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HomeScience AdvancesVol. 8, No. 47Successful targeting of PD-1/PD-L1 with
chimeric antigen receptor-natural killer cells and nivolumab in a humanized
mouse cancer model
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SUCCESSFUL TARGETING OF PD-1/PD-L1 WITH CHIMERIC ANTIGEN RECEPTOR-NATURAL KILLER
CELLS AND NIVOLUMAB IN A HUMANIZED MOUSE CANCER MODEL
Wai Nam Liu https://orcid.org/0000-0002-4323-4566, Wing Yan So
https://orcid.org/0000-0001-9647-3686, [...] , Sarah L. Harden
https://orcid.org/0000-0002-1029-8440, Shin Yie Fong, [...] , Melissa Xin Yu
Wong https://orcid.org/0000-0002-6217-5258, Wilson Wei Sheng Tan
https://orcid.org/0000-0003-3027-1162, Sue Yee Tan, Jessica Kai Lin Ong
https://orcid.org/0000-0002-6684-6370, Ravisankar Rajarethinam
https://orcid.org/0000-0001-9249-7840, [...] , Min Liu, Jia Ying Cheng, Lisda
Suteja https://orcid.org/0000-0002-4548-2766, Joe Poh Sheng Yeong
https://orcid.org/0000-0002-6674-7153, N. Gopalakrishna Iyer, Darren Wan-Teck
Lim https://orcid.org/0000-0002-4655-0206 qchen@imcb.a-star.edu.sg, and Qingfeng
Chen https://orcid.org/0000-0001-6437-1271 qchen@imcb.a-star.edu.sg+13 authors
+11 authors +6 authors fewerAuthors Info & Affiliations
Science Advances
23 Nov 2022
Vol 8, Issue 47
DOI: 10.1126/sciadv.add1187
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ABSTRACT
In recent decades, chimeric antigen receptor (CAR)–engineered immune effector
cells have demonstrated promising antileukemic activity. Nevertheless, their
efficacy remains unsatisfactory on solid cancers, plausibly due to the influence
of tumor microenvironments (TME). In a novel mouse cancer model with a humanized
immune system, tumor-infiltrating immunosuppressive leukocytes and exhausted
programmed death protein-1 (PD-1)high T cells were found, which better mimic
patient TME, allowing the screening and assessment of immune therapeutics.
Particularly, membrane-bound programmed death ligand 1 (PD-L1) level was
elevated on a tumor cell surface, which serves as an attractive target for
natural killer (NK) cell–mediated therapy. Hematopoietic stem cell–derived
CAR-NK (CAR pNK) cells targeting the PD-L1 showed enhanced in vitro and in vivo
anti-solid tumor function. The CAR pNK cells and nivolumab resulted in a
synergistic anti-solid tumor response. Together, our study highlights a robust
platform to develop and evaluate the antitumor efficacy and safety of previously
unexplored therapeutic regimens.
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INTRODUCTION
Conventional therapies for patients with cancer include chemotherapy and/or
radiotherapy, which lack specificity and are usually accompanied by side effects
(1). Various innovative approaches, such as targeted therapy and gene therapy,
are currently under evaluation (1). The early success of immune checkpoint
inhibitors (ICI) has revolutionized cancer treatment (2), which may partially
restore the cytotoxic function of tumor-infiltrating T cells (3). For instance,
ipilimumab, an anti-cytotoxic T lymphocyte–associated protein 4 (CTLA-4)
monoclonal antibody, was the first ICI approved by the Food and Drug
Administration (FDA) to treat patients with late-stage melanoma in 2011. Dual
therapy using ipilimumab and nivolumab [anti–programmed death protein-1 (PD-1)
antibody] was subsequently FDA-approved for the frontline treatment of advanced
melanoma in 2015. However, the response rates for most solid tumors are still
far from satisfactory, and most patients are refractory to this treatment (4).
In addition, there are immune-related adverse events (irAE) observed in patients
who are treated differently from classical chemotherapy-related toxicity (5).
Apart from the ICI, cell-mediated therapy using T cell receptor–engineered T
cells and chimeric antigen receptor (CAR)–T cells represents another class of
manipulating the immune system to treat patients with cancer (6, 7). To date,
more than 500 CAR-T clinical trials have been conducted, and CAR-T cell
products, such as Kymriah, Yescarta, Tecartus, and Breyanzi, are available on
the market (8). Nevertheless, CAR-T cell therapy usually requires gene editing
of autologous T cells from patients with cancer, which can be time-consuming and
delay emergent treatment (9). T cell–mediated therapy is also potentially
associated with graft-versus-host disease, cytokine release syndrome (CRS),
immune effector cell–associated neurotoxicity syndrome, and cytopenias (10, 11).
Natural killer (NK) cells are capable of killing tumor cells directly by
releasing granzyme and perforin or producing interferon-γ (IFN-γ) to mount an
adaptive immune response (12, 13). Contrary to T cell–mediated therapy, NK
cell–mediated therapy serves as a safer and more effective option for cancer
immunotherapy (14). NK cell function is independent of human leukocyte antigen
matching, thus allowing allogeneic infusion in patients with cancer, and banking
“off-the-shelf” CAR-NK cells may reduce time and cost for patients with cancer
(15). To achieve this purpose, the NK cells can be differentiated from human
embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), where
these NK cells may be gene-edited to generate target-specific CAR-NK cells (16,
17).
To date, CAR-NK cell–mediated therapy has represented an emerging paradigm for
antileukemic treatment, and clinical trials are being conducted globally (8).
Similar to T cell–mediated therapy, the antitumor efficacy of NK cell–mediated
therapy is less optimistic in solid cancers, plausibly due to poor infiltration
and exhaustion of effector cells in the tumor inflammatory milieu (18, 19).
Tumor microenvironments (TME) consist of tumor cells, endothelial cells, stromal
cells, and fibroblasts, with a unique cytokine and chemokine milieu for
recruiting and modulating the phenotype of human immune cells, particularly
those cells of myeloid lineage (20, 21).
Conventional in vivo models, such as nude mice and nonobese diabetic-severe
combined immunodeficiency interleukin-2 (IL-2) receptor gamma chain-null (NSG)
mice, lack an immune background that limits the understanding of immunological
response during the progression of cancers, as well as the evaluation of
antitumor efficacy of treatments, particularly immunotherapy. In addition,
immune-related toxicities are mediated by the host immune system after receiving
immune cell therapy (11), hence the lack of an in vivo humanized immune system
may hamper their assessment. In this regard, our group has recently established
a novel humanized mouse nasopharyngeal carcinoma–patient-derived xenografts
(NPC-PDX) model for testing immunotherapy (22). Similar to primary tumors,
tumor-infiltrating lymphocytes (TIL) were found in the NPC-PDX in humanized mice
but not in the PDX in NSG mice (22, 23), and pharmacodynamic modulation of the
immune checkpoint proteins was observed.
In the present study, we first delineated transcriptomic differences in NPC-PDX
between humanized mice and NSG mice. In addition to TIL, human cytokines and
chemokines were detected in the tumors from humanized mice. These elements are
crucial to remodel TME and better mimic the TME in patients with cancer. In
particular, plasma soluble programmed death ligand 1 (sPD-L1) was found in
tumor-bearing humanized mice, and membrane-bound PD-L1 was highly expressed in
these tumors, thus serving as an excellent target for immune cell therapy. From
our results, CAR-expressing NK92 cells and hematopoietic stem cell (HSC)–derived
primary NK (CAR pNK) cells targeting PD-L1 could inhibit the growth of different
solid cancers that express PD-L1, both in vitro and in vivo, while exhibiting
minimal cytotoxicity on primary human hepatocytes (PHH) and side effects in
humanized mice. Intriguingly, NPC-PDX showed an increase in the antigen
processing and presentation pathway in CAR pNK cell–injected humanized mice.
Because tumor-infiltrating T cells displayed exhaustion phenotypes, combination
therapy involving the CAR pNK cells and nivolumab was applied to the mice, which
resulted in a synergistic anti-solid tumor response, suggesting that restoration
of T cell function by specific ICI could further inhibit the growth of tumors
after sensitization by the NK cell–mediated therapy.
In summary, HSC-derived CAR pNK cells highlight a potential off-the-shelf
therapeutic option for patients with solid cancers. By combining NK
cell–mediated therapy and ICI therapy, we demonstrated better antitumor efficacy
in our humanized mouse NPC-PDX model. As other exhaustion markers, such as T
cell immunoreceptor with immunoglobulin (Ig) and ITIM domains (TIGIT) and T cell
Ig and mucin-domain containing-3 (TIM-3) were up-regulated in the
tumor-infiltrating T cells and NK cells, other combination regimens can be
assessed using this robust preclinical platform. This will enable preclinical
evaluation of previously unexplored therapeutic strategies to treat cancers and
may potentially predict and improve patients’ clinical outcomes.
RESULTS
THE PRESENCE OF A HUMANIZED IMMUNE SYSTEM CHANGES THE PROFILE OF TME
TIL play a pivotal role to shape TME and influence clinical outcomes of cancer
treatments (24). Therefore, our mouse cancer model with a humanized immune
system serves as a better platform to screen for potential biomarkers and
evaluate the efficacy of therapeutics when compared to traditional
immunocompromised mouse cancer model (22). To gain insights into the
transcriptomic and proteomic difference of the tumors, NPC-PDX were engrafted in
NSG mice and humanized mice. Four weeks after the transplant, the tumors were
harvested and subjected to RNA sequencing, and their sequencing data were
compared (Fig. 1A). Heatmap analysis of differentially expressed (DE) genes
demonstrated that their transcriptomic profile was distinct (Fig. 1B),
indicating that the PDX was greatly influenced by the infiltration of CD45+
humanized immune cells (fig. S1A). Several molecular pathways, including T cell
receptor (PTPRC, ICOS, and CD3E), cytokine-related (IL2RB, CCR5, and CD40LG),
and chemokine-related signaling (ITK, CCR5, and PIK3CG) were significantly
up-regulated in the tumors from humanized mice (Fig. 1C). Proteomic analysis
further revealed that the expressions of C-X-C motif chemokine ligand 10
(CXCL10), IL-8, and plasminogen activator inhibitor-1 (PAI-1) were augmented in
the tumors from humanized mice (Fig. 1D), which are known to be positively
correlated with the recruitment of regulatory T cells (25, 26), myeloid-derived
suppressor cells (MDSC), and M2 macrophages (27–29). Notably, the CXCL10 could
also promote the recruitment of CD8+ T cells in the tumors (30). Flow cytometry
analysis confirmed that CD8+ T cells (hCD45+hCD3+hCD8+), regulatory T cells
(hCD45+hCD3+hCD4+hCD25+hCD127low), and two subsets of MDSC, including
polymorphonuclear (PMN)–MDSC (hCD45+hCD11b+hCD33+hHLA-DR−hCD14−hCD15+) and
early-stage (E)–MDSC (hCD45+hCD11b+hCD33+hHLA-DR−hCD14−hCD15−), infiltrated the
tumors in humanized mice but not in NSG mice (fig. S1, B to D). Apart from local
immune responses in the TME, various inflammatory cytokines (IFN-γ, IL-8, and
IL-18), chemokine (MCP-1), and immune checkpoint biomarkers [Galectin-9 (Gal-9),
sPD-L1, sCD25, and transforming growth factor–β1 (TGF-β1)] were found in the
periphery of PDX-bearing humanized mice, while these soluble mediators were
minimally detected in NSG mice (fig. S2).
Fig. 1. TME in humanized mice is distinct from that in immunocompromised NSG
mice.
(A) Schematic description showing the methodology to generate humanized mice and
workflow for RNA sequencing using NPC-PDX from humanized mice and NSG mice. (B)
Heatmap of DE genes from NPC-PDX between humanized mice (n = 3) and NSG mice (n
= 3) is shown. (C) Signaling pathway analysis of DE genes. (D) Relative protein
expressions of CXCL-10, IL-8, and PAI-1 in tumor lysate from NSG mice (n = 4)
and humanized mice (n = 4). Data are expressed as means ± SEM. **P < 0.01,
two-tailed unpaired t test. (E) Volcano plot of selected DE genes between
humanized mice and NSG mice is shown. (F and G) Surface expressions of PD-1 on
CD3+CD8+ T cells and CD3−CD16+CD56+ NK cells in circulation (blue histogram) and
in tumor (red histogram) were analyzed by flow cytometry. Data are expressed as
means ± SEM. **P < 0.01 and ***P < 0.001, two-tailed unpaired t test (n = 4 for
both groups). MFI, mean fluorescence intensity. (H) Relative mRNA expression
level of human PD-L1 on tumor cells isolated from NPC-PDX from NSG mice (n = 5)
and humanized mice (n = 5). Data are expressed as means ± SEM. ***P < 0.001,
two-tailed unpaired t test. (I) Surface expression of human PD-L1 on tumor
cells. Isotype control (black histogram), cells harvested from NSG mice (blue
histogram), and humanized mice (red histogram) were analyzed by flow cytometry.
(J) Representative photomicrographs showing immunohistochemical staining of
human PD-L1 on NPC-PDX from NSG mice and humanized mice. Scale bars, 100 μm. n =
3 per group.
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From the RNA sequencing data, it was shown that gene expression levels of
several exhaustion markers, including CTLA4 and PDCD1, were highly up-regulated
in the NPC-PDX from humanized mice (Fig. 1E). By flow cytometry, it was
validated that the expression of PD-1 was more pronounced in tumor-infiltrating
CD8+ T cells and CD56+ NK cells (hCD45+hCD3−hCD16+hCD56+) than those in
circulating CD8+ T cells and CD56+ NK cells (Fig. 1, F and G). Intriguingly,
other exhaustion markers, such as TIGIT and TIM-3, were consistently
up-regulated in the T cells and NK cells in the PDX (fig. S3). As revealed by
multiplex immunohistochemical images obtained from paired archival tissue of the
donor patients, CD8+ T cells and FOXP3+ regulatory T cells were found, and these
cells expressed PD-1 and/or CTLA-4 in the primary NPC tumors (fig. S4),
suggesting that the composition of immune cell infiltration is comparable
between PDX and primary tumors.
As sPD-L1 was found in circulation, we also examined its membrane-bound
expression level in the tumors from NSG mice and humanized mice. It was found
that transcriptional and surface protein levels of PD-L1 were significantly
higher in the isolated tumor cells from humanized mice (Fig. 1, H and I). This
result was validated by in situ immunohistochemistry staining of PD-L1 in tumors
(Fig. 1J). Notably, some of the tumor-infiltrating CD45+ immune cells expressed
PD-L1 (fig. S5), and it has been suggested that other nonimmune epithelial cells
and stromal cells could also express the PD-L1 (31, 32). The increment in the
expression level of PD-L1 possibly results in T cell and NK cell exhaustion in
the tumors via PD-1/PD-L1 axis (33, 34), which have been targeted by respective
ICI, such as anti–PD-1 or anti–PD-L1 monoclonal antibodies. We further explored
the possibility of using cell-mediated therapy to target the elevated PD-L1
expressed on solid tumor cell surface.
CAR-ENGINEERED NK92 CELLS ENHANCE THE KILLING OF PD-L1–EXPRESSING CANCERS
Third-generation CAR technology was applied in this study to generate NK cells
targeting PD-L1, which consists of an anti–PD-L1 single-chain fragment variable
(ScFv), a spleen focus-forming virus promoter to enhance the gene expression
level, a CD8 hinge (H) and transmembrane (TM) domain, two costimulatory domains
(CD28 and 4-1BB), and a CD3ζ activation domain. In addition, an endogenous green
fluorescence protein (eGFP) reporter gene was inserted, and a puromycin
resistance gene allowed us to establish a stable CAR NK92 cell line for robust
validation of its antitumor efficacy, both in vitro and in vivo (Fig. 2A).
Successful transduction was demonstrated by immunofluorescence staining (fig.
S6A), and expression of CAR on the NK92 cell surface was further confirmed by
flow cytometry (fig. S6B). In the absence of IFN-γ, HepG2 cells expressed a
lower level of PD-L1 on their cell surface (Fig. 2B), and the cytotoxicity of
CAR NK92 cells on untreated HepG2 cells was minimal (Fig. 2C). Pretreatment of
the HepG2 cells with IFN-γ induced PD-L1 expression, and the killing ability of
CAR NK92 cells on the IFN-γ–treated cells was increased drastically (Fig. 2, B
and C). In addition, various human cancer cell lines, including A549, C666-1,
and Panc 08.13 cells that constitutively expressed the PD-L1, were cocultured
with the CAR NK92 cells (Fig. 2B). Our results demonstrated that the CAR NK92
cells were able to lyse their target cells more effectively when compared to
those nontransduced wild-type (WT) NK92 cells (Fig. 2C), suggesting that our
CAR-NK cells can target a broad spectrum of cancer types as long as they express
PD-L1. Apart from direct killing, NK cells are also known to lyse their target
cells through apoptosis (35). Without prior stimulation of the IFN-γ, there were
no significant changes in the apoptotic rate in the HepG2 cells (Fig. 2D). In
contrast, the CAR NK92 cells could induce apoptosis in the IFN-γ–treated HepG2
cells and other human cancer cells (Fig. 2D), accompanied by an increase in NK
cell CD107a expression, which indicates a release of cytotoxic granule (Fig.
2E). By enzyme-linked immunosorbent assay (ELISA), it was further revealed that
IFN-γ was secreted from the CAR NK92 cells upon stimulation with those
PD-L1–expressing cancer cells (Fig. 2F).
Fig. 2. Enhanced in vitro antitumor activity in CAR-expressing NK92 cells.
(A) Design of CAR construct. Puro, puromycin. SFFV, spleen focus-forming virus.
(B) Surface expression of human PD-L1 on HepG2 (untreated or IFN-γ–treated),
A549, C666-1, and Panc 08.13 cells. Cells stained with isotype control (blue
histogram) and anti–PD-L1 antibody (red histogram) were examined by flow
cytometry. WT NK92 cells (n = 4) and CAR NK92 cells (n = 4) were cocultured with
various human cancer cell lines at the indicated E:T ratios (C) or at a 10:1
ratio of E:T (D to F). (C) The percentages of NK cell cytotoxicity on different
cancer cell lines are shown. (D) The extent of apoptosis as indicated by
enrichment factor. (E) CD107a expression was determined in anti-CD56–labeled NK
cells after stimulation by different cancer cell lines. (F) Net release of IFN-γ
after stimulation by different cancer cell lines. Data from (D) to (F) are
expressed as means ± SEM. **P < 0.01 and ***P < 0.001, two-tailed unpaired t
test.
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To evaluate in vivo anti-solid tumor efficacy of CAR NK92 cells, C666-1 cells,
and NPC-PDX were inoculated into NSG mice. Upon the formation of visible tumors,
which took approximately 1 to 2 weeks, the mice received scheduled NK
cell–mediated therapy (Fig. 3A). Compared to saline-injected and WT NK92
cell–injected mice, the tumor size and weight of C666-1 were diminished in the
CAR NK92 cell–treated group (Fig. 3, B to D). Similarly, the in vivo antitumor
effect of CAR NK92 cells was demonstrated on the PD-L1–expressing NPC-PDX (Fig.
3, E to G), where the PDX were established from primary tumors of three
patients. Apart from the NPC, it was found that the CAR NK92 cells could also
exhibit growth inhibitory effects on PD-L1–expressing A549 tumors (fig. S7, A to
D) and lung adenocarcinoma (LAC)–PDX, respectively, in the NSG mice (fig. S7, A
and E to G). These results demonstrated that the CAR-NK cells are efficient in
killing various solid cancer types, both in vitro and in vivo.
Fig. 3. CAR-expressing NK92 cells exhibit better anti-solid tumor effect in NSG
mice.
(A) Schematic description of tumor inoculation and cell-mediated therapy in NSG
mice. The mice were either injected with C666-1 cells or transplanted with
NPC-PDX, which usually took 1 to 2 weeks to form visible tumors. (B to D) NSG
mice were inoculated with C666-1 cells. After the formation of visible tumors,
the mice were injected with saline (n = 15), WT NK92 cells (n = 15), and CAR
NK92 cells (n = 18). (B) Representative image of tumors. (C) Tumor volumes are
shown. Data are expressed as means ± SEM. *P < 0.05 and **P < 0.01, one-way
analysis of variance (ANOVA) followed by multiple comparisons, when CAR NK92
cell–injected group is compared to saline-injected group. (D) Tumor weights are
shown. Data are combined from two independent experiments and expressed as means
± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA followed by multiple comparisons.
(E to G) NSG mice were transplanted with NPC-PDX. After the formation of visible
tumors, the mice were injected with saline (n = 20), WT NK92 cells (n = 20), and
CAR NK92 cells (n = 21). (E) Representative image of tumors. (F) Tumor volumes
are shown. Data are expressed as means ± SEM. **P < 0.01, one-way ANOVA followed
by multiple comparisons, when CAR NK92 cell–injected group is compared to
saline-injected group. (G) Tumor weights are shown. Data are combined from three
independent experiments using PDX derived from three patients and expressed as
means ± SEM. **P < 0.01, one-way ANOVA followed by multiple comparisons.
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HSC-DERIVED PRIMARY CAR-NK CELLS ARE EFFICIENT IN KILLING PD-L1–EXPRESSING SOLID
CANCERS
Before infusing cells of malignant origin into patients, NK92 cells need to be
irradiated to avoid in vivo proliferation (36). To minimize safety concerns,
primary NK cells are preferably used for CAR-NK cell–mediated therapy in clinics
(37). In recent years, it has been demonstrated that hESCs and iPSCs could be
differentiated into NK cells, which showed similar phenotypes and functions to
those mature NK cells in peripheral blood (38, 39). In the present study, CD34+
HSC in human umbilical cord blood were expanded and differentiated into primary
NK (pNK) cells in a feeder cell-free system (fig. S8, A and B). By flow
cytometric analysis, it was found that the phenotype of HSC-derived pNK cells
was similar to that of cord blood NK (CB-NK) cells (Fig. 4A), despite the
expression level of CD16 of the latter being higher. In terms of their
functions, CB-NK cells and pNK cells exhibited similar cytotoxicity toward K-562
cells (Fig. 4B) and induced apoptosis of the K-562 cells (Fig. 4C). In addition,
their expression levels of CD107a and release of IFN-γ were comparable after the
coculture (Fig. 4, D and E). Next, CAR pNK cells were generated by lentiviral
transduction. The efficiency varied from 20 to 40%, subject to batch-to-batch
variation of the stem cells (fig. S8, C and D). Notably, the transduction was
more efficient than infecting the CB-NK cells directly (fig. S8D), thus
resulting in a much higher yield of the CAR pNK cells. Apart from killing C666-1
cells, the CAR pNK cells could also lyse tumor cells isolated from the NPC-PDX
(Fig. 4F) and trigger apoptosis in the tumor cells by increasing the NK cell
CD107a expression and IFN-γ release (Fig. 4, G to I). In addition, the CAR pNK
cells were capable of killing other PD-L1–expressing cancer cell lines (fig.
S8E).
Fig. 4. Phenotypic and functional characterization of CAR-expressing HSC-derived
NK cells.
(A) Surface expressions of NK cell–specific markers under the gate of human
CD45+ and CD56+ subset. Cells stained with isotype control (blue histogram) and
indicated PE-conjugated antibodies (red histogram) were examined by flow
cytometry. CB-NK cells (n = 4) and pNK cells (n = 4) were cocultured with K-562
cells at the indicated E:T ratios (B) or at a 10:1 ratio of E:T (C to E). (B)
The percentages of NK cell cytotoxicity on K-562 cells are shown. (C) The extent
of apoptosis as indicated by enrichment factor. (D) CD107a expression was
determined in anti-CD56–labeled NK cells after stimulation by K-562 cells. (E)
Net release of IFN-γ after stimulation by K-562 cells. WT pNK cells (n = 4) and
CAR pNK cells (n = 4) were cocultured with C666-1 cells or tumor cells isolated
from NPC-PDX at the indicated E:T ratios (F) or at a 10:1 ratio of E:T (G to I).
(F) The percentages of NK cell cytotoxicity on tumor cells are shown. (G) The
extent of apoptosis. (H) CD107a expression on NK cells after stimulation by
tumor cells. (I) Net release of IFN-γ after stimulation by tumor cells. Data
from (C) to (E) and (G) to (I) are expressed as means ± SEM. *P < 0.05, **P <
0.01, and ***P < 0.001, two-tailed unpaired t test.
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In vivo function of the CAR pNK cells were then assessed alongside nontransduced
pNK (WT pNK) cells and CAR-expressing primary T (CAR pT) cells to compare their
antitumor efficacy, following the treatment schedule as shown in Fig. 3A. A
reduction of tumor burden was observed in NSG mice treated with the CAR pNK
cells, but not the CAR pT cells, suggesting that the former displayed a higher
antitumor potency in our NPC-PDX model (Fig. 5, A to C). The tumors were
harvested 2 days after the last cell-mediated therapy. Fluorescence-activated
cell sorting (FACS) analysis and histological examinations revealed the presence
of tumor-infiltrating NK cells and T cells (Fig. 5, D to F). Despite a greater
number of CAR pT cells persisting in the PDX, the results from terminal
deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end
labeling (TUNEL) illustrated that more tumor cells underwent apoptosis after the
injection of CAR pNK cells (Fig. 5, G and H).
Fig. 5. CAR-expressing primary NK cells suppress in vivo growth of NPC-PDX more
effectively.
NSG mice were transplanted with NPC-PDX. After the formation of visible tumors,
the mice were injected with saline (n = 5), WT pNK cells (n = 5), CAR pNK cells
(n = 5), and CAR pT cells (n = 5). (A) Representative image of tumors. (B) Tumor
volumes are shown. Data are expressed as means ± SEM, and comparative
statistical analysis were done by one-way ANOVA followed by multiple
comparisons. *P < 0.05, when CAR pNK cell–injected group is compared to
saline-injected group; #P < 0.05 and ##P < 0.01, when CAR pNK cell–injected
group is compared to CAR pT cell–injected group. (C) Tumor weights are shown.
Data are expressed as means ± SEM. *P < 0.05 and ***P < 0.001, one-way ANOVA
followed by multiple comparisons. (D and E) Representative FACS plots showing
the infiltration of CD3−CD56+ NK cells (D) and CD3+CD56− T cells (E) in tumors.
(F) Representative photomicrographs showing immunohistochemical staining in
tumors. The presence of NK and T cells was indicated by the expression of NCR1
and CD3, respectively. Scale bars, 100 μm. n = 5 per group. (G) Representative
photomicrographs showing TUNEL in tumors. Apoptotic cells were labeled by red
fluorescence. Scale bars, 100 μm. n = 5 per group. DAPI,
4′,6-diamidino-2-phenylindole. (H) Signal intensity of TUNEL is quantified from
the images, and data are expressed as means ± SEM. ***P < 0.001, one-way ANOVA
followed by multiple comparisons.
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As mentioned earlier, the immunosuppressive TME in humanized mouse model was
found to more closely resemble that in patients as compared to NSG mice.
Therefore, we further assessed the efficacy and immunotoxicity of CAR-NK
cell–mediated therapy in humanized mice transplanted with NPC-PDX (Fig. 6A).
After the injection of CAR pNK cells and CAR pT cells, there was an increase in
the plasma IFN-γ level, and the elevation was more pronounced in the CAR pT
cell–treated mice (fig. S9A). Because the expression level of PD-L1 could be
induced by IFN-γ in vitro, we further examined the modulation of the plasma
sPD-L1 level in the mice. Accompanied by the increase in IFN-γ levels, the
sPD-L1 levels in circulation were increased in both groups, particularly in the
CAR pT cell–treated mice (fig. S9B). To demonstrate that there were no premature
death of the CAR pNK cells and CAR pT cells in circulation, these cells were
treated with human sPD-L1 recombinant protein in vitro. Our results showed that
there were no significant changes in their viability (fig. S9C) while increasing
their release of IFN-γ (fig. S9D), suggesting that these cells might also be
activated in the circulation after binding to the sPD-L1 before reaching the
tumor sites in humanized mice. Apart from the plasma IFN-γ and sPD-L1, it was
also found that the transcriptome expression levels of IFN-γ and PD-L1 were
augmented more drastically in the PDX after the CAR pT cell therapy (fig. S9, E
and F). Intriguingly, tumor burden was reduced after the mice were injected with
the CAR pNK cells (Fig. 6, B to D) despite the presence of tumor-infiltrating
regulatory T cells and MDSC and the influence of the immunosuppressive niche.
Nevertheless, mice injected with CAR pT cells did not show significant reduction
in tumor weight when compared to the saline control group. Minimal CAR pNK cells
and CAR pT cells persisted in the PDX 2 days after the last injection (fig.
S10). Of equal importance, the in vitro and in vivo safety of CAR pNK cells was
evaluated. As shown in fig. S11A, there was a subtle increase in PD-L1
expression after PHH were treated with IFN-γ, similar to the observation in
HepG2 cells (Fig. 2B). Nevertheless, the CAR pNK cells showed a limited increase
in cytotoxicity, if any, on the IFN-γ–treated PHH in vitro (fig. S11B), while
IFN-γ–treated HepG2 cells were more susceptible to the CAR pNK cells (fig. S8E).
By immunofluorescence stating, it was found that some of the tumor-infiltrating
CD45+ immune cells expressed PD-L1 (fig. S5). Nonetheless, the CAR pNK cells
showed minimal increase in cytotoxicity when they were cocultured with the TIL,
possibly due to the low expression level of PD-L1 on the immune cells (fig. S11,
C and D). Besides, the CAR pNK cells only exhibited mild cytotoxicity on the
nonimmune stromal cells while showing better killing effects on the tumor cells
(fig. S11, C and D). Recently, it has been reported that the amino acid sequence
between human PD-L1 and mouse PD-L1 is well conserved, and their structures are
almost identical (40). By flow cytometry, we proved that mouse PD-L1 recombinant
protein could bind with our CAR (fig. S12A). As shown in fig. S12 (B and C), the
mouse PD-L1 expression level in liver and lung was up-regulated after
PDX-bearing humanized mice were treated with CAR pNK cells and CAR pT cells.
These cells might also bind to the mouse PD-L1–expressing normal cells in the
organs. Intriguingly, the survival rate of NSG mice among different treatment
groups was comparable, while a significant reduction in the survival rate was
observed in the CAR pT cell–injected humanized mice (Fig. 6, E and F) but not in
the CAR pNK cell–treated group. Hence, the decrease in survivability of the CAR
pT cell–treated mice might also be contributed by the release of soluble
mediators. By LEGENDplex, it was found that plasma concentrations of IFN-γ,
IL-6, and IL-10 were significantly elevated in the humanized mice treated with
the CAR pT cells but not CAR pNK cells (Fig. 6, G to I). This suggested that the
former might elicit CRS in the presence of the humanized immune system. To
confirm whether the T cell–mediated therapy led to organ damage, liver and lungs
were subjected to pathological evaluation. From the results, the liver and lungs
from saline (fig. S13, A and E) and CAR pNK cell–treated groups (fig. S13, B and
F) showed normal histology. However, after the treatment of CAR pT cells, there
were widespread multifocal aggregates of mononuclear cells that include
lymphocytes or histiocytes present in hepatic parenchyma. The mononuclear cells
were mostly confined to perivascular area, and their presence was associated
with multinucleated giant cells exhibiting a granulomatous inflammation (fig.
S13, C and D). Besides, alveoli were mildly hyperplastic and infiltrated with
the moderate presence of mixed inflammatory cells, such as mononuclear cells
and, to some extent, few PMN cells in the lungs (fig. S13, G and H). Together,
the surge of various cytokines under circulation and pathological conditions in
the liver and lung might lead to the reduction in mouse body weight (fig. S13I)
and survivability after CAR pT cell–mediated therapy.
Fig. 6. Evaluation of antitumor efficacy and safety of CAR pNK cells in
NPC-PDX–bearing humanized mice.
(A) Schematic description of PDX engraftment and cell-mediated therapy in
humanized mice. (B to D) Humanized mice were transplanted with NPC-PDX. After
the formation of visible tumors, the mice were injected with saline (n = 9), WT
pNK cells (n = 10), CAR pNK cells (n = 13), and CAR pT cells (n = 7). (B)
Representative image of tumors. (C) Tumor volumes are shown. Data are expressed
as means ± SEM. (D) Tumor weights are shown. Data are expressed as means ± SEM.
*P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by multiple
comparisons. (E) Survival curve of NSG mice after treatment with saline (n =
36), WT pNK cells (n = 34), CAR pNK cells (n = 38), and CAR pT cells (n = 20).
Data are combined from at least three independent experiments. (F) Survival
curve of humanized mice after treatment with saline (n = 20), WT pNK cells (n =
12), CAR pNK cells (n = 28), and CAR pT cells (n = 22). Data are combined from
at least three independent experiments. The survival rate of mice between CAR
pNK cell–injected group and CAR pT cell–injected group is compared by
Kaplan-Meier analysis via a log-rank test. **P < 0.01. (G to I) Blood samples
were collected from humanized mice 2 days after the last treatment with saline
(n = 12 for IL-6 and IL-10; n = 19 for IFN-γ), WT pNK cells (n = 11), CAR pNK
cells (n = 16), and CAR pT cells (n = 11). Plasma concentrations of IFN-γ (G),
IL-6 (H), and IL-10 (I) were determined by LEGENDplex. Data are expressed as
means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by
multiple comparisons.
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COMBINATION THERAPY USING CAR PNK CELLS AND NIVOLUMAB SHOWS A SYNERGISTIC
ANTITUMOR RESPONSE IN A HUMANIZED MOUSE NPC-PDX MODEL
To investigate the transcriptomic changes in NPC-PDX after cell-mediated therapy
and identify potential combination regimens, the tumors were sent for bulk RNA
sequencing. Compared to saline-injected group, there was an up-regulation of NK
cell–mediated cytotoxicity pathway (TYROBP, KLRD1, and FCER1G), as well as
antigen processing and presentation pathway (CTSL, KLRD1, and CD8A) after CAR
pNK treatment (Fig. 7, A and B). Because T cells were the predominant immune
cell type in the tumors (fig. S1B), we postulated that the presented antigens
might be recognized by the tumor-infiltrating T cells for further antitumor
effect. Nonetheless, these T cells exhibited exhaustion phenotypes (Fig. 1, F
and G), which might impede their functions. To overcome this issue and assay the
effect of combination therapy with different ICI, ipilimumab or nivolumab was
used along with CAR pNK to block the immune inhibitory signals in TME (Fig. 7C).
In line with our previous findings (22) and paired outcomes from donor patients
treated with immunotherapy (fig. S14), treatments involving these antibodies
alone did not reduce the tumor burden of NPC-PDX in humanized mice (Fig. 7, D,
F, and G). However, it was found that more CD3+ T cells were present in the PDX
when the mice were treated with ipilimumab, and a reduction in CD4/CD8 ratio was
observed when the mice received nivolumab treatment (fig. S15). Next, the CAR
pNK cells were administered into the mice, in combination with the ICI (Fig.
7C). From our results, the combination of NK cell–mediated therapy and
ipilimumab did not further reduce the PDX burden. In contrast, dual therapy
using the CAR pNK cells and nivolumab resulted in a synergistic antitumor
response in vivo (Fig. 7, E to G), which might partially due to the restoration
of host T cell function. Collectively, the NK cell–mediated therapy could
sensitize the tumors, increase antigen processing and presentation, and enhance
the killing ability of tumor-infiltrating immune cells after the treatment of
specific ICI.
Fig. 7. Dual therapy using CAR pNK cells and nivolumab results in a synergistic
antitumor response on NPC-PDX in humanized mice.
(A) Heatmap of DE genes from NPC-PDX between CAR pNK cell–injected mice (n = 3)
and saline-injected mice (n = 3) is shown. (B) Signaling pathway analysis of DE
genes. (C) Schematic description of PDX engraftment, ICI therapy alone, or in
combination with CAR pNK cell–mediated therapy in humanized mice. (D to G)
Humanized mice were transplanted with NPC-PDX. After the formation of visible
tumors, the mice were injected with saline (n = 23), ipilimumab (n = 15),
nivolumab (n = 13), CAR pNK cells (n = 22), CAR pNK cells plus ipilimumab (n =
22), and CAR pNK cells plus nivolumab (n = 26). (D and E) Representative image
of tumors. (F) Tumor volumes are shown. Data are expressed as means ± SEM. **P <
0.01 and ***P < 0.001, when CAR pNK cells plus nivolumab–treated group is
compared to saline-injected group, @P < 0.05, when CAR pNK cells plus
ipilimumab–treated groups are compared to saline-injected group, #P < 0.05, when
CAR pNK cell–treated groups are compared to saline-injected group, one-way ANOVA
followed by multiple comparisons. (G) Tumor weights are shown. Data are combined
from at least three independent experiments using PDX derived from three
patients and expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001,
one-way ANOVA followed by multiple comparisons.
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DISCUSSION
Cancer immunotherapy using genetically modified immune effector cells represents
an emerging treatment option for patients. In particular, CAR expression on NK
cells facilitates the recognition of tumor-associated antigens, thus enhancing
NK cell cytotoxicity on tumor cells (8). Despite their astonishing activity in
the treatment of hematological malignancies, the antitumor efficacy remains
unsatisfactory in solid cancers (18, 19), likely due to the unique
immunosuppressive TME. In the presence of a humanized immune system, our model
better simulates the TME in patients when compared to immunocompromised NSG mice
(22). Hence, we aimed to screen for potential targets for solid cancer
treatments based on the TME difference between NSG mice and humanized mice. Our
RNA sequencing data revealed that the transcriptomic profile of NPC-PDX in
humanized mice is distinct from that in NSG mice, suggesting that the PDX is
influenced by the infiltration of CD45+ humanized immune cells, which is
congruent with findings from other studies (41, 42). Besides, protein
expressions of CXCL-10, IL-8, and PAI-1 were found to be increased in PDX
lysate. These mediators are responsible for recruiting tumor-infiltrating
regulatory T cells (25, 26) and MDSC (27, 28). Immunosuppressive cell subsets,
such as regulatory T cells, PMN-MDSC, and E-MDSC, as well as exhausted T cells
and NK cells, were found in the PDX from humanized mice. This is reflective of
subpopulations that have appeared in the NPC microenvironment from paired
archival tissue of the donor patients and in patients from other study (43).
Besides, it has been proposed that localized immune responses in the TME cannot
exist without continuous coordination with the peripheral immune system (44). In
addition to the presence of humanized immune cells, various inflammatory
cytokines (IFN-γ, IL-8, and IL-18), chemokine (MCP-1), and immune checkpoint
biomarkers (Gal-9, sPD-L1, sCD25, and TGF-β1) were found in the circulation in
PDX-bearing humanized mice. These peripheral immune cells and soluble mediators
play a crucial role in tumor development, while these elements were either
absent or minimally detected in the immunocompromised NSG mice. Because the
coordination of the peripheral immune system and TME more closely resembles that
in patients, the humanized mouse model can improve our understanding of systemic
immunity during tumor progression and provide a more accurate platform to
develop and assess antitumor efficacy and safety of different treatment
regimens, particularly immunotherapies.
In our study, we found that sPD-L1 was present in the circulation of
NPC-PDX–bearing humanized mice, and the membrane-bound PD-L1 expression level in
the PDX from humanized mice was significantly higher when compared to that from
NSG mice, where tumor cells, nonimmune stromal cells, and a minor population of
CD45+ TIL expressed the PD-L1 on their cell surface. It is possible that the
presence of IFN-γ in humanized mice induces the PD-L1 expression on the tumor
cells (45), which serves as an excellent target for both cell-mediated therapy
and ICI therapy. As a proof of concept, our third-generation stable CAR NK92
cell line that targets PD-L1 showed superior cytotoxicity and induced apoptosis
in various human cancer cell lines that express the PD-L1, and the results are
comparable to those aforementioned studies using high-affinity NK92 cells
(46–48). Upon coculturing with the PD-L1–expressing cancer cells, the CAR NK92
cells showed augmented degranulation, as reflected by increasing their CD107a
expression and releasing more IFN-γ. Besides, the CAR NK92 cells could suppress
the growth of C666-1 cells in NSG mice. Moreover, PD-L1–expressing NPC-PDX
established from different donors were transplanted into the NSG mice. From our
results, it was found that the CAR NK92 cells could hinder the growth of these
PDX more effectively when compared to WT NK92 cells, suggesting that the
antitumor effect of the CAR NK92 cells was not PDX specific. Similarly, the CAR
NK92 cells could exhibit growth inhibitory effects on lung cancer A549 cells and
LAC-PDX that express the PD-L1. These results demonstrated that our CAR-NK cells
are efficient in killing different solid cancer types, both in vitro and in
vivo.
To date, NK92 is the only human NK cell line that has been approved for NK
cell–mediated therapy in clinical trials (36). These cells were derived from a
progressive non-Hodgkin’s lymphoma (49), and irradiation of these cells is
essential to reduce their proliferative ability before infusing them into
patients (36). Apart from the NK92 cells, primary NK cells from human peripheral
blood or cord blood can be isolated and genetically engineered with CAR for
subsequent cell therapy. However, one of the major caveats is that the primary
NK cells are resistant to gene transfer, where vesicular stomatitis virus type G
lentiviral vectors that are typically used to generate CAR-T cells, calcium
phosphate precipitation, liposome reagents, and electroporation techniques
showed a limited gene transduction rate (50). Until more recently, baboon
envelope pseudotyped lentiviral vectors have been shown to improve the
transduction efficiency (51). Alternatively, primary NK cells can be
differentiated from multipotent progenitor cells and pluripotent stem cells,
such as hESCs, iPSCs, mobilized bone marrow stem cells, and umbilical cord blood
stem cells (17, 39). Compared to mature NK cells, these progenitor and stem
cells demonstrated higher transduction efficiency (52). In the current study,
HSC obtained from human cord blood samples and differentiated into NK cells
presents a viable method to develop CAR-NK cells for clinical use. We first
illustrated that pNK cells showed similar phenotypes and functions when compared
to CB-NK cells, despite the latter expressing a higher level of CD16. In clinic,
large numbers of CAR-NK cells, ranging from 2 × 105 to 4 × 109 cells/kg, are
needed for single or multiple infusions into patients (52, 53). Compared to the
CB-NK cells, HSC showed a better expansion capacity and transduction efficiency.
Hence, it is feasible to generate an ample number of CAR pNK cells for single or
multiple adoptive transfers. Apart from the cytotoxicity on PD-L1–expressing
human cancer cell lines, the CAR pNK cells, but not CAR pT cells, could inhibit
the growth of NPC-PDX in NSG mice. Despite there was a higher number of
tumor-infiltrating CAR pT cells, the CAR pNK cell–mediated therapy could elicit
more apoptosis in the tumors. One of the possibilities is that the NK cells can
lyse tumor cells without prior priming, while the T cell function is major
histocompatibility complex (MHC)–restricted (54). Further, we showed that our
current CAR pNK cell–mediated therapy retained its antitumor potency on NPC-PDX
despite the presence of tumor-infiltrating immunosuppressive leukocytes and the
influence of TME in humanized mice. Accompanied by an increase in IFN-γ after
cell-mediated therapy, the level of plasma sPD-L1 and membrane-bound PD-L1
expression in the PDX was enhanced, particularly in the mice treated with CAR pT
cells. However, there were no significant changes in the tumor burden after the
injection of CAR pT cells, suggesting that other factors, such as the release of
soluble mediators upon binding to the PD-L1–expressing tumor cells, might affect
their antitumor potency. Minimal CAR pNK cells and CAR pT cells persisted in the
PDX 2 days after the last injection. It has been reported that CAR-NK cells
generally had a short life span in vivo (55), while a hypoxia TME might disturb
the cytotoxicity of NK cells and the recruitment of CD8+ T cells into tumor
areas (56, 57). Further improvements of CAR design might be beneficial to the
infiltration, cytotoxicity, and persistence of the CAR-expressing immune cells
for better antitumor outcomes.
Administration of ICI targeting PD-1 and other immune checkpoints is now an
established clinical treatment standard for NPC, as several exhausted and
immunosuppressive cell subsets, including HAVCR2+PD-1+ T cells,
CD25+FOXP3+CTLA-4+ regulatory T cells, and CD68+ myeloid-derived cells, are
found in patient TME (31, 43). However, the reported objective response rates
using anti–PD-1 monoclonal antibody and other ICI were around 20% in different
single-arm trials (23). Consistent with the lack of response in donor patients
in the clinic, treatments involving ipilimumab or nivolumab alone did not reduce
the burden of NPC-PDX in humanized mice, although more CD3+ T cells were
infiltrated into the PDX after ipilimumab treatment and CD4/CD8 ratio was
reduced after nivolumab treatment when compared to saline-treated group. As
demonstrated by our previous findings, the ICI treatment mainly restored the
function of tumor-infiltrating T cells, which was indicated by an increase in
their activation marker HLA-DR and cytokine production (22). However, the
restoration of T cell function alone was not sufficient to reduce the tumor
burden. In recent years, it has been proposed that other modes of cancer
therapy, including radiotherapy, chemotherapy, and gene-targeted therapies,
could modulate the immunosuppressive TME and potentially synergize with ICI
therapy (58). Our RNA sequencing data revealed an increment in antigen
processing and presentation pathway after the injection of CAR pNK cells in
humanized mice, suggesting that the host immune system was also stimulated after
the treatment, despite the existence of immunosuppressive and exhausted
tumor-infiltrating immune cells. Thus, blocking inhibitory signals in these
cells, in combination with the NK cell–mediated therapy, might further improve
their antitumor functions. Encouragingly, treatment regimens involving CAR pNK
cells and nivolumab, but not ipilimumab, resulted in a synergistic antitumor
response in the humanized mice. It is anticipated that PD-L1–expressing tumor
cells were first targeted and lysed by the CAR pNK cells, and the presented
antigens were further recognized by tumor-infiltrating T cells to regress local
tumors, where these cells could respond to specific ICI and reinvigorate their
functions in our model. Further examinations of mRNA differential expression in
NPC-PDX among different treatment regimens, such as CAR pNK cell–treated group,
nivolumab-treated group, and CAR pNK cell and nivolumab-treated group, either in
synchronous or sequential manner, might provide insights to identify potential
therapeutic targets in the future, and the sequential administration of CAR pNK
cells and nivolumab might result in different antitumor efficacy. Notably, the
CAR pNK cells might acquire PD-1–expressing exhaustion phenotype when they
infiltrated into the tumors, but considering the strong activation motifs
carried by the CAR pNK cells and their short life span, the nivolumab treatment
might not have remarkable impact on the tumor-infiltrating CAR pNK cells when
compared to the host tumor-infiltrating T cells.
Apart from antitumor efficacy, the safety of NK cell–mediated therapy was
investigated. After treatment with IFN-γ, there was an increase in PD-L1
expression on both PHH and HepG2 cell surface. However, CAR pNK cells showed
limited in vitro cytotoxicity on the former, whereas the latter were more
susceptible to the CAR pNK cells. Similarly, the CAR pNK cells exhibited minimal
cytotoxicity on the CD45+ TIL when compared to the tumor cells isolated from the
same PDX. One of the possibilities is that NK cells recognize the absence of
self-antigens on tumor cells via the interaction between killer cell Ig-like
receptors on NK cell surface and MHC-I molecules on cancer cell surface. In
contrast, normal healthy cells express MHC-I molecules on their surface, which
serve as ligands for inhibitory receptors on NK cells and contribute to NK cell
tolerance (53). Tumor-bearing humanized mice injected with CAR pT cells resulted
in a drastic reduction in their survivability, when compared to that after CAR
pNK treatment. It is conceivable that one of the side effects of T cell–mediated
therapy is the induction of CRS, which is featured by a massive release of
inflammatory cytokines, including IFN-γ, tumor necrosis factor–α, IL-6, and
IL-10 (11, 59). Consistently, we found that plasma concentrations of IFN-γ,
IL-6, and IL-10 were significantly up-regulated in tumor-bearing humanized mice
after CAR pT treatment but not in the CAR pNK cell–treated mice. In particular,
the increase in IFN-γ might further induce the expression of mouse PD-L1 in
liver and lung. Pathological conditions were observed in these organs, which
might partially explain the reduction in mouse body weight and survivability
after the CAR pT cell–mediated therapy. Notably, it has been suggested that host
immune cells play a crucial role in the pathogenesis of CRS, and depletion of
macrophages and monocytes might reduce the severity of the CRS (11). Hence,
further use of the humanized mouse model is warranted to identify mechanisms
that lead to CRS and other irAE and to assess the safety of different cancer
treatments, including but not limiting to cell-mediated therapy and ICI therapy.
Together, we illustrated that TME in the humanized mouse model more closely
resembles that in patients. This provides a better platform to identify
molecular targets, develop therapeutic drugs, and assess their antitumor
efficacy and immunotoxicity in the presence of the humanized immune system.
Pursuing an unmet need in the treatment of solid cancers, dual therapy using
HSC-derived CAR pNK cells and nivolumab was tested in this study, and we
observed a notable growth inhibitory effect on NPC-PDX in humanized mice.
Besides, the expression levels of other immune checkpoint receptors, including
but not limiting to TIGIT and TIM-3, were also up-regulated in the
tumor-infiltrating T cells and NK cells in humanized mice, and their signaling
is strongly associated with immune-response regulation and tumor progression
(60). Hence, further evaluations of combination regimens in the humanized mice
are highly warranted to provide insights for developing next-generation cancer
immunotherapies and improve clinical outcomes in patients.
MATERIALS AND METHODS
STUDY DESIGN
TIL play a crucial role to shape TME and influence clinical outcomes of cancer
treatments. In the presence of a humanized immune system, our model better
simulates the TME in patients when compared to immunocompromised NSG mice.
Hence, this study aimed to identify and validate potential targets for solid
cancer treatments based on the TME difference between NSG mice and humanized
mice. Taken as an example, we observe that membrane-bound PD-L1 was highly
expressed in the PDX in humanized mice, thus serving as an excellent target for
immune cell therapy. Hence, we established a CAR NK92 cell line targeting the
PD-L1 to investigate in vitro and in vivo CAR-NK cell–mediated anti-solid tumor
effects and the action mechanisms, if any. To minimize potential safety concern
using the NK92 cell line, we further generated HSC-derived CAR pNK cells. The in
vitro cytotoxicity of CAR pNK cells toward human cancer cells, in vivo
anti-solid tumor efficacy and safety, action mechanisms, and transcriptomic
changes were assessed. Notably, the immunosuppressive TME in humanized mice
might lead to immune effector cells exhaustion, which hamper their antitumor
functions. Hence, the anti-solid tumor efficacy of CAR pNK cells, either alone
or in combination with different ICI, was evaluated. In our study, mice were
randomly divided into different treatment groups once they showed tumor growth,
and no animals were excluded because of humane end point. Researchers were not
blinded to the treatments.
MOUSE STRAIN AND GENERATION OF HUMANIZED MICE
NSG mice were purchased from The Jackson Laboratory and bred in a specific
pathogen–free condition at Biological Resource Centre in Agency for Science,
Technology and Research (A*STAR), Singapore. Humanized mice were generated as
described elsewhere (22). Briefly, human cord blood CD34+ HSC (2 × 105 per pup;
Lonza Bioscience, Basel, Switzerland) were inoculated into NSG pups by
intrahepatic injection after the pups were sublethally irradiated at 1 Gy. Ten
to 12 weeks after the injection, blood samples were collected from the mice, and
human immune cell engraftment (chimerism) in the mice was determined by flow
cytometry. The chimerism was calculated by [% human CD45+/(% human CD45+ + %
mouse CD45+)], and humanized mice with the chimerism between 20 and 50% were
used for subsequent experiments.
SingHealth and National Health Care Group Research Ethics Committees Singapore
specifically approved this study [Institutional Review Board (IRB) number:
2020-040]. All animal experiments were conducted in strict accordance with the
guidelines on Care and Use of Animals for Scientific Purposes, which are
released by the National Advisory Committee on Laboratory Animal Research,
Agri-Food and Veterinary Authority of Singapore, and International Animal Care
and Use Committee (IACUC) at A*STAR. The IACUC specifically approved this study
(IACUC numbers: 181367 and 191440).
NASOPHARYNGEAL CARCINOMA AND LAC XENOGRAFTS
Primary tumors of NPC and LAC were collected from National Cancer Center
Singapore (NCCS), with written consent obtained from patients and in strict
accordance with the institutional ethical guidelines of NCCS. SingHealth and
National Health Care Group Research Ethics Committees Singapore specifically
approved this study (IRB numbers: 2016/2887 and 2007/444/B). NPC-PDX and LAC-PDX
were established in NSG mice as described previously (22).
RNA SEQUENCING AND DATA ANALYSIS
NPC-PDX in NSG mice and humanized mice were harvested, and total RNA was
extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The
total RNA of each sample was then quantified and qualified by NanoDrop (Thermo
Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo
Alto, CA, USA). Next, 1 μg of total RNA was used for library preparations
according to the manufacturer’s protocol (VAHTS mRNA-seq V3 Library Prep Kit for
Illumina). The poly(A) mRNA isolation was performed using the Poly(A) mRNA
Magnetic Isolation Module. The mRNA fragmentation and priming were performed
using First-Strand Synthesis Reaction Buffer and Random Primers, where first-
and second-strand cDNAs were synthesized using ProtoScript II Reverse
Transcriptase and Second Strand Synthesis Enzyme Mix, respectively. The
double-stranded cDNA was purified by beads and further treated with End Prep
Enzyme Mix. Size selection of adaptor-ligated DNA was then performed using
beads, and fragments of ~400 bp (with an approximate insert size of 300 bp) were
recovered. The samples were amplified by polymerase chain reaction (PCR) using
P5 and P7 primers. Subsequently, PCR products were cleaned up using beads,
validated using a Qsep100 (Bioptic, Taiwan), and quantified by a Qubit 3.0
Fluorometer (Invitrogen, Carlsbad, CA, USA). Libraries with different indices
were multiplexed and loaded on an Illumina Novaseq instrument according to the
manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was
performed using a 2 × 150 paired-end configuration. Image analysis and base
calling were conducted by NovaSeq Control Software + OLB + GAPipeline-1.6
(Illumina) on the NovaSeq instrument. After sequencing, the reads were analyzed
on Partek Flow software (Partek Inc., St. Louis, MO, USA). In essence, the reads
were mapped to the hg38 reference genome using STAR aligner. The aligned reads
were quantified and annotated based on RefSeq transcripts (version 99).
Differential gene expression analysis was performed using the Partek GSA
algorithm, and overrepresentation enrichment analysis was performed using Kyoto
Encyclopedia of Genes and Genomes Pathways (Homo sapiens).
FLOW CYTOMETRIC ANALYSIS
Before surface marker staining, all cells were stained with a dye provided in
the LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Sigma-Aldrich, Saint Louis, MO,
USA) at room temperature for 15 min. To examine the presence of CAR on NK cell
surface, biotinylated protein L (GeneScript, Piscataway, NJ, USA) and
Allophycocyanin (APC)–conjugated streptavidin (BioLegend, San Diego, CA, USA)
were used. In essence, the cells were stained with the biotinylated protein L
(10 μg/ml) at 4°C for 15 min. Afterward, the cells were washed with
phosphate-buffered saline (PBS) and incubated with the APC-conjugated
streptavidin (5 μg/ml) at 4°C for 30 min.
To determine the binding between mouse PD-L1 recombinant protein and CAR on NK
cell surface, the cells were incubated with mouse PD-L1 protein-His tag (10
μg/ml; Sino Biological Inc., China) at room temperature for 1 hour, followed by
an incubation of APC-conjugated anti-His tag (R&D Systems, Minneapolis, MN, USA)
at 4°C for 30 min.
The expression level of human PD-L1 on different cells was examined using
anti-human PD-L1 (clone 29E.2A3) and isotype control antibodies (mouse IgG2b,
clone MG2b-57). All FACS antibodies are purchased from BioLegend, unless
otherwise specified. The following antibodies were used in this study: (i)
anti-human antibodies: CD3 (clone UCHT1, BD Biosciences, Franklin Lakes, NJ,
USA), CD4 (clone SK3, BD Biosciences), CD8 (clone SK1), CD11b (clone ICRF44),
CD14 (clone HCD14), CD15 (clone W6D3), CD16 (clone 3G8), CD19 (clone HIB19),
CD25 (clone 2A3, BD Biosciences), CD33 (clone WM53, BD Biosciences), CD34 (clone
581), CD45 (clone HI30, BD Biosciences), CD56 (clone HCD56), CD57 (clone
QA17A04), CD94 (clone 18d3), CD107a (clone H4A3), CD127 (clone A019D5), CD133/2
(clone 293C3, Miltenyi Biotec, Germany), BTLA (clone MIH16), CTLA-4 (clone
BNI3), HLA-DR (clone L243, BD Biosciences), LAG-3 (clone 3DS223H, Thermo Fisher
Scientific), NKG2A (clone REA110, Miltenyi Biotec), NKG2D (clone REA797,
Miltenyi Biotec), NKp30 (clone REA823, Miltenyi Biotec), NKp44 (clone 2.29,
Miltenyi Biotec), NKp46 (clone 9E2/NKp46, BD Biosciences), PD-1 (clone EH12.1,
BD Biosciences), TIGIT (clone MBSA43, Thermo Fisher Scientific), and TIM-3
(clone F38-2E2); (ii) anti-mouse antibody: CD45 (clone A20); (iii) isotype
control antibodies: mouse G1 (MOPC-21) and rat IgG2a (clone RTK2758).
Flow cytometric analysis was performed on a LSRII flow cytometer using FACSDiva
software (Becton Dickinson, Sparks, MD, USA). Hundred thousand events were
collected per sample and analyzed using FlowJo software version 10 (TreeStar,
Ashland, OR, USA).
CYTOKINE ARRAY
The relative protein expressions of intratumoral cytokines from NSG and
humanized mice were analyzed using the Proteome Profiler Human Cytokine Array
Kit (R&D Systems) according to the manufacturer’s instructions. Briefly, NPC-PDX
were harvested and tumor fragments were digested in radioimmunoprecipitation
assay buffer (Thermo Fisher Scientific). Protein concentration was determined by
Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules,
CA, USA), and 200 μg of protein was applied to each membrane. The cytokines were
detected using streptavidin–horseradish peroxidase and chemiluminescent reagent
mix. Subsequently, the pixel density of each dot was determined using ImageJ
software (National Institutes of Health, Bethesda, MD, USA), and the readout
from each sample dot was normalized using the average reading of six positive
control dots.
QUANTIFICATION OF PLASMA CYTOKINES, CHEMOKINES, AND IMMUNE CHECKPOINT MOLECULES
Plasma cytokines and chemokines levels and immune checkpoint biomarkers from NSG
and humanized mice were determined by LEGENDplex Human CD8/NK Panel, Human
Inflammation Panel 1, and HU Immune Checkpoint Panel 1 (BioLegend) according to
the manufacturer’s instructions. The profile was analyzed by flow cytometry and
LEGENDplex data analysis software (BioLegend).
QUANTITATIVE REAL-TIME PCR
NPC-PDX in NSG mice and humanized mice were harvested. Tumors were first
digested into single-cell suspension using the Tumor Dissociation Kit, Human
(Miltenyi Biotec). Viable human cells in the cell suspensions were then selected
using the Dead Cell Removal Kit (Miltenyi Biotec) and the Mouse Cell Depletion
Kit (Miltenyi Biotec), followed by incubating with CD45 (TIL) MicroBeads, Human
(Miltenyi Biotec). The eluted tumor cells were collected. Total RNA from the
tumor cells was extracted using TRIzol Reagent and subjected to reverse
transcription using the QuantiTect Reverse Transcription Kit (Qiagen, Germany).
Gene expressions of human PD-L1 and human IFN-γ were quantified by real-time PCR
using SsoFast EvaGreen supermix (Bio-Rad Laboratories). The reaction was
performed on CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories).
Relative gene expressions of the human PD-L1 and human IFN-γ were analyzed using
the 2(-ΔΔCt) method and normalized relative to human β-actin. Primer sequences
used are listed as follows: human PD-L1 forward, 5′-GCTGCACTAATTGTCTATTGGGA-3′;
human PD-L1 reverse, 5′-AATTCGCTTGTAGTCGGCACC-3′; human IFN-γ forward,
5′-TCGGTAACTGACTTGAATGTCCA-3′; human IFN-γ reverse, 5′-TCGCTTCCCTGTTTTAGCTGC-3′;
human β-actin forward, 5′-GGGTCAGAAGGATTCCTATG-3′; human β-actin reverse,
5′-GGTCTCAAACATGATCTGGG-3′.
Similarly, total RNA from liver and lung from PDX-bearing humanized mice was
extracted using TRIzol Reagent and subjected to reverse transcription using the
QuantiTect Reverse Transcription Kit. Gene expression of mouse PD-L1 was
quantified by real-time PCR, and relative gene expression of the mouse PD-L1 was
analyzed as described above. Primer sequences used are listed as follows: mouse
PD-L1 forward, 5′-GCTCCAAAGGACTTGTACGTG-3′; mouse PD-L1 reverse,
5′-TGATCTGAAGGGCAGCATTTC-3′; mouse β-actin forward, 5′-ACAGAGCCTCGCCTTTGCC-3′;
mouse β-actin reverse, 5′-GATATCATCATCCATGGTGAGCTGG-3′.
IMMUNOHISTOCHEMICAL STAINING
Tumors were formalin-fixed (Sigma-Aldrich) at room temperature for 24 hours,
paraffin-embedded (Leica, Germany), sliced into 5-μm sections, and stained for
human PD-L1, human CD3, and human NCR1 using their respective antibodies
purchased from Abcam (Cambridge, MA, USA), followed by hematoxylin counterstain
(Thermo Fisher Scientific). The stained images were captured using a ZEN
fluorescence microscope (Zeiss, Germany) with ZEN 2 acquisition software (Zen
Blue Version, Zeiss).
IMMUNOFLUORESCENCE STAINING
Tumors were embedded and frozen. Fixed cryostat sections were incubated with
anti-human CD3 antibody conjugated with Phycoerythrin (PE) (Miltenyi Biotec),
anti-human CD45 antibody conjugated with PE (Miltenyi Biotec), anti-human CD56
antibody conjugated with PE (Miltenyi Biotec), and/or unconjugated anti-human
PD-L1 antibody. After washing, slides were probed with Alexa Fluor 488
dye–conjugated secondary antibodies (Thermo Fisher Scientific), whenever
indicated, mounted with ProLong Diamond Antifade Mountant (Thermo Fisher
Scientific), and the signals were examined using an Eclipse Ti-E fluorescence
microscope (Nikon, Tokyo, Japan).
To confirm successful transduction of CAR in NK92 cells, nontransduced WT NK92
and CAR NK92 cells (1 × 104 cells in 100 μl of PBS) were seeded onto a
microscopic slide by cytocentrifugation at 500 rpm for 5 min using Shandon
Cytospin centrifuge (Shandon Scientific Ltd., UK). The cells were then
air-dried, fixed with 4% paraformaldehyde for 15 min, and mounted with ProLong
Diamond Antifade Mountant. GFP signal in the cells was examined using an Eclipse
Ti-E fluorescence microscope.
CELL CULTURE
Human non-Hodgkin’s lymphoma NK92 cells were cultured in complete RPMI 1640
medium (Cytiva Life Sciences, Marlborough, MA, USA) containing human IL-2
recombinant protein (200 IU/ml; Miltenyi Biotec) and 10% fetal bovine serum
(FBS; Gibco, Grand Island, NY, USA). Human hepatocellular carcinoma HepG2 cells,
human embryonic kidney (HEK) 293T cells, and lung carcinoma A549 cells were
cultured in complete Dulbecco’s modified Eagle’s medium (Cytiva Life Sciences)
containing 10% FBS. Human IFN-γ (Miltenyi Biotec) was supplemented at a
concentration of 10 μg/ml medium, whenever indicated, to induce the expression
of PD-L1 on the HepG2 cells or PHH. Human NPC C666-1 cells, erythroleukemic
K-562 cells, and pancreatic carcinoma Panc 08.13 cells were cultured in complete
RPMI 1640 medium containing 10% FBS. All the cell lines were purchased from
American Type Culture Collection.
Primary NK cells were differentiated from human CD34+ HSC using the StemSpan NK
Cell Generation Kit according to the manufacturer’s instructions (STEMCELL
Technologies). The purity and phenotype of the cells were examined by flow
cytometry after 28-day differentiation (fig. S8B). Human sPD-L1 recombinant
protein (Thermo Fisher Scientific) was supplemented at a concentration of 500
pg/ml medium, whenever indicated, to treat the primary NK cells.
Primary T cells isolated from cord blood (Lonza Biosciences) were seeded in a
24-well plate and maintained in TexMACS medium (Miltenyi Biotec) supplemented
with human IL-2 (20 IU/ml). Human sPD-L1 recombinant protein (500 pg/ml medium)
and/or T cell TransAct (20 μl/ml medium; Miltenyi Biotec) was supplemented at a
concentration of 500 pg/ml medium, whenever indicated, to treat the primary T
cells. All the cell lines and primary cells were incubated in a humidified
incubator containing 5% CO2 at 37°C and passaged, whenever necessary, using
aseptic technique.
DESIGN OF CAR CONSTRUCT AND LENTIVIRAL TRANSDUCTION
The construct consisted of a ScFv domain, which targeted PD-L1, two
costimulatory domains (CD28 and 4-1BB), and a CD3ζ activation domain. Lentivirus
was obtained from the supernatant of HEK293T cells, followed by cotransfection
of the CAR construct and third-generation viral packaging plasmids using
Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA).
To transduce NK92 cells, 5 × 105 cells [in 400 μl of RPMI 1640, supplemented
with polybrene (8 μg/ml)] were seeded in a 24-well plate, and 100 μl of viral
supernatant was added. The plate was centrifuged at 2000g at 32°C for 2 hours,
and the cells were incubated in a humidified incubator containing 5% CO2 at
37°C. Puromycin (10 μg/ml; Gibco) was added to the NK92 cells after 3 days of
transduction to establish a stable CAR-expressing NK92 cell line. For the
generation of CAR-expressing primary NK cells, differentiated NK cells were
transduced similar to NK92. To transduce primary T cells, the cells were seeded
in a 24-well plate and activated in TexMACS medium containing T cell TransAct
(20 μl/ml) and human IL-2 (20 IU/ml) according to the manufacturer’s
instructions. Two days after activation, lentiviral particle (100 μl) and
polybrene (8 μg/ml) were added to the cells, and the plate was centrifuged at
2000g at 32°C for 2 hours. The cells were harvested for downstream applications
3 days after transduction. The expression of CAR was verified by flow cytometry
before each experiment.
CELL LYSIS ASSAY
To determine NK cell cytotoxicity on their target cells, CytoTox 96
Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) was performed
according to the manufacturer’s instructions. Briefly, NK92 cells or primary NK
cells (effector cells) were cocultured with HepG2, A549, C666-1, K-562, and Panc
08.13, isolated cells from NPC-PDX and PHH (target cells) at the indicated
effector cell to target cell ratio (E:T) ratio at 37°C for 4 hours. After
incubation, 50 μl of the cell-free supernatant was incubated with 50 μl of
substrate solution at room temperature for 30 min, followed by the addition of
stop solution. The absorbance at 490 nm was measured using an Infinite M200
plate reader (Tecan, Switzerland). The percentage of cytotoxicity was calculated
using the following formula: % cytotoxicity = 100 × (corrected reading from test
well − corrected reading from untreated well)/(corrected maximum release of
lactate dehydrogenase control).
MEASUREMENT OF DNA FRAGMENTATION
The induction of apoptosis was determined using the Cell Death Detection
ELISAPLUS Kit (Sigma-Aldrich), and the experiments were conducted according to
the manufacturer’s instructions. NK92 cells or primary NK cells (5 × 105/ml)
were cocultured with HepG2, A549, C666-1, K-562, and Panc 08.13 cells and
isolated tumor cells from NPC-PDX (5 × 105/ml) in a flat-bottomed 96-well plate
at 37°C for 4 hours. The cell-free supernatant was transferred to a
streptavidin-coated 96-well microtiter plate, and the absorbance at 405 nm was
recorded by an Infinite M200 plate reader. The degree of apoptosis was expressed
as an enrichment factor, which was calculated as follows: Enrichment factor =
Absorbance of the well with CAR-NK cells and cancer cells/absorbance of the well
with WT-NK cells and cancer cells.
CD107A EXPRESSION ON NK CELLS AND ELISA FOR HUMAN IFN-Γ
NK92 cells or primary NK cells (5 × 105/ml) were cocultured with HepG2, A549,
C666-1, K-562, and Panc 08.13 cells and isolated tumor cells from NPC-PDX (5 ×
105/ml) in a flat-bottomed 96-well plate at 37°C for 4 hours. After incubation,
the cells and the supernatant were collected to examine the expression level of
CD107a on the NK cells and the release of IFN-γ, respectively. In short, the
cells were stained with anti-human CD16, CD45, CD56, and CD107a antibodies at
4°C for 15 min before FACS analysis. The supernatant was centrifuged to remove
cell debris, and the release of IFN-γ was quantified by ELISA MAX Deluxe Set
Human IFN-γ (BioLegend) according to the manufacturer’s instructions. The
absorbance at 450 nm was recorded using an Infinite M200 plate reader.
IN VIVO NK CELL–MEDIATED THERAPY
To evaluate antitumor efficacy of cell-mediated therapy, 5 × 105 C666-1 cells or
A549 cells, a piece of NPC-PDX or LAC-PDX (3 mm by 3 mm fragment) was inoculated
into the right flank of NSG mice and humanized mice. Once the tumor grew, 2.5 ×
106 WT NK92, CAR NK92, WT pNK, CAR pNK, or CAR pT cells and nivolumab or
ipilimumab (5 mg/kg mouse body weight) were injected intravenously into the mice
every week for four consecutive weeks. Human IL-2 (1 × 104 IU per mouse) was
administered together with the NK92 cells to promote NK cell survival and
maintain their cytotoxicity, as described in an earlier study (17, 61). Tumor
measurement was performed weekly using a caliper as reported previously (22).
Two days after the last injection, tumors were harvested and tumor weight was
measured. Blood samples were collected in EDTA tubes (Greiner Bio-One, Monroe,
NC, USA) through cardiac puncture.
TUNEL ASSAY
Tumors were formalin-fixed, paraffin-embedded, and sliced as described above,
and the presence of apoptotic cells was visualized using the In Situ Cell Death
Detection Kit, TMR red (Sigma-Aldrich) according to the manufacturer’s
instructions. Briefly, tumor sections were dewaxed, rehydrated, and incubated
with citrate buffer (pH 6) to achieve heat-induced epitope retrieval. After
incubation, the sections were probed with the TUNEL reaction mixture at 4°C
overnight. The slides were washed with PBS, mounted with ProLong Diamond
Antifade Mountant, and imaged using an Eclipse Ti-E fluorescence microscope. The
relative intensity of the signal was quantified with ImageJ software.
HEMATOXYLIN AND EOSIN STAINING
Liver and lungs from PDX-bearing humanized mice injected with saline, CAR pNK
cells, or CAR pT cells were harvested 2 days after the last treatment. The
samples were formalin-fixed, paraffin-embedded, sliced into 5-μm sections, and
stained with hematoxylin and eosin (Thermo Fisher Scientific). Pathological
evaluations were performed by an experienced research veterinary pathologist at
the Advanced Molecular Pathology Laboratory from Institute of Molecular and Cell
Biology (IMCB).
STATISTICAL ANALYSIS
All statistical analyses were performed using GraphPad Prism version 9 (GraphPad
Software Inc., La Jolla, CA, USA), and data are expressed as means ± SEs.
Unpaired Student’s t test, one-way analysis of variance (ANOVA) with post hoc
Tukey’s multiple comparisons test and two-way ANOVA were used, whenever
appropriate, and Kaplan-Meier survival curves were analyzed by a log-rank test.
The differences are considered statistically significant when P < 0.05.
ACKNOWLEDGMENTS
Funding: This work was supported by National Research Foundation Singapore
Fellowship NRF-NRFF2017-03 (to Q.C.), A*STAR BMRC Central Research Fund (ATR)
(to Q.C.), 2020 A*STAR Career Development Award 20701/C210112003 (to W.N.L.),
NRF-ISF joint grant NRF2019-NRF-ISF003-3127 (to Q.C.), Industry Alignment
Fund–Industry Collaboration Projects (IAF-ICP) Grant ICP-2000120 (to Q.C.),
National Medical Research Council Clinician Scientist Individual Research Grant
CIRG19may-0051 (to D.W.-T.L.), Industry Alignment Fund–Industry Pre-positioning
Program (IAF-PP) Grant H18/01/a0/022 (to Q.C.), and National Medical Research
Council-OF-LCG OFLCG19May-0038 (to Q.C.).
Author contributions: Conceptualization: W.N.L. and Q.C. Methodology: W.N.L. and
Q.C. Investigation: W.N.L., W.Y.S., S.L.H., S.Y.F., M.X.Y.W., W.W.S.T., S.Y.T.,
J.K.L.O., R.R., M.L., J.Y.C., L.S., and J.P.S.Y. Visualization: W.N.L. and
J.K.L.O. Funding acquisition: W.N.L., D.W.-T.L., and Q.C. Project
administration: N.G.I., D.W.-T.L., and Q.C. Supervision: D.W.-T.L. and Q.C.
Writing—original draft: W.N.L. Writing—review and editing: W.N.L., W.Y.S.,
S.L.H., N.G.I., D.W.-T.L., and Q.C.
Competing interests: Q.C. is the scientific founder of two biotech companies.
The other authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in
the paper are present in the paper and/or the Supplementary Materials.
SUPPLEMENTARY MATERIALS
THIS PDF FILE INCLUDES:
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Science Advances
Volume 8 | Issue 47
November 2022
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Association for the Advancement of Science. No claim to original U.S. Government
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Accepted: 5 October 2022
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ACKNOWLEDGMENTS
Funding: This work was supported by National Research Foundation Singapore
Fellowship NRF-NRFF2017-03 (to Q.C.), A*STAR BMRC Central Research Fund (ATR)
(to Q.C.), 2020 A*STAR Career Development Award 20701/C210112003 (to W.N.L.),
NRF-ISF joint grant NRF2019-NRF-ISF003-3127 (to Q.C.), Industry Alignment
Fund–Industry Collaboration Projects (IAF-ICP) Grant ICP-2000120 (to Q.C.),
National Medical Research Council Clinician Scientist Individual Research Grant
CIRG19may-0051 (to D.W.-T.L.), Industry Alignment Fund–Industry Pre-positioning
Program (IAF-PP) Grant H18/01/a0/022 (to Q.C.), and National Medical Research
Council-OF-LCG OFLCG19May-0038 (to Q.C.).
Author contributions: Conceptualization: W.N.L. and Q.C. Methodology: W.N.L. and
Q.C. Investigation: W.N.L., W.Y.S., S.L.H., S.Y.F., M.X.Y.W., W.W.S.T., S.Y.T.,
J.K.L.O., R.R., M.L., J.Y.C., L.S., and J.P.S.Y. Visualization: W.N.L. and
J.K.L.O. Funding acquisition: W.N.L., D.W.-T.L., and Q.C. Project
administration: N.G.I., D.W.-T.L., and Q.C. Supervision: D.W.-T.L. and Q.C.
Writing—original draft: W.N.L. Writing—review and editing: W.N.L., W.Y.S.,
S.L.H., N.G.I., D.W.-T.L., and Q.C.
Competing interests: Q.C. is the scientific founder of two biotech companies.
The other authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in
the paper are present in the paper and/or the Supplementary Materials.
AUTHORS
AFFILIATIONSEXPAND ALL
WAI NAM LIU HTTPS://ORCID.ORG/0000-0002-4323-4566
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition,
Investigation, Methodology, Project administration, Resources, Validation,
Visualization, Writing - original draft, and Writing - review & editing.
View all articles by this author
WING YAN SO HTTPS://ORCID.ORG/0000-0001-9647-3686
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation and Resources.
View all articles by this author
SARAH L. HARDEN HTTPS://ORCID.ORG/0000-0002-1029-8440
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation, Writing - original draft, and Writing - review & editing.
View all articles by this author
SHIN YIE FONG
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation and Methodology.
View all articles by this author
MELISSA XIN YU WONG HTTPS://ORCID.ORG/0000-0002-6217-5258
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation, Methodology, and Writing - review & editing.
View all articles by this author
WILSON WEI SHENG TAN HTTPS://ORCID.ORG/0000-0003-3027-1162
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation, Methodology, and Resources.
View all articles by this author
SUE YEE TAN
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation, Methodology, and Resources.
View all articles by this author
JESSICA KAI LIN ONG HTTPS://ORCID.ORG/0000-0002-6684-6370
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation and Methodology.
View all articles by this author
RAVISANKAR RAJARETHINAM HTTPS://ORCID.ORG/0000-0001-9249-7840
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation and Methodology.
View all articles by this author
MIN LIU
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation, Methodology, Project administration, and Resources.
View all articles by this author
JIA YING CHENG
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Investigation and Methodology.
View all articles by this author
LISDA SUTEJA HTTPS://ORCID.ORG/0000-0002-4548-2766
Duke-NUS Medical School, 169857, Singapore.
Roles: Methodology, Validation, and Visualization.
View all articles by this author
JOE POH SHENG YEONG HTTPS://ORCID.ORG/0000-0002-6674-7153
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Roles: Formal analysis, Investigation, Methodology, Resources, Visualization,
and Writing - review & editing.
View all articles by this author
N. GOPALAKRISHNA IYER
Duke-NUS Medical School, 169857, Singapore.
Department of Head and Neck Surgery, National Cancer Centre Singapore, 169610,
Singapore.
Roles: Methodology, Resources, and Writing - review & editing.
View all articles by this author
DARREN WAN-TECK LIM* HTTPS://ORCID.ORG/0000-0002-4655-0206
QCHEN@IMCB.A-STAR.EDU.SG
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Division of Medical Oncology, National Cancer Center Singapore, 169610,
Singapore.
Roles: Funding acquisition, Investigation, Project administration, Resources,
Supervision, Validation, and Writing - review & editing.
View all articles by this author
QINGFENG CHEN* HTTPS://ORCID.ORG/0000-0001-6437-1271 QCHEN@IMCB.A-STAR.EDU.SG
Institute of Molecular and Cell Biology, Agency for Science, Technology and
Research, 138673, Singapore.
Department of Microbiology and Immunology, Yong Loo Lin School of Medicine,
National University of Singapore, 117593, Singapore.
Singapore Immunology Network, Agency for Science, Technology and Research,
138648, Singapore.
Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition,
Investigation, Methodology, Project administration, Resources, Supervision,
Validation, Visualization, Writing - original draft, and Writing - review &
editing.
View all articles by this author
FUNDING INFORMATION
National Research Foundation Singapore Fellowship: NRF-NRFF2017-03
A*STAR BMRC Central Research Fund (ATR)
2020 A*STAR Career Development Award: 20701/C210112003
NRF-ISF joint grant: NRF2019-NRF-ISF003-3127
Industry Alignment Fund-Industry Collaboration Projects (IAF-ICP) Grant:
ICP-2000120
National Medical Research Council Clinician Scientist Individual Research Grant:
CIRG19may-0051
Industry Alignment Fund-Industry Pre-positioning Program (IAF-PP) Grant:
H18/01/a0/022
National Medical Research Council- OF-LCG: OFLCG19May-0038
NOTES
*
Corresponding author. Email: qchen@imcb.a-star.edu.sg (Q.C.);
darren.lim.w.t@singhealth.com.sg (D.W.-T.L.)
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* Wai Nam Liu et al.
,
Successful targeting of PD-1/PD-L1 with chimeric antigen receptor-natural killer
cells and nivolumab in a humanized mouse cancer model.Sci.
Adv.8,eadd1187(2022).DOI:10.1126/sciadv.add1187
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CITED BY
1. * John Maher,
* David M. Davies,
CAR Based Immunotherapy of Solid Tumours—A Clinically Based Review of Target
Antigens, Biology, 12, 2, (287),
(2023).https://doi.org/10.3390/biology12020287
Crossref
2. * Martin J. Raftery,
* Alexander Sebastian Franzén,
* Gabriele Pecher,
CAR NK Cells: The Future Is Now, Annual Review of Cancer Biology, 7, 1,
(229-246), (2023).https://doi.org/10.1146/annurev-cancerbio-061521-082320
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FiguresMultimedia
FIGURES
Fig. 1. TME in humanized mice is distinct from that in immunocompromised NSG
mice.
(A) Schematic description showing the methodology to generate humanized mice and
workflow for RNA sequencing using NPC-PDX from humanized mice and NSG mice. (B)
Heatmap of DE genes from NPC-PDX between humanized mice (n = 3) and NSG mice (n
= 3) is shown. (C) Signaling pathway analysis of DE genes. (D) Relative protein
expressions of CXCL-10, IL-8, and PAI-1 in tumor lysate from NSG mice (n = 4)
and humanized mice (n = 4). Data are expressed as means ± SEM. **P < 0.01,
two-tailed unpaired t test. (E) Volcano plot of selected DE genes between
humanized mice and NSG mice is shown. (F and G) Surface expressions of PD-1 on
CD3+CD8+ T cells and CD3−CD16+CD56+ NK cells in circulation (blue histogram) and
in tumor (red histogram) were analyzed by flow cytometry. Data are expressed as
means ± SEM. **P < 0.01 and ***P < 0.001, two-tailed unpaired t test (n = 4 for
both groups). MFI, mean fluorescence intensity. (H) Relative mRNA expression
level of human PD-L1 on tumor cells isolated from NPC-PDX from NSG mice (n = 5)
and humanized mice (n = 5). Data are expressed as means ± SEM. ***P < 0.001,
two-tailed unpaired t test. (I) Surface expression of human PD-L1 on tumor
cells. Isotype control (black histogram), cells harvested from NSG mice (blue
histogram), and humanized mice (red histogram) were analyzed by flow cytometry.
(J) Representative photomicrographs showing immunohistochemical staining of
human PD-L1 on NPC-PDX from NSG mice and humanized mice. Scale bars, 100 μm. n =
3 per group.
GO TO FIGUREOPEN IN VIEWER
Fig. 2. Enhanced in vitro antitumor activity in CAR-expressing NK92 cells.
(A) Design of CAR construct. Puro, puromycin. SFFV, spleen focus-forming virus.
(B) Surface expression of human PD-L1 on HepG2 (untreated or IFN-γ–treated),
A549, C666-1, and Panc 08.13 cells. Cells stained with isotype control (blue
histogram) and anti–PD-L1 antibody (red histogram) were examined by flow
cytometry. WT NK92 cells (n = 4) and CAR NK92 cells (n = 4) were cocultured with
various human cancer cell lines at the indicated E:T ratios (C) or at a 10:1
ratio of E:T (D to F). (C) The percentages of NK cell cytotoxicity on different
cancer cell lines are shown. (D) The extent of apoptosis as indicated by
enrichment factor. (E) CD107a expression was determined in anti-CD56–labeled NK
cells after stimulation by different cancer cell lines. (F) Net release of IFN-γ
after stimulation by different cancer cell lines. Data from (D) to (F) are
expressed as means ± SEM. **P < 0.01 and ***P < 0.001, two-tailed unpaired t
test.
GO TO FIGUREOPEN IN VIEWER
Fig. 3. CAR-expressing NK92 cells exhibit better anti-solid tumor effect in NSG
mice.
(A) Schematic description of tumor inoculation and cell-mediated therapy in NSG
mice. The mice were either injected with C666-1 cells or transplanted with
NPC-PDX, which usually took 1 to 2 weeks to form visible tumors. (B to D) NSG
mice were inoculated with C666-1 cells. After the formation of visible tumors,
the mice were injected with saline (n = 15), WT NK92 cells (n = 15), and CAR
NK92 cells (n = 18). (B) Representative image of tumors. (C) Tumor volumes are
shown. Data are expressed as means ± SEM. *P < 0.05 and **P < 0.01, one-way
analysis of variance (ANOVA) followed by multiple comparisons, when CAR NK92
cell–injected group is compared to saline-injected group. (D) Tumor weights are
shown. Data are combined from two independent experiments and expressed as means
± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA followed by multiple comparisons.
(E to G) NSG mice were transplanted with NPC-PDX. After the formation of visible
tumors, the mice were injected with saline (n = 20), WT NK92 cells (n = 20), and
CAR NK92 cells (n = 21). (E) Representative image of tumors. (F) Tumor volumes
are shown. Data are expressed as means ± SEM. **P < 0.01, one-way ANOVA followed
by multiple comparisons, when CAR NK92 cell–injected group is compared to
saline-injected group. (G) Tumor weights are shown. Data are combined from three
independent experiments using PDX derived from three patients and expressed as
means ± SEM. **P < 0.01, one-way ANOVA followed by multiple comparisons.
GO TO FIGUREOPEN IN VIEWER
Fig. 4. Phenotypic and functional characterization of CAR-expressing HSC-derived
NK cells.
(A) Surface expressions of NK cell–specific markers under the gate of human
CD45+ and CD56+ subset. Cells stained with isotype control (blue histogram) and
indicated PE-conjugated antibodies (red histogram) were examined by flow
cytometry. CB-NK cells (n = 4) and pNK cells (n = 4) were cocultured with K-562
cells at the indicated E:T ratios (B) or at a 10:1 ratio of E:T (C to E). (B)
The percentages of NK cell cytotoxicity on K-562 cells are shown. (C) The extent
of apoptosis as indicated by enrichment factor. (D) CD107a expression was
determined in anti-CD56–labeled NK cells after stimulation by K-562 cells. (E)
Net release of IFN-γ after stimulation by K-562 cells. WT pNK cells (n = 4) and
CAR pNK cells (n = 4) were cocultured with C666-1 cells or tumor cells isolated
from NPC-PDX at the indicated E:T ratios (F) or at a 10:1 ratio of E:T (G to I).
(F) The percentages of NK cell cytotoxicity on tumor cells are shown. (G) The
extent of apoptosis. (H) CD107a expression on NK cells after stimulation by
tumor cells. (I) Net release of IFN-γ after stimulation by tumor cells. Data
from (C) to (E) and (G) to (I) are expressed as means ± SEM. *P < 0.05, **P <
0.01, and ***P < 0.001, two-tailed unpaired t test.
GO TO FIGUREOPEN IN VIEWER
Fig. 5. CAR-expressing primary NK cells suppress in vivo growth of NPC-PDX more
effectively.
NSG mice were transplanted with NPC-PDX. After the formation of visible tumors,
the mice were injected with saline (n = 5), WT pNK cells (n = 5), CAR pNK cells
(n = 5), and CAR pT cells (n = 5). (A) Representative image of tumors. (B) Tumor
volumes are shown. Data are expressed as means ± SEM, and comparative
statistical analysis were done by one-way ANOVA followed by multiple
comparisons. *P < 0.05, when CAR pNK cell–injected group is compared to
saline-injected group; #P < 0.05 and ##P < 0.01, when CAR pNK cell–injected
group is compared to CAR pT cell–injected group. (C) Tumor weights are shown.
Data are expressed as means ± SEM. *P < 0.05 and ***P < 0.001, one-way ANOVA
followed by multiple comparisons. (D and E) Representative FACS plots showing
the infiltration of CD3−CD56+ NK cells (D) and CD3+CD56− T cells (E) in tumors.
(F) Representative photomicrographs showing immunohistochemical staining in
tumors. The presence of NK and T cells was indicated by the expression of NCR1
and CD3, respectively. Scale bars, 100 μm. n = 5 per group. (G) Representative
photomicrographs showing TUNEL in tumors. Apoptotic cells were labeled by red
fluorescence. Scale bars, 100 μm. n = 5 per group. DAPI,
4′,6-diamidino-2-phenylindole. (H) Signal intensity of TUNEL is quantified from
the images, and data are expressed as means ± SEM. ***P < 0.001, one-way ANOVA
followed by multiple comparisons.
GO TO FIGUREOPEN IN VIEWER
Fig. 6. Evaluation of antitumor efficacy and safety of CAR pNK cells in
NPC-PDX–bearing humanized mice.
(A) Schematic description of PDX engraftment and cell-mediated therapy in
humanized mice. (B to D) Humanized mice were transplanted with NPC-PDX. After
the formation of visible tumors, the mice were injected with saline (n = 9), WT
pNK cells (n = 10), CAR pNK cells (n = 13), and CAR pT cells (n = 7). (B)
Representative image of tumors. (C) Tumor volumes are shown. Data are expressed
as means ± SEM. (D) Tumor weights are shown. Data are expressed as means ± SEM.
*P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by multiple
comparisons. (E) Survival curve of NSG mice after treatment with saline (n =
36), WT pNK cells (n = 34), CAR pNK cells (n = 38), and CAR pT cells (n = 20).
Data are combined from at least three independent experiments. (F) Survival
curve of humanized mice after treatment with saline (n = 20), WT pNK cells (n =
12), CAR pNK cells (n = 28), and CAR pT cells (n = 22). Data are combined from
at least three independent experiments. The survival rate of mice between CAR
pNK cell–injected group and CAR pT cell–injected group is compared by
Kaplan-Meier analysis via a log-rank test. **P < 0.01. (G to I) Blood samples
were collected from humanized mice 2 days after the last treatment with saline
(n = 12 for IL-6 and IL-10; n = 19 for IFN-γ), WT pNK cells (n = 11), CAR pNK
cells (n = 16), and CAR pT cells (n = 11). Plasma concentrations of IFN-γ (G),
IL-6 (H), and IL-10 (I) were determined by LEGENDplex. Data are expressed as
means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by
multiple comparisons.
GO TO FIGUREOPEN IN VIEWER
Fig. 7. Dual therapy using CAR pNK cells and nivolumab results in a synergistic
antitumor response on NPC-PDX in humanized mice.
(A) Heatmap of DE genes from NPC-PDX between CAR pNK cell–injected mice (n = 3)
and saline-injected mice (n = 3) is shown. (B) Signaling pathway analysis of DE
genes. (C) Schematic description of PDX engraftment, ICI therapy alone, or in
combination with CAR pNK cell–mediated therapy in humanized mice. (D to G)
Humanized mice were transplanted with NPC-PDX. After the formation of visible
tumors, the mice were injected with saline (n = 23), ipilimumab (n = 15),
nivolumab (n = 13), CAR pNK cells (n = 22), CAR pNK cells plus ipilimumab (n =
22), and CAR pNK cells plus nivolumab (n = 26). (D and E) Representative image
of tumors. (F) Tumor volumes are shown. Data are expressed as means ± SEM. **P <
0.01 and ***P < 0.001, when CAR pNK cells plus nivolumab–treated group is
compared to saline-injected group, @P < 0.05, when CAR pNK cells plus
ipilimumab–treated groups are compared to saline-injected group, #P < 0.05, when
CAR pNK cell–treated groups are compared to saline-injected group, one-way ANOVA
followed by multiple comparisons. (G) Tumor weights are shown. Data are combined
from at least three independent experiments using PDX derived from three
patients and expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001,
one-way ANOVA followed by multiple comparisons.
GO TO FIGUREOPEN IN VIEWER
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CURRENT ISSUE
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HomeScience AdvancesVol. 8, No. 47Successful targeting of PD-1/PD-L1 with
chimeric antigen receptor-natural killer cells and nivolumab in a humanized
mouse cancer model
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FiguresTables
View figure
Fig. 1
Fig. 1. TME in humanized mice is distinct from that in immunocompromised NSG
mice.
(A) Schematic description showing the methodology to generate humanized mice and
workflow for RNA sequencing using NPC-PDX from humanized mice and NSG mice. (B)
Heatmap of DE genes from NPC-PDX between humanized mice (n = 3) and NSG mice (n
= 3) is shown. (C) Signaling pathway analysis of DE genes. (D) Relative protein
expressions of CXCL-10, IL-8, and PAI-1 in tumor lysate from NSG mice (n = 4)
and humanized mice (n = 4). Data are expressed as means ± SEM. **P < 0.01,
two-tailed unpaired t test. (E) Volcano plot of selected DE genes between
humanized mice and NSG mice is shown. (F and G) Surface expressions of PD-1 on
CD3+CD8+ T cells and CD3−CD16+CD56+ NK cells in circulation (blue histogram) and
in tumor (red histogram) were analyzed by flow cytometry. Data are expressed as
means ± SEM. **P < 0.01 and ***P < 0.001, two-tailed unpaired t test (n = 4 for
both groups). MFI, mean fluorescence intensity. (H) Relative mRNA expression
level of human PD-L1 on tumor cells isolated from NPC-PDX from NSG mice (n = 5)
and humanized mice (n = 5). Data are expressed as means ± SEM. ***P < 0.001,
two-tailed unpaired t test. (I) Surface expression of human PD-L1 on tumor
cells. Isotype control (black histogram), cells harvested from NSG mice (blue
histogram), and humanized mice (red histogram) were analyzed by flow cytometry.
(J) Representative photomicrographs showing immunohistochemical staining of
human PD-L1 on NPC-PDX from NSG mice and humanized mice. Scale bars, 100 μm. n =
3 per group.
View figure
Fig. 2
Fig. 2. Enhanced in vitro antitumor activity in CAR-expressing NK92 cells.
(A) Design of CAR construct. Puro, puromycin. SFFV, spleen focus-forming virus.
(B) Surface expression of human PD-L1 on HepG2 (untreated or IFN-γ–treated),
A549, C666-1, and Panc 08.13 cells. Cells stained with isotype control (blue
histogram) and anti–PD-L1 antibody (red histogram) were examined by flow
cytometry. WT NK92 cells (n = 4) and CAR NK92 cells (n = 4) were cocultured with
various human cancer cell lines at the indicated E:T ratios (C) or at a 10:1
ratio of E:T (D to F). (C) The percentages of NK cell cytotoxicity on different
cancer cell lines are shown. (D) The extent of apoptosis as indicated by
enrichment factor. (E) CD107a expression was determined in anti-CD56–labeled NK
cells after stimulation by different cancer cell lines. (F) Net release of IFN-γ
after stimulation by different cancer cell lines. Data from (D) to (F) are
expressed as means ± SEM. **P < 0.01 and ***P < 0.001, two-tailed unpaired t
test.
View figure
Fig. 3
Fig. 3. CAR-expressing NK92 cells exhibit better anti-solid tumor effect in NSG
mice.
(A) Schematic description of tumor inoculation and cell-mediated therapy in NSG
mice. The mice were either injected with C666-1 cells or transplanted with
NPC-PDX, which usually took 1 to 2 weeks to form visible tumors. (B to D) NSG
mice were inoculated with C666-1 cells. After the formation of visible tumors,
the mice were injected with saline (n = 15), WT NK92 cells (n = 15), and CAR
NK92 cells (n = 18). (B) Representative image of tumors. (C) Tumor volumes are
shown. Data are expressed as means ± SEM. *P < 0.05 and **P < 0.01, one-way
analysis of variance (ANOVA) followed by multiple comparisons, when CAR NK92
cell–injected group is compared to saline-injected group. (D) Tumor weights are
shown. Data are combined from two independent experiments and expressed as means
± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA followed by multiple comparisons.
(E to G) NSG mice were transplanted with NPC-PDX. After the formation of visible
tumors, the mice were injected with saline (n = 20), WT NK92 cells (n = 20), and
CAR NK92 cells (n = 21). (E) Representative image of tumors. (F) Tumor volumes
are shown. Data are expressed as means ± SEM. **P < 0.01, one-way ANOVA followed
by multiple comparisons, when CAR NK92 cell–injected group is compared to
saline-injected group. (G) Tumor weights are shown. Data are combined from three
independent experiments using PDX derived from three patients and expressed as
means ± SEM. **P < 0.01, one-way ANOVA followed by multiple comparisons.
View figure
Fig. 4
Fig. 4. Phenotypic and functional characterization of CAR-expressing HSC-derived
NK cells.
(A) Surface expressions of NK cell–specific markers under the gate of human
CD45+ and CD56+ subset. Cells stained with isotype control (blue histogram) and
indicated PE-conjugated antibodies (red histogram) were examined by flow
cytometry. CB-NK cells (n = 4) and pNK cells (n = 4) were cocultured with K-562
cells at the indicated E:T ratios (B) or at a 10:1 ratio of E:T (C to E). (B)
The percentages of NK cell cytotoxicity on K-562 cells are shown. (C) The extent
of apoptosis as indicated by enrichment factor. (D) CD107a expression was
determined in anti-CD56–labeled NK cells after stimulation by K-562 cells. (E)
Net release of IFN-γ after stimulation by K-562 cells. WT pNK cells (n = 4) and
CAR pNK cells (n = 4) were cocultured with C666-1 cells or tumor cells isolated
from NPC-PDX at the indicated E:T ratios (F) or at a 10:1 ratio of E:T (G to I).
(F) The percentages of NK cell cytotoxicity on tumor cells are shown. (G) The
extent of apoptosis. (H) CD107a expression on NK cells after stimulation by
tumor cells. (I) Net release of IFN-γ after stimulation by tumor cells. Data
from (C) to (E) and (G) to (I) are expressed as means ± SEM. *P < 0.05, **P <
0.01, and ***P < 0.001, two-tailed unpaired t test.
View figure
Fig. 5
Fig. 5. CAR-expressing primary NK cells suppress in vivo growth of NPC-PDX more
effectively.
NSG mice were transplanted with NPC-PDX. After the formation of visible tumors,
the mice were injected with saline (n = 5), WT pNK cells (n = 5), CAR pNK cells
(n = 5), and CAR pT cells (n = 5). (A) Representative image of tumors. (B) Tumor
volumes are shown. Data are expressed as means ± SEM, and comparative
statistical analysis were done by one-way ANOVA followed by multiple
comparisons. *P < 0.05, when CAR pNK cell–injected group is compared to
saline-injected group; #P < 0.05 and ##P < 0.01, when CAR pNK cell–injected
group is compared to CAR pT cell–injected group. (C) Tumor weights are shown.
Data are expressed as means ± SEM. *P < 0.05 and ***P < 0.001, one-way ANOVA
followed by multiple comparisons. (D and E) Representative FACS plots showing
the infiltration of CD3−CD56+ NK cells (D) and CD3+CD56− T cells (E) in tumors.
(F) Representative photomicrographs showing immunohistochemical staining in
tumors. The presence of NK and T cells was indicated by the expression of NCR1
and CD3, respectively. Scale bars, 100 μm. n = 5 per group. (G) Representative
photomicrographs showing TUNEL in tumors. Apoptotic cells were labeled by red
fluorescence. Scale bars, 100 μm. n = 5 per group. DAPI,
4′,6-diamidino-2-phenylindole. (H) Signal intensity of TUNEL is quantified from
the images, and data are expressed as means ± SEM. ***P < 0.001, one-way ANOVA
followed by multiple comparisons.
View figure
Fig. 6
Fig. 6. Evaluation of antitumor efficacy and safety of CAR pNK cells in
NPC-PDX–bearing humanized mice.
(A) Schematic description of PDX engraftment and cell-mediated therapy in
humanized mice. (B to D) Humanized mice were transplanted with NPC-PDX. After
the formation of visible tumors, the mice were injected with saline (n = 9), WT
pNK cells (n = 10), CAR pNK cells (n = 13), and CAR pT cells (n = 7). (B)
Representative image of tumors. (C) Tumor volumes are shown. Data are expressed
as means ± SEM. (D) Tumor weights are shown. Data are expressed as means ± SEM.
*P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by multiple
comparisons. (E) Survival curve of NSG mice after treatment with saline (n =
36), WT pNK cells (n = 34), CAR pNK cells (n = 38), and CAR pT cells (n = 20).
Data are combined from at least three independent experiments. (F) Survival
curve of humanized mice after treatment with saline (n = 20), WT pNK cells (n =
12), CAR pNK cells (n = 28), and CAR pT cells (n = 22). Data are combined from
at least three independent experiments. The survival rate of mice between CAR
pNK cell–injected group and CAR pT cell–injected group is compared by
Kaplan-Meier analysis via a log-rank test. **P < 0.01. (G to I) Blood samples
were collected from humanized mice 2 days after the last treatment with saline
(n = 12 for IL-6 and IL-10; n = 19 for IFN-γ), WT pNK cells (n = 11), CAR pNK
cells (n = 16), and CAR pT cells (n = 11). Plasma concentrations of IFN-γ (G),
IL-6 (H), and IL-10 (I) were determined by LEGENDplex. Data are expressed as
means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA followed by
multiple comparisons.
View figure
Fig. 7
Fig. 7. Dual therapy using CAR pNK cells and nivolumab results in a synergistic
antitumor response on NPC-PDX in humanized mice.
(A) Heatmap of DE genes from NPC-PDX between CAR pNK cell–injected mice (n = 3)
and saline-injected mice (n = 3) is shown. (B) Signaling pathway analysis of DE
genes. (C) Schematic description of PDX engraftment, ICI therapy alone, or in
combination with CAR pNK cell–mediated therapy in humanized mice. (D to G)
Humanized mice were transplanted with NPC-PDX. After the formation of visible
tumors, the mice were injected with saline (n = 23), ipilimumab (n = 15),
nivolumab (n = 13), CAR pNK cells (n = 22), CAR pNK cells plus ipilimumab (n =
22), and CAR pNK cells plus nivolumab (n = 26). (D and E) Representative image
of tumors. (F) Tumor volumes are shown. Data are expressed as means ± SEM. **P <
0.01 and ***P < 0.001, when CAR pNK cells plus nivolumab–treated group is
compared to saline-injected group, @P < 0.05, when CAR pNK cells plus
ipilimumab–treated groups are compared to saline-injected group, #P < 0.05, when
CAR pNK cell–treated groups are compared to saline-injected group, one-way ANOVA
followed by multiple comparisons. (G) Tumor weights are shown. Data are combined
from at least three independent experiments using PDX derived from three
patients and expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001,
one-way ANOVA followed by multiple comparisons.
Reference #1