, as very few off-target edits were declined in frequency until cell harvest. The translocations
identified in over 7000 sites of cleavage, and there were very and the TRBC1:TRBC2 deletion were evident in the three pa-
few off-target reads identified at the TRAC1 and TRAC2 loci tients between 10 days after infusion and 30 to 170 days after
(Fig. 4C). infusion (Fig. 5B). However, the rearrangements declined in
The genomic localization of identified DNA cleavage sites frequency in vivo suggesting that they conferred no evidence
was as expected, given the chromosomal location of the three of a growth advantage over many generations of expansion
targeted genes on chromosomes 2, 7 and 14 (Fig. 4A). The dis- in the patients on this trial (Fig. 3, A and B). At day 30, 150
tribution of the incorporation of the dsODN label around on- and 170 in patients UPN07, UPN35 and UPN39, chromosomal
target sites, based on pileups within a window of 100 base translocations were at the limits of detection or not detected
pairs, is shown in Fig. 4B and fig. S4. While most mutations for all rearrangements except for the 9.3 kB deletion for
were on target, there were off-target mutations identified TRBC1:TRBC2.
(Fig. 4C and fig. S5). For the TRAC sgRNA, there were low
abundance mutations within the transcriptional unit of Single-cell RNA sequencing analysis reveals evolution
CLIC2 (chloride intracellular channel 2); however disruption of CRISPR-Cas9 engineered NYCE cells
of CLIC2 in T cells is not expected to have negative conse- We used single cell RNA sequencing (scRNAseq) to compre-

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quences since it is not reported to be expressed in T cells. For hensively characterize the transcriptomic phenotype of the
the TRBC sgRNA, off-target edits were identified in genes en- NYCE T cells and their evolution over time in patient UPN39
coding a transcriptional regulator (ZNF609) and a long inter- (fig. S7). UPN39 was chosen because they had the highest
genic non-protein coding RNA (LINC00377) (table S3). In level of cell engraftment and because this patient had evi-
addition to the above post-hoc investigations of multiplex ed- dence of tumor regression. CRISPR-Cas9 engineered T cells
iting specificity, all products were shown not to have cellular were infused to patient UPN39 and recovered after infusion
transformation by virtue of the absence of long-term growth from the blood on day 10 (D10) and at ~4 months (day, D113)
prior to infusion (table S1). and were analyzed by scRNAseq, as described in the materi-
als and methods. For each sample (infusion product, D10 and
Detection of chromosomal translocations in CRISPR- D113), T cells were sorted based on expression of CD4 or CD8
Cas9 engineered T cells and processed using droplet-based 5′ scRNAseq. From the
In addition to the above detection of repair of double-strand gene expression libraries, PCR was used to further amplify
DNA breaks by NHEJ, on-target mutagenesis by engineered cellular cDNA corresponding to the NY-ESO-1 TCR transgene,
nucleases can result in deletions, duplications, inversions, as well as TRAC, TRBC and PDCD1 target sequences, allowing
and translocations, and can also lead to complex chromoso- us to genotype single cells as wild-type or mutant. In the in-
mal rearrangements under some conditions (37). CRISPR- fusion product, cells were identified that contained muta-
Cas9 has been used to intentionally create oncogenic chro- tions in all three target sequences (Fig. 6, A and B). The most
mosomal rearrangements (38). In preclinical studies with hu- commonly mutated gene was TRAC. Approximately 30% of
man T cells, simultaneous gene editing of TRAC and CD52 cells had no mutations identified, while ~40% had 1 muta-
using transcription activator-like effector nucleases tion, and ~20% and ~10% of the T cells in the manufactured
(TALENs) led to translocations that were detected at frequen- product were double-mutated and triple-mutated respec-
cies of 10−4 to 10−2 (39). In a subsequent clinical report using tively at the target sequences. Of the transgenic TCR+ cells in
dual-gene editing with TALENs, chromosomal rearrange- the infusion product, monogenic mutations were less fre-
ments were observed in 4% of infused cells (40). In order to quent than di-genic and tri-genic mutations (Fig. 6A). Single-
study the safety and genotoxicity of multiplex CRISPR-Cas9 cell genotyping of UPN39 cells at 10 days and 4 months after
genome editing on three chromosomes, we employed strin- infusion showed a decline in the frequency of gene-edited
gent release criteria of the manufactured cells and assays to T cells from the levels in the infusion product, and this de-
detect translocations (fig. S6). We developed and qualified cline occurred regardless of whether the cells were trans-
qPCR assays to quantify the 12 potential translocations that duced with the NY-ESO-1 TCR (Fig. 6C). The frequency of
could occur with the simultaneous editing of four loci: TRAC, gene-edited cells was quite stable between day 10 and
TRBC1, TRBC2, PDCD1 (see materials and methods). We ob- 4 months post-infusion, and remarkably, approximately 40%
served translocations in all manufactured products, however of the peripheral blood circulating T cells in this patient
the translocations were at the limit of detection for the assay 4 months after infusion were mutated at any one of the tar-
in patient UPN39 (Fig. 5A). TRBC1:TRBC2 was the most geted genes (Fig. 6, B and C, and table S4).
abundant rearrangement (Fig. 5A), resulting in a 9.3 kB dele- Of particular interest is the frequency and evolution of
tion (supplementary materials). The deletion and transloca- PD-1 deficient T cells due to the previous mention that ge-
tions peaked on days 5 to 7 of manufacturing and then netic disruption of PDCD1 in CAR and TCR T cells enhances

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antitumor efficacy in preclinical models (19, 21–24). We found engineered T cells (Fig. 7D).
that ~25% of the T cells expressing the NY-ESO-1 TCR in the
infusion product had mutations in the PDCD1 locus (fig. S8). Discussion
It is interesting that the frequency of cells with edits in the Our phase I first-in-human pilot study demonstrates the ini-
PDCD1 locus decreased to ~5% of the cells expressing the tial safety and feasibility of multiplex CRISPR-Cas9 T cell hu-
transgenic TCR at 4 months after infusion. This would be man genome engineering in patients with advanced,
consistent with mouse studies of chronic infection where refractory cancer. In one patient analyzed at depth, a fre-
PD-1 deficient T cells are less able to establish memory (18). quency of 30% of di-genic and tri-genic editing was achieved
Figure 6D shows the distribution of engineered T cells in in the infused cell population, and 20% of the TCR transgenic
patients expressing the NY-ESO-1 TCR transgene over time, T cells in circulation 4 months later had persisting di-genic
as they evolved from the infusion product at baseline and and tri-genic edits. We chose to redirect specificity of the
then again at 4 months in vivo. In the heat map (Fig. 6E), the fT cells with a T cell receptor, rather than a chimeric antigen
most differentially expressed genes in the cells expressing the receptor, in order to avoid the chimeric antigen receptor-as-
NY-ESO-1 transcript at the various time points are shown in sociated potential toxicities such as cytokine release syn-
table S5. It is notable that this patient had increases in ex- drome (31). This provided a lower baseline toxicity profile,

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pression of genes associated with central memory (IL7R, thus enhancing the ability to detect toxicity specifically asso-
TCF7) over time (Fig. 6, D and E, and table S4). This is in ciated with the CRISPR-Cas9 engineering process. We ob-
marked contrast to the recently published results with NY- served mild toxicity and most of the adverse events were
ESO-1 T cells in the absence of genome editing, where the in- attributed to the lymphodepleting chemotherapy. We note
fused transgenic T cells evolved to a terminally differentiated that while the initial clinical results have acceptable safety,
phenotype and displayed characteristics of T cell exhaustion experience with more patients given infusions of CRISPR-
in cancer patients (12). engineered T cells with higher editing efficiencies, and longer
observation after infusion, will be required to fully assess the
Clinical observations safety of this approach.
The clinical course of the three infused cancer patients is Our large-scale product manufacturing process resulted
shown in Fig. 7 (and described in the supplementary materi- in gene editing efficiencies similar to our preclinical studies
als and methods). No patient experienced cytokine release (24). A surprising finding was the high-level engraftment and
syndrome or overt side effects attributed to the cell infusion long-term persistence of the infused CRISPR-Cas9 engi-
(table S5). The best clinical responses were stable disease in neered T cells. In previous clinical studies testing adoptively
two patients. UPN39 had a mixed response with a ~50% de- transferred NY-ESO-1 transgenic T cells, the engrafted cells
crease in a large abdominal mass that was sustained for four and had an initial decay half-life of approximately 1 week (10–
months (Fig. 7D), although other lesions progressed. As of De- 12). The explanation for the extended survival that we ob-
cember 2019, all patients have progressed: two are receiving served remains to be determined, and could include the edit-
other therapies and UPN07 died from progressive myeloma. ing of the endogenous TCR, PD-1 and/or the choice of the
Biopsies of bone marrow and tumor showed trafficking of TCR and vector design.
the NYCE engineered T cells to the sites of tumor in all three The use of scRNAseq technology permitted the analysis of
patients (Fig. 3A). It is interesting to note that even though the transcriptome of the infused NY-ESO-1 specific T cells
the tumor biopsies revealed residual tumor, in both patients (i.e., CRISPR-Cas9 engineered T cells) at baseline and for up
with myeloma there was a reduction in the target antigens to four months in vivo. The results shown for UPN39 revealed
NY-ESO-1 and/or LAGE-1 (fig. S9). The reduction of target an- that the infused cells evolved to a state consistent with cen-
tigen was transient in patient UPN07 and persistent in pa- tral memory. These results are in contrast to a recent study
tient UPN35. This result is consistent with an on-target effect where the infused NY-ESO-1 T cells evolved to a state con-
of the infused cells, likely resulting in tumor editing (41). sistent with T cell exhaustion (12). A limitation of our in vivo
To determine whether the NYCE cells retained antitumor single cell analysis is that for purposes of feasibility, it is lim-
activity after infusion, samples of blood obtained from pa- ited to the one patient who had the highest level of engraft-
tients 3 to 9 months after infusion were expanded in culture ment. Another limitation is that we were not able to compare
in the presence of NY ESO-1 peptide and assessed for cytotox- the transcriptional state of the modified cells in the tumor
icity against tumor cells (Fig. 7E and fig. S10). Antigen-spe- microenvironment to circulating NYCE T cells.
cific cytotoxicity was observed in all three patients. It is Analysis of the manufacturing process in vitro demon-
interesting to note that the most potent anti-tumor cytotoxi- strated monochromosomal translocations and rearrange-
city was observed in UPN39, as UPN39 was the only patient ments, and some of these persisted in vivo. The translocations
to have tumor regression after infusion of the CRISPR-Cas9 were not random in occurrence and occurred most frequently

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between PDCD1:TRAC and TRBC1:TRBC2. The frequency of gRNA was prepared from linearized DNA (Aldevron) using
translocations that we observed with tri-genic editing is sim- Bulk T7 Megascript 5X (Ambion) and purified using RNeasy
ilar to that reported for di-genic editing using transcription Maxi Kit (Qiagen).
activator-like effector nuclease (TALEN)-mediated gene edit-
ing in preclinical and clinical studies, where rearrangements Recombinant Cas9 protein
were detected in approximately 4% of cells (39, 40). It is im- Cas9 recombinant protein derived from Streptococcus
portant to note that healthy individuals often harbor onco- pyogenes was TrueCut Cas9 v2 (Catalogue # A36499, Ther-
genic translocations in B and T cells (42–44). T cells bearing moFisher). Cas9 RNP was made by incubating protein with
translocations can persist for months to years without evi- gRNA at a molar ratio of 1:1 at 25°C for 10 minutes immedi-
dence of pathogenicity (45–47). ately prior to electroporation.
Antagonism of the PD-1:PD-L1 costimulatory pathway can
result in organ-specific and systemic autoimmunity (17, 48). Lentiviral vector manufacturing
PD-1 has been reported to function as a haploinsufficient tu- The 8F TCR recognizes the HLA-A*0201 SLLMWITQC
mor suppressor in mouse T cells (49). Our patients have had epitope on NY-ESO-1 and LAGE-1. The 8F TCR was isolated
engraftment with PD-1 deficient T cells and to date, there is from a T cell clone obtained from patient after vaccination

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no evidence of autoimmunity or T cell genotoxicity. with NY-ESO-1 peptide. The TCR sequences were cloned into
In conclusion, our phase I human pilot study has con- a transfer plasmid that contains the EF-1α promoter, a cPPT
firmed that multiplex CRISPR-Cas9 editing of the human ge- sequence, a rev response element and a woodchuck hepatitis
nome is possible at clinical scale. We note that while the virus posttranscriptional regulatory element (WPRE) as
initial clinical results are safe, experience with more patients shown in fig. S1B. Plasmid DNA was manufactured at Pure-
given infusions with higher editing efficiencies and longer syn, Inc. Lentiviral vector was produced at the University of
observation after infusion will be required to fully assess the Pennsylvania Center for Advanced Retinal and Ocular Ther-
safety of this approach. The potential rejection of infused apeutics using transient transfection with four plasmids ex-
cells due to pre-existing immune responses to Cas9 (28, 29) pressing the transfer vector, Rev, VSV-G and gag/pol, in 293T
does not appear to be a barrier to the application of this cells.
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3 January 2020; accepted 28 January 2020

Published online 6 February 2020
10.1126/science.aba7365

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Fig. 1. Feasibility of CRISPR-Cas9 NYCE T cell engineering. (A) Schematic representation of
CRISPR-Cas9 NYCE T cells. (B) Large-scale expansion of NYCE T cells. Autologous T cells were
transfected with Cas9 protein complexed with single guide RNAs (ribonucleoprotein; RNP complex)
against TRAC, TRBC (i.e., endogenous TCR deletion) and PDCD1 (i.e., PD-1 deletion) and
subsequently transduced with a lentiviral vector to express a transgenic NY-ESO-1 cancer-specific
TCR. Cells were expanded in dynamic culture for 8 to 12 days. On the final day of culture, NYCE
T cells were harvested and cryopreserved in infusible medium. The total number of enriched T cells
during culture is plotted for all four subjects (UPN07, UPN27, UPN35 and UPN39). (C) NY-ESO-1
TCR transduction efficiency was determined in harvested infusion products by flow cytometry. Data
are gated on live CD3-expressing and Vβ8.1 or dextramer positive lymphocytes and further gated on
CD4 and CD8 positive cells. (D) The frequencies of TRAC, TRBC and PDCD1 gene-disrupted total
cells in NYCE infusion products were measured using chip-based digital PCR. All data are
representative of at least two independent experiments. Error bars represent mean +/− SEM.

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Fig. 2. Potency and immunogenicity of CRISPR-Cas9 engineered T cells. (A) Cytotoxicity of NYCE T cells co-
cultured with HLA-A*0201 positive Nalm-6 tumor cells engineered to express NY-ESO-1 and luciferase. Patient
T cells transduced with the NY-ESO-1 TCR without CRISPR-Cas9 editing (NY-ESO-1 TCR) and untransduced T cells
with CRISPR-Cas9 editing of TRAC, TRBC and PDCD1 (denoted as here as CRISPR) were included as controls (n = 4
patient T cell infusion products). Asterisks indicate statistical significance determined by paired t-tests between
groups (*P < 0.05). Error bars represent SEM. (B) Levels of soluble interferon-γ produced by patient NYCE T cell
infusion products (denoted as NYCE) following a 24-hour co-culture with anti-CD3/CD28 antibody-coated beads or
NY-ESO-1-expressing Nalm-6 target cells. Patient NY-ESO-1 TCR transduced T cells (NY-ESO-1 TCR) and
untransduced, CRISPR-Cas9 edited T cells (denoted as CRISPR) served as controls. Error bars indicate SEM.
(C) Quantification of residual Cas9 protein in NYCE T cell infusion products in clinical-scale manufacturing is shown
over time. Asterisks depict statistical significance determined by paired t-tests between time points (*P < 0.05).
(D) Results from the fluorescence-based indirect ELISA screen performed to detect antibodies against Cas9 protein
in the sera of three patients treated with NYCE T cells. Each dot represents the amount of anti-Cas9 signals detected
in patient serum prior to T cell infusion (denoted by a vertical black arrow) and at various time points following NYCE
T cell transfer. RFU = relative fluorescent units. (E) Immunoreactive Cas9-specific T cells in baseline patient
leukapheresis samples were detected. Representative flow cytometry plots (left panel) from two patients whose
T cells were positive for interferon-γ in response to Cas9 peptide stimulation. Unstimulated T cells treated with
vehicle alone (dimethyl sulfoxide, DMSO) served as a negative control, while matched T cells stimulated with phorbol
myristate acetate (denoted as PMA)/Ionomycin served as a positive control. Bar graphs (right panel) show the
frequency of ex vivo CD4+ and CD8+ T cells from patients or healthy donor controls (n = 6) that secrete interferon-γ
in response to stimulation with three different Cas9 peptide pools. The background frequency of interferon-γ-
expressing T cells (unstimulated control group, DMSO alone) is subtracted from the values shown in the bar graph.
Error bars depict SD.

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Fig. 3. Sustained in vivo expansion and persistence of CRISPR-Cas9 engineered T cells in
patients. (A) The total number of vector copies per microgram of genomic DNA of the NY-ESO-1 TCR
transgene in the peripheral blood (UPN07, UPN35 and UPN39), bone morrow (UPN07 and UPN35;
multiple myeloma), and tumor (UPN39; sarcoma) is shown pre- and post-NYCE T cell infusion.
(B) Calculated absolute numbers of NY-ESO-1 TCR-expressing T cells per microliter of whole blood
from the time of infusion to various post-infusion time points in the study are shown. The limit of
detection is approximately 2.5 cells/μl of whole blood. (C) Frequencies of CRISPR-Cas9-edited
T cells (TRAC, TRBC and PDCD1 knockout) before and following adoptive cell transfer are depicted.
Error bars indicate SD.

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Fig. 4. Fidelity of CRISPR-Cas9 gene editing. (A) Genomic distribution of oligonucleotide (dsODN)
incorporation sites, which mark locations of double strand breaks. The ring indicates the human
chromosomes aligned end-to-end, plus the mitochondrial chromosome (labeled M). The targeted
cleavage sites are on chromosomes 2, 7, and 14. The frequency of cleavage and subsequent dsODN
incorporation is shown on a log scale on each ring (pooled over 10 Mb windows). The purple inner
most ring plots all alignments identified. The green ring shows “pile ups” of three or more overlapping
sequences; the blue ring shows alignments extending along either strand from a common dsODN
incorporation site (“flanking pairs”); the red ring shows reads with matches to the gRNA (allowing
<6 mismatches) within 100 bp (“target matched”). (B) Distribution of inferred positions of cleavage
and dsODN incorporation at an on-target locus. Incorporations in different strand orientations are
shown on the positive (red) and negative (blue) y-axis. The percentage in the bottom right corner is
an estimate of the number of incorporations associated with the on-target site (based on pileups)
captured within the allowed window of 100 bps. (C) Sequences of sites of cleavage and dsODN
incorporation are shown, annotated by whether they are on target or off target (“Target”); the total
number of unique alignments associated with the site (“Abund”); and an identifier indicating the
nearest gene (“Gene ID”). *Symbols after the gene name indicate that the site is within the
transcription unit of the specific gene, whereas the ~ symbol indicates the gene appears on the
allOnco cancer-associated gene list.

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Fig. 5. Detection of chromosomal translocations in engineered T cells following CRISPR-Cas9
gene editing. (A) Evaluation of chromosomal translocations in NYCE T cell infusion products during
the course of large-scale culture is shown. For the 12 monocentromeric translocation assays
conducted, a positive reference sample that contains 1 × 103 copies of the synthetic template plasmid
was evaluated as a control and the percent difference between expected and observed marking was
calculated. The absence of amplification from the 12 reactions that correspond to the different
chromosomal translocations indicates assay specificity (see supplemental methods).
(B) Longitudinal analysis of chromosomal translocations in vivo in three patients pre- and post-NYCE
T cell product infusion is displayed. In (A) and (B), error bars denote SD. For graphical purposes, the
proportions of affected cells were plotted on a log scale; a value of 0.001% indicates that
translocations were not detected.

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Fig. 6. Single-cell RNA sequencing of patient UPN39 CRISPR-Cas9 engineered NYCE T cells pre-
and post-infusion. (A) Venn diagram showing relative numbers of NY-ESO-1 TCR-positive cells with
TRAC, TRBC and/or PDCD1 mutations in the NYCE T cell infusion product (Day 0). (B) Proportions
of pre-infusion (Day 0) and post-infusion (Days 10 and 113) T cells without mutations (wild-type),
with TRAC, TRBC, PDCD1 mutations or expressing the NY-ESO-1 TCR transgene. Numbers of cells
belonging to each of these categories are listed below the graph. (C) Analysis of NY-ESO-1 TCR-
positive (right) and TCR–negative (left) cells without mutations (wild-type) or with single, double or
triple mutations at Day 0 (NYCE T cell infusion product) and Day 113 post-NYCE T cell infusion.
(D) UMAP plots of gene expression data. Analysis was performed on all T cells integrated across time
points, but only NY-ESO-1 TCR-expressing cells, split by time point, are shown (top panel). The
increase in TCF7 expression is indicative of an acquired central memory phenotype (bottom panel,
same cells). (E) Heat map showing scaled expression of differentially expressed genes in NY-ESO-1
TCR-positive T cells across time points. Color scheme is based on scaled gene expression from –2
(yellow) to 2 (purple).

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Fig. 7. Clinical responses and patient outcome following infusion of CRISPR-Cas9 engineered
NYCE T cells. (A) Swimmer’s plot describing time on study for each patient, duration of follow-up off
study (defined as survival beyond progression or initiation of other cancer therapy) and present
status (differentially colored) is shown. Arrows indicate ongoing survival. (B) Changes in kappa light
chain levels (mg/L × 103) in patient UPN07 following NYCE T cell product infusion are depicted.
Vertical black arrow indicates initiation of a D-ACE salvage chemotherapy regimen (defined as
intravenous infusion of cisplatin, etoposide, cytarabine and dexamethasone). (C) Longitudinal M-
Spike levels (g/dL) in patient UPN35 post-NYCE T cell product administration are shown. Vertical
black arrows denote administration of combination therapy with elotuzumab, pomalidomide and
dexamethasone. (D) Computed tomography scans demonstrating tumor regression in patient
UPN39 following administration of an autologous NYCE T cell infusion product. Radiologic studies
were obtained before therapy and after adoptive transfer of NYCE T cells. Tumor is indicated by red
X. SD, stable disease; PD, progressive disease. (E) Cytolytic capacity of NY-ESO-1-specific CD8+
T cells recovered at the indicated month after infusion and expanded from patients is shown. PBMC
samples collected after NYCE T cell product infusion were expanded in vitro in the presence of
NY-ESO-1 peptide and IL-2. The ability of expanded effector cells to recognize antigen and elicit
cytotoxicity was tested in a 4-hour 51CR release assay incorporating Nalm-6 NY-ESO-1+, parental
Nalm-6 (NY-ESO-1-) and A375 melanoma cells (NY-ESO-1+). All target cell lines were HLA-A*02
positive. Assays were performed in triplicate and error bars represent SD.

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Table 1. Patient demographics and date of engineered T cell infusion. UPN, unique patient number; MM, multiple
myeloma; BM, bone marrow; XRT, radiation therapy; ASCT, autologous hematopoietic stem cell transplant; ND, not
done.

Subject ID
Prior LAGE-1* /
(UPN) / Gender / Clinical
Diagnosis Prior Therapy Transplant / NY-ESO-1* /
Infusion Age Sites
Surgery NY-ESO-1**
date

Lenalidomide,
pomalidomide,
bortezomib,
carfilzomib,
UPN35 Female / IgG kappa BM, lytic
daratumumab, ASCT x 3 Pos/Pos/Neg
1/7/19 66 years MM 2008 bone lesions
panobinostat

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(8 lines; see
supplementary
materials)

Doxorubicin, Resection/
Myxoid/ ifosfamide, debulking × 2,
Abdominal/
UPN39 Male / round cell XRT 60-Gy, left nephrec-
pelvic ND/ND/Pos
3/18/19 66 years liposarcoma trabectedin, tomy/ partial
masses
2012 gemcitabine, sigmoid
taxol, XRT resection

Lenalidomide,
pomalidomide,
bortezomib,
carfilzomib,
Kappa light
UPN07 Female / BM, lytic daratumumab,
chain MM ASCT x 2 Pos/Pos/Neg
8/5/19 62 years bone lesions anti-CD38
2009
immunoconjugate
(6 lines; see
supplementary
materials)

*qPCR
**Immunohistochemistry

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Table 2. List of adverse events on the study.

All
AE Category Toxicity Grade 1/2 Grade 3/4
Grades

Anemia 2 1 1
Leukopenia 4 - 4
Hematologic Neutropenia 4 1 3
Thrombocytopenia 6 3 3
Lymphopenia 1 - 1

Upper Respiratory 1 1 -
Infection
Febrile Neutropenia 2 - 2

Hypercalcemia 1 1 -

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Hyperphosphatemia 1 1 -
Hypoalbuminemia 1 1 -
Hypocalcemia 3 2 1
Electrolyte
Hypokalemia 1 1 -
Hypomagnesemia 1 1 -
Hyponatremia 1 1 -
Hypophosphatemia 1 - 1

Dysgeusia 1 1 -
Headache 1 1 -
Neurologic Paresthesia 2 2 -
Syncope 1 - 1
Pain 3 3 -

Acute kidney injury 1 1 -

Renal
Urinary obstruction 1 - 1

Aspiration 1 - 1
Respiratory Nasal congestion 1 1 -
Cough 2 2 -

Lower GI bleed 1 1 -
Gastrointestinal
Vomiting 1 1 -

Alopecia 1 1 -
Other Phlebitis 1 1 -
LE edema 1 1 -

Total 50 30 20

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Table 3. iGUIDE measurement of on-target editing efficiency for each gene by final product.

Manufactured NYCE T cell Product

PDCD1 TRAC TRBC
(Subject ID)

UPN07 100.0% 99.6% 96.1%

UPN27 99.6% 99.1% 96.8%
UPN35 99.8% 99.1% 97.0%
UPN39 98.2% 96.7% 93.5%
Average ± SD 99.4% ± 0.8% 98.6% ± 1.3% 95.8% ± 1.6%

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CRISPR-engineered T cells in patients with refractory cancer
Edward A. Stadtmauer, Joseph A. Fraietta, Megan M. Davis, Adam D. Cohen, Kristy L. Weber, Eric Lancaster, Patricia A. Mangan,
Irina Kulikovskaya, Minnal Gupta, Fang Chen, Lifeng Tian, Vanessa E. Gonzalez, Jun Xu, In-young Jung, J. Joseph Melenhorst,
Gabriela Plesa, Joanne Shea, Tina Matlawski, Amanda Cervini, Avery L. Gaymon, Stephanie Desjardins, Anne Lamontagne,
January Salas-Mckee, Andrew Fesnak, Donald L. Siegel, Bruce L. Levine, Julie K. Jadlowsky, Regina M. Young, Anne Chew,
Wei-Ting Hwang, Elizabeth O. Hexner, Beatriz M. Carreno, Christopher L. Nobles, Frederic D. Bushman, Kevin R. Parker, Yanyan
Qi, Ansuman T. Satpathy, Howard Y. Chang, Yangbing Zhao, Simon F. Lacey and Carl H. June

published online February 6, 2020

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