NK cells engineered with a IL-15/IL-15Rα complex and CD19-targeted CAR show in vivo

expansion and tumor regression in a murine xenograft model of B-cell leukemia in vivo

+Renata Nacasaki Silvestre1, +Jiri Eitler2,3,4, Julia Teixeira Cottas de Azevedo1, Mariane Cariati
Tirapelle1, Daianne Maciely Carvalho Fantacini1, Lucas Eduardo Botelho de Souza1, Kamilla
Swiech1, Dimas Tadeu Covas1, Rodrigo T Calado1, Kelen Cristina Ribeiro Malmegrim3, Marxa L.
Figueiredo4, Torsten Tonn2, 3, 4 , Virginia Picanço-Castro*1

1
Center for Cell-based Therapy CTC, Regional Blood Center of Ribeirão Preto, University of São
Paulo, Ribeirão Preto, São Paulo, Brazil
2
2 Experimental Transfusion Medicine, Faculty of Medicine Carl Gustav Carus, Dresden University
of Technology, 01307 Dresden, Germany.
3Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, 01307
Dresden, Germany.
4German Cancer Consortium (DKTK), Partner Site Dresden, Dresden, Germany

3
Department of Clinical Analyses, Toxicology and Food Science, School of Pharmaceutical Sciences
of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
4
Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA .
+
These authors contributed equally to this work

* Correspondence:
Virginia Picanço-Castro
virginia.picanco@hemocentro.fmrp.usp.br
Center for Cell-based Therapy CTC, Regional Blood Center of Ribeirão Preto, University of São
Paulo, Ribeirão Preto, São Paulo, Brazil
Av. Tenente Catão Roxo 2505, Monte Alegre, Ribeirao-Preto-SP, Brazil

Keywords: CAR-NK cells, NK-92, adoptive cell therapy, IL-15, IL-15 receptor, B-cell
lymphoma, allogeneic therapy.
Abstract

Natural Killer (NK) cells are an attractive therapeutic approach as carriers for chimeric antigen
receptors (CAR) in allogeneic settings. However, sufficient ex vivo CAR-NK cell expansion remains
challenging. Adoptive transfer of CAR-NK cells has shown significant therapeutic efficacy without
serious side effects in many preclinical models and recent clinical studies. Although transgenic
expression of cytokines in CAR-NK cells partially improves their survival and proliferative abilities,
the optimal cytokine combination for expansion and function of CAR-NK cells remains unclear to
date. Here, we demonstrated that a fourth generation CD19-targeted CAR (CAR.19) co-expressing
IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly promotes NK-92
cell proliferation, secretion of proinflammatory cytokines, and their cytotoxic activity against B cell
cancer cell lines. Moreover, NK-92-CAR.19-IL-15/IL-15Rα cells can overcome growth limitations
of NK cells without supplementation of exogenous IL-2. In addition, NK-92-CAR.19-IL-15/IL-15Rα
cells demonstrated significant ability to control cancer cells in a murine xenograft model of B cell
lymphoma. This innovative approach supports further clinical evaluation of CAR-NK cells in
hematological neoplasms and solid tumors.
1 Introduction

NK cells are an attractive alternative to CAR-T cells. CAR-NK therapy is less likely to cause
cytokine release syndrome (CRS), due to the lower levels of pro-inflammatory cytokines, such as
interleukin-6 (IL-6) secreted by NK cells. This makes CAR-NK therapy generally considered to be
safer than CAR-T therapy (DOI: 10.4161/onci.28147). CAR-NK cells may be used in a non-
autologous manner due to the absence of GvHD reactivity and may thus serve as universal cell
products, easily available off-the-shelf for clinical applications (1). Allogeneic CAR-NK cells may be
constructed, expanded, cryopreserved and supplied to patients on demand at significantly decreased
manufacturing costs.
However, NK cells do pose several challenges for their translation into clinical applications.
These challenges include difficulties in transducing and transfecting NK cells, limited expansion
capacity both in vitro and in vivo, and difficulty in cryopreservation. Hence, translation of CAR-NK
cell-based therapies are limited by the demand for high vector copy numbers at clinical scale (10-100
times higher compared to CAR-T cells) (doi: 10.1155/2018/4054815) and the lack of in vivo
expansion which mandates high cell dosages (https://doi.org/10.1186/s40364-022-00364-6).
IL-15 has been suggested to allow expansion of NK cells in vivo
(https://doi.org/10.1038/leu.2017.226,). IL-15 is a cytokine physiologically released by dendritic
cells, monocytes and macrophages that plays a key role in the development, homeostasis, activation,
and survival of T, natural killer (NK), and NK-T cells (10.1146/annurev.immunol.17.1.19). IL-15
cytokine has at least three functional forms: (i) a soluble monomeric IL-15 (sIL-15), (ii) the soluble
IL-15/IL-15Rα complex, and (iii) the membrane-bound IL-15/IL-15Rα (doi.org/10.1182/blood-2011-
10-384362, doi: 10.1084/jem.20041389, doi.org/10.1182/blood-2005-01-0064). IL-15 as well as IL-2
are members of the γ chain family of cytokines and are responsible for activation and proliferation of
NK cells. Both cytokines share the γc and IL2/IL15Rβ chains and specific subunits, IL-2RC or IL-
15Rα, which form the IL-2 and/or IL-15 receptors, respectively (https://doi.org/10.1038/nri1901).

Although the IL-15/IL-15Rα complex has greater potency, bioavailability, and stability
compared to soluble monomeric IL-15 when administered to mice and humans
(doi.org/10.1016/j.cyto.2011.09.028, doi.org/10.1074/jbc.M113.461756 e
doi.org/10.4049/jimmunol.2100066, https://doi.org/10.1073/pnas.1911678117) the soluble
monomeric IL-15 presentation is the most common form incorporated in the expression vectors used
in CAR-NK cells to improve NK potency in vivo, including in some recent clinical trials
(https://doi.org/10.1038/leu.2017.226, DOI: 10.1056/NEJMoa1910607).

In this context, to identify the best CAR-NK constructs for clinical use, different forms of
recombinant IL-15 currently should be evaluated for their potential to improve NK cell activation and
proliferation. IL-15 and IL-15/IL-15Rα can promote significant increase in NK cell antitumor
cytotoxic activity (https://doi.org/10.1016/j.it.2022.08.004). Here, using NK-92 as a model, we
investigated the different effects of soluble IL-15 and a membrane bound protein consisting of human
IL-15 and human IL-15Rα fused by flexible linkers (IL-15/IL-15Ra) to overcome NK potency and
expansion capacity of NK cells in vitro and in a murine xenograft model for CD19 hematologic
malignancies. We have generated two fourth generation CD19-targeted CAR (CAR.19) co-
expressing soluble IL-15 (CAR.19-IL-15) or IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) and compared
them with a second-generation CAR.19 for their capacity to promote NK cell survival, activation,
proliferation, and in vitro cytotoxic activity against B-cell lines. Moreover, we have evaluated the
transcriptional profile of the NK-92-CAR.19 cell variants to elucidate the pathways involved in IL-15
and IL-15/IL-15Rα signaling. Finally, the therapeutic efficacy of CAR-NK92 constructs was
evaluated in a murine xenograft model of B cell lymphoma.

2 Methods

2.1 Cells and culture medium

Natural Killer 92 (NK-92) cells were maintained in serum free X-VIVO 10 medium (Lonza,
Cologne, Germany) containing 5% human heat inactivated AB plasma (German Red Cross Blood
Donation Service Baden Württemberg–Hessen, Frankfurt, Germany), 100 μg/ml
penicillin/streptomycin (Life Technologies), supplemented with 500 IU/ml IL-2 (Clinigen), as
previously described (3,6). Human leukemia cell lines K562 (ATCC CCL-243), Raji (ATCC CCL-
86) and NALM-6 (CRL-3273) were cultured in RPMI medium (ThermoFisher Scientific) with 10%
Fetal Bovine Serum (FBS). All cell lines have been tested for mycoplasma using MycoAlertPLUS
Mycoplasma Detection Kit (Lonza) and were negative (reference value < 1.0).

2.2. STR profiling

For genotypic identification of the working master bank of NK-92, HEK293T, Raji and Nalm-6 cells,
we analyzed the tandem repeat (STR) DNA profile. This methodology uniquely identifies human cell
lines derived from the tissue of a single individual, allowing verification that the cell culture is not
contaminated. A minimum of eight major STR loci are required to uniquely identify a human cell
line. STR loci were amplified using the AmpFlSTR™ Identifiler® Plus PCR Amplification Kit
(Applied Biosystems, USA) (detecting D5S818, D7S820, CSF1PO, vWA, D16S539, TPOX, THO,
and D13S317). Electrophoretic analysis was performed using a 3730/3130xl DNA Analyzer (Applied
Biosystems, USA). After electrophoresis, data were analyzed with Gene Mapper® ID-X Software
v1.2 (Applied Biosystems, USA) to categorize peaks by size in relation to an internal standard allelic
ladder. The following STR profile: NK-92 cell (CSF1PO: 11,12; D13S317: 9,12; D16S539: 11,12;
D5S818: 12,13; D7S820: 10, 11; THO: 6, 9.3; TPOX: 8; vWA: 18), K562 cell line (CSF1PO: 9,10;
D13S317: 8; D16S539: 11,12; D5S818: 11,12; D7S820: 9, 11; THO: 9.3; TPOX: 8,9 ; vWA: 16),
HEK293T: (CSF1PO: 11, 12; D13S317: 12, 14; D16S539: 9, 13; D5S818: 8, 9; D7S820: 11; THO1:
7, 9.3; TPOX: 11; vWA: 16, 18, 19), Nalm6: CSF1PO: 12, 13; D13S317: 9, 12, 13; D16S539: 9, 10,
11; D5S820: 8,9; THO1: 8, 10; TPOX: 8, 10; vWa: 15,16). The values found were in accordance
with the description of cell lines from the ATCC bank.

2.3 Construction of lentiviral vectors and transduction of NK-92 cells

The lentiviral vectors encoding anti-CD19 CAR (CAR.19) were constructed based on a previously
described vector (22). CAR.19 consists of murine anti-CD19 single chain fragment variable (scFv)
from the antibody clone FMC63, CD8 hinge and transmembrane region, 4-1BB costimulatory
domain, and CD3ζ cytoplasmic region. CAR.19-IL-15 and CAR.19-IL-15/IL-15Rα contain
additionally IL-15 or IL-15/IL-15Rα separated by a T2A peptide. IL-15 was linked to IL-15Rα as
previously described (23). Lentiviral production was generated by the transient cotransfection of
HEK 293 T cells with the three-plasmid system previously described (22). The titer of lentiviral stock
was determined in a K562 cell line. NK-92 cells were transduced in the presence of 8 µg/ml
polybrene and 1000 IU/mL IL-2 for 5 hours at 37°C.

2.4 Enrichment of NK-92-CAR cells

NK-92-CAR cells were enriched by positive selection with magnetic beads. NK-92-CAR cells were
incubated with Biotin-SP-conjugated anti-mouse F(ab')2 antibodies (Jackson Immunoresearch)
followed by incubation with anti-biotin MicroBeads (Miltenyi Biotec). Separation was done with
MACS separation columns (Miltenyi Biotec) according to the manufacturer’s instructions. The
procedure was repeated 7 days after the first selection.

2.5 Flow cytometry

For flow cytometry, NK cells were labeled with monoclonal antibodies specific for CD56, CD3,
CD45, CD28, CD16, CD11a, CD2, NKG2D, NKp30, NKp46, DNAM-1, CD95 (BD Bioscience) for
30 minutes on ice. CAR expression on the surface of NK cells was detected using an anti-mouse
F(ab')2 antibody (Jackson Immunoresearch). For checkpoint molecules, antibodies specific for PD-1
(BD Biosciences), LAG- 3 (BD Biosciences) and TIM-3-specific (BD Biosciences) were used. Dead
cells were excluded by 7-AAD viability dye (BD Biosciences). Samples were acquired by using a
LSRFortessa cell analyzer (BD Biosciences), and data analyzed using the FlowJo 10 software (BD
Biosciences).

2.6 Cytotoxicity assays

Cytotoxicity of NK-92 cells toward established cancer B cell lines was analyzed by a flow
cytometry-based assay as described (22). Briefly, target cells were labeled with PKH67 (Sigma–
Aldrich) and incubated with effector cells at various effector to target (E:T) ratios (2:1 and 10:1) for 5
h at 37°C. 7-AAD solution was added to each sample before flow cytometric analysis. Cells were
acquired with LSRFortessa cell analyzer (BD Biosciences). Dead target cells were identified as
double positive for PKH67 and 7-AAD. Cell cytotoxicity was analyzed measuring the difference in
the percentage of live cells in time points 0h and 5h after cocultivation. The percent of cell death (%
cytotoxicity) was calculated using the following equation:

Cytotoxicity ( % )=100− ( )
% live B cells(5 h)
% live B cells(0 h)
*100

2.7 Cytokine release assay

For the cytokine release assay, 2.5x 105 NK cells were co-cultured with 0.25x105 cancer cells for 5
hours at 37°C. Supernatants were harvested and IL-15, IFN-γ, TNFα and IL-10 were measured by
multiplex assay using the MILLIPLEX MAP Human CD8+ T-Cell magnetic Bead Panel kit and
MILLIPLEX Human Cytokine/Chemokine/Growth factor Panel A (Merck-Millipore). The MAGPIX
® System (Luminex Corporation) was used for data analysis according to manufacturer’s
specifications. Cytokine concentrations were quantified using the Milliplex Analyst software.

2.8 RNAseq

NK cells were cocultured with Raji cell 10:1 ratio (E:T) for 24h at 37°C, enough time to eliminate
target cells. Total RNA from NK cells was isolated from two biological replicates. In short, RNA
was isolated using RNeasy kit (Qiagen) as per the manufacturer's protocol. Poly(A) RNA sequencing
library was prepared following Illumina's TruSeq-stranded-mRNA protocol and conducted by LC
Sciences (Houston, TX, USA). Poly(A) tail-containing mRNAs were purified using oligo-dT Briefly,
magnetic beads with two rounds of purification, and fragmented using divalent cation buffer at
elevated temperature. Quality control analysis and quantification of the sequencing library were
performed using an Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-
ended sequencing was performed on Illumina’s NovaSeq 6000. For transcript assembly and
estimating transcript expression levels, software is described by Figueiredo et al. (2020)
(https://pubmed.ncbi.nlm.nih.gov/34209203/, DOI: 10.3390/bioengineering8070090) For differential
expression analysis of mRNAs, StringTie was used by calculating fragments per kilobase million
(FPKM). The differentially expressed mRNAs were selected with log2 (fold change) > 1.5 or log2
(fold change) < -1.5 and with statistical significance (p < 0.05) by edgeR. The datasets generated in
this study will be submitted to the Gene Expression Omnibus (GEO) repository upon acceptance with
assistance by the Purdue Bioinformatics Core.

2.9 In vivo B-cell lymphoma model

NSG mice, 8- to 10-week-old male, were intravenously injected with 2 × 10 4 Raji/Luc cells at day 0.
At days 0, 3, 7, 10 and 14, after tumor cell inoculation, 7 × 10 6 NK-92-CAR.19 or parental NK-92
cells were injected intravenously. Control mice received PBS. Disease development was monitored
by an in vivo imaging system (IVIS, Perkin Elmer) after intraperitoneal injection of 150 mg/kg of D-
luciferin (Perkin Elmer). For in vivo experiments all applicable guidelines for the care and use of
animals were followed and animal experiments were approved by the responsible government
committee in Brazil (n. 124/2017).

2.10 Statistical analysis

All statistical analyses were done using Graphpad Prism 8.0 software. Where not indicated
differently, data were analyzed by two-tailed unpaired Student’s t-test. P values <0.05 were
considered statistically significant. ANOVA and post-hoc analyses (Tukey’s multiple comparisons
test) were applied to compare mean transduction efficiencies, cytotoxicity, proliferation, cytokine
secretion levels and inhibitory molecule expression. The survival data were assessed using a log-rank
analysis.

3. Results

3.1 Generation of NK-92-CAR.19-IL-15 and NK-92-CAR.19-IL-15/IL-15Rα cells

We have constructed lentiviral vectors carrying the second-generation CAR.19, which

contains the anti-CD19-specific scFv fragment derived from the antibody clone FMC63, CD8 both
hinge and transmembrane region, and the intracellular signaling domains of human 4-1BB and the
CD3ζ motif in tandem. A 2A linker sequence was inserted directly downstream of CARs followed by
IL-15 or IL-15/IL-15Rα molecules (Figure 1A). The CAR sequences were inserted into the self-
inactivating lentiviral vector pSEW and VSV-G pseudotyped lentiviral vector particles were
produced and used for transduction of human NK-92 cells. NK-92-CAR cells were enriched by two
rounds of positive selection with magnetic beads resulting in homogeneous CAR-positive NK92
cells, which demonstrate stable CAR expression after 30 days in culture (Figure 1 B and C).
After the generation and expansion of NK-92-CAR.19 cells, we have characterized
immunophenotypically the different engineered NK-92-CAR.19 cells (Figure 2). The expression
levels of CD56, CD45, CD28, CD11a (ITGAL), CD2 (LFA-2), NKG2D, NKp30, NKp46, CD95 all
were very similar among the engineered NK-92-CAR.19 and parental NK-92 cells (Supplemental
Figure 1).

3.3 NK-92-CAR.19-IL-15/IL-15Rα cells specifically eliminate CD19+ cancer cell lines

We evaluated the target specificity of our NK-92-CAR.19 cells using two CD19-positive cell
lines (Raji and NALM-6 cells) and one CD19-negative cell line (K562) (Figure 2). In co-culture
assays, all NK-92-CAR cells showed enhanced cytotoxicity relative to unmodified NK-92 cells under
identical conditions in 10:1 effector:target cell ratio, both for NALM-6 and Raji. NK-92 cells
expressing IL-15 or IL-15/IL-15Rα were significantly more cytotoxic than NK-92-CAR.19 in
cocultures with NALM-6 cells (Figure 2A, B). There was no difference between NK-92-CAR versus
unmodified NK cells regarding the cytotoxicity against K562 cells, confirming the specific CAR-
based cytotoxicity of the NK-92-CAR cells (Figure 2C).
3.4 IL-15/IL-15Rα expression in NK-92-CAR.19 cells leads to higher secretion of INF-γ and
TNF-α after stimulation with Raji cells
Further, we evaluated cytokines and other molecules involved in the cytotoxic function of
NK-92-CAR cells. We have quantified levels of IFN-γ and TNF-α in the supernatants of the
cocultures of NK-CAR cells with target cells (CD19+ or CD19-) (Figure 3). NK-CAR
CD19-IL-15/IL-15Rα cells secreted higher amounts of IFN-γ (Figure 3A,B) and TNF-α (Figure 3D,
E) when co-cultured with target cells, indicating their greater antitumor potential.

3.5 Differential transcriptomic profiling of NK-92 cells expressing different CAR.19 constructs
We conducted bioinformatic analysis for all NK92-CAR.19 cell variants to examine
similarities and differences between transcriptional changes after activation with target cells. Cluster
analysis showed that NK-92-CAR.19-IL15/IL-15Rα cells have distinct gene signatures from the
other NK-92 cells (Figure 5A). As illustrated in the circos plot (Figure 5B), there is a large overlap of
genes and biological processes among the different NK-92-CAR.19 cell variants. However, there are
critical differences in the signaling pathways of CAR-NK-CAR.19-IL15/IL-15Rα cells, compared to
the other CAR-NK-CAR.19 cell variants (Figure 4A).
Metascape analysis identified six protein-protein interaction subnetworks (Figure 4C)
including the PI3K-AKT pathway, which seems to be critical for the IL-15-mediated activation of
NK cells. The heatmap representation of different expressed transcription factors (Figure 4D)
demonstrates higher degree of functional overlap, as these in vitro studies likely captured different
parts of the same biological processes as they are related to cell death and apoptosis pathways, for
example.

3.6 IL-15 or IL-15/IL-15Rα expression downregulates inhibitory molecules in NK-92-CAR.19

cells
To assess the effects of repeated stimulation of NK-CAR.19 with target CD19+ cells, we co-
cultured NK-92-CAR.19, NK-92-CAR.19-IL-15 or NK-92-CAR.19-IL15/IL-15Rα cells with Raji
cells for 3 days at an initial E/T ratio of 1:2, and repeated stimulation by adding fresh target cells
after 24 and 48 h (Figure 5). Surface expression was examined for the inhibitory molecules PD-1,
LAG-3 and TIM-3, which are associated with exhaustion of lymphocytes
(10.1016/j.immuni.2016.05.001). The expression of these markers in the restimulated NK-92 cells
was determined by flow cytometry and compared with unstimulated NK-92-CAR cells.
All inhibitory markers showed similar expression in unstimulated NK-92, NK-92-CAR.19,
NK-92-CAR.19-IL-15, and NK-92-CAR.19-IL-15/IL-15Rα, indicating that CAR.19 expression did
not affect their expression in the absence of the target cells (Figure 5).
Expression of PD-1 and LAG-3 increased after repeated stimulation in all engineered NK-92-
CAR cells (Figure 6 A, B). However, NK-92-CAR.19 expressing IL-15 or IL-15/IL-15Rα showed
lower expression of these markers relative to NK-92 cells expressing only CAR.19. All NK-92-
CAR.19 cells showed high TIM-3 expression before stimulation. Nevertheless, TIM-3 expression
decreased in all cell constructs after repeated cell stimulation with the target CD19+ cells (Figure 5C)

3.7 NK-92-CAR.19-IL-15/IL-15Rα cells proliferate independently of IL-2

Next, we evaluated whether the cytokine produced by the vectors would be sufficient to
replace addition of exogenous IL-2 in the cultures and allow cell expansion. NK-92-CAR.19 cells
with different vector constructions were cultured in the presence or absence of IL-2 and cell
proliferation was evaluated for 21 days. In the absence of IL-2, NK-92 and NK-92-CAR.19 cells
stopped proliferating after 9 days of culture and then cell numbers started to decline. Besides, NK-92-
CAR.19-IL-15 cells expanded until day 12, then subsequently began to decrease in number. In
contrast, NK-92-CAR.19-IL-15/IL-15Rα cells successfully expanded throughout the 21-day culture
period (Figure 6A). Notably, cell viability was maintained between 80-95% in the 21 days of culture
without IL-2 (Figure 6B).
IL-15 was measured in the cell culture supernatants by Luminex assay (Figure 6C). NK-92-
CAR.19-IL-15 cells secreted the highest amount of IL-15 (Figure 6C). As expected, only low levels
of IL-15 were detected in NK-CAR.19-IL-15/I-15Rα, because in this case IL-15 remained bound to
its high-affinity Rα receptor on the cell surface and will not be detected by the anti-IL-15 antibodies.

3.6 NK-92-CAR.19-IL-15/IL-15Rα cells have enhanced in vivo cytotoxic function against B cell
lymphoma than the other constructs
We challenged the anti-lymphoma activity of NK-92-CAR cells in a Raji xenograft
immunodeficient mouse model. Mice received one intravenous infusion (2x 104/mouse) of Raji-Luc+
cell line (D0). Mice were treated with six infusions of control ex vivo-expanded NK-92 cells, NK-92-
CAR.19, NK-92-CAR.19-IL15 or NK-92-CAR.19-IL15/IL-15Rα cells (7x106/mouse; five mice per
group) (Figure 7A). Tumor growth was monitored by measuring changes in tumor bioluminescence
over time. As expected, tumor bioluminescence increased rapidly in mice treated with control NK-92
cells (Figure 7B and C). By contrast, NK-92-CAR.19-IL15/IL-15Rα cells led to improved tumor
control and significant prolongation of tumor-free survival compared with NK-92 cells (p= 0.0209,
Figure 7B and C). Thus, NK cells coexpressing IL15/IL15RA showed superior in vivo antitumor
activity against lymphoma cells compared with NK-92 cells, underscoring the clinical potential of
CAR-NK-based adoptive therapy using this innovative strategy.

4. Discussion

Clinical use of off-the-shelf therapeutic NK-92 cells may help more patients to get access to
NK-CAR cell-based therapy due to logistics and costs. Here, we use the NK-92 cell line due to its
excellent in vitro expansion and high cytotoxic potential against cancer cells. Several in vitro studies
and preclinical models have shown the effectiveness of CAR-NK-92 cells against a variety of
cancers, including hematological and solid tumors (24,25). CAR-NK-92 cells are capable of
bypassing inhibitory signals from the tumor microenvironment (26). Therefore, these cells have
become an attractive option for clinical translation, as a truly off-the-shelf therapeutic cell with a
great expanding capacity (27). At present, several clinical trials using CAR-NK-92 cells are ongoing,
which will provide valuable insights for improving their further application in cancer therapy
(https://doi.org/10.1016/j.jcyt.2022.12.003).
IL-15 is an essential cytokine for NK development, survival, proliferation, and function (28).
This cytokine has different functional forms in vivo, and the best IL-15 form for activation and
expansion of NK cells in vitro has not been determined yet. Soluble IL-15 is currently the most
common form incorporated in engineered NK-CAR cells used in basic studies and clinical trials
(https://pubmed.ncbi.nlm.nih.gov/32023374/, DOI: 10.1056/NEJMoa1910607;
https://pubmed.ncbi.nlm.nih.gov/34999097/. DOI: 10.1053/j.gastro.2021.12.281).

In this work, we have engineered an innovative vector expressing IL-15 linked to its high
affinity receptor alpha IL-15Rα via a flexible SG-link, inspired by the natural trans-presentation of
IL-15 (23), in combination with a CAR construct to enhance the cytotoxicity potential of NK-92 cells
towards CD19+ target cells. In trans-presentation, IL-15 and IL-15Rα may form a heterodimeric
complex, which is subsequently guided to the cell membrane and interacts with cells expressing the
receptor IL-2/IL-15Rβ–γc such as other NK cells (33–35).

We also demonstrated that NK-92-CAR.19 cells expressing IL-15 or IL-15/IL-15Rα showed

significantly enhanced in vitro antitumor activity compared to NK-92 cells expressing only the
CAR.19 construct. Although the CAR.19 construct could also mediate an antitumor response, it is
clear that soluble IL-15 or IL-15 combined with its receptor (Rα) improved the cytotoxicity potential
of NK-92.CAR.19 cells. We examined the cytokines and other proteins involved in the cytotoxic
potential of NK-92 modified with CAR.19 constructs. NK-92-CAR.19-IL-15/IL-15Rα was able to
secrete the highest amounts of TNF-α and IFN-γ, indicating its enhanced anti-tumor activity. These
findings are in line with recent publications investigating the effect of IL-15 on NK cells in cancer
patient therapy (39–41). The authors described a massive expansion of NK cells in patients who
received continuous infusions of IL-15. Furthermore, IL-15 treatment resulted in increased secretion
of granzymes A and B and perforin in NK cells, indicating that IL-15 infusions may potentiate their
cytotoxic antitumor response. It has been shown recently that improper polarization of lytic granules
towards immunological synapses in NK cells, following contact with resistant cancers, leads to
inefficient cytotoxicity (42). It was also shown that soluble IL-2 can increase lytic granule
convergence (43). The role of IL-15 or IL-15/IL-15Rα in the engineered CAR-NK’s lytic granule
machinery is unknown. Further studies are required to clarify whether IL-15/IL-15Rα could induce
stronger or faster convergence or polarization of lytic granules in CAR-NK cells.

In order to unravel genes and/or pathways expressed by the soluble and transmembrane forms
of IL-15 signaling, RNA sequencing was performed on the activated NK-92-CAR.19 cells.
Bioinformatics analysis demonstrated that NK-92-CAR.19-IL-15 and NK-92-CAR.19-IL-15/IL-
15Rα cells had distinct gene expression profiles. Both soluble IL-15 and IL-15/IL-15Ra triggered a
cascade of intracellular events that ultimately activated the PI3K/AKT signaling pathway, however,
by recruiting different players. The PI3K/AKT signaling pathway is one of the well-described
mechanisms promoting NK survival, proliferation, and their effector functions
(https://doi.org/10.3389/fimmu.2015.00355).

PD-1, LAG-3 and TIM-3 are molecules associated with inhibitory activity or exhaustion in
NK cells and other lymphocytes (44). Although we have found comparable levels of these checkpoint
molecules in unstimulated NK-92-CAR cells, repeated stimulation with CD19+ target cells
differentially affected the expression of these inhibitory molecules on the NK-92-CAR.19 cell
variants. Most strikingly, PD-1 and LAG-3 were less expressed in NK-92-CAR.19-IL-15 and NK-
92-CAR.19-IL-15/IL-15Rα as compared with NK-92-CAR.19 cells. Therefore, our data suggest that
NK-92-CAR.19 cells producing IL-15 or IL-15/IL-15Rα may be more robust and less sensitive to
regulation by checkpoint inhibition.

Our data suggest that IL-15/IL-15Rα is sufficient to confer enhanced proliferation even
without exogenous IL-2. Similar to our data, Xu et al. has shown that NK-92 modified with an
interleukin (IL)-15Rα-sushi/IL-15 complex and a Programmed cell death-1 (PD1) signal
inverter could maintain sustained proliferation under low IL-2 concentration (10 UI/mL) (38). On the
other hand, NK-92-CAR.19 cells expressing soluble IL-15 can grow only for 9 to 12 days in culture
without IL-2. This could represent an advantageous strategy for NK expansion with less (or without)
IL-2, thereby significantly reducing the manufacturing costs.

Due to their increased cytotoxic activity in vitro, NK-92-CAR.19-IL-15/IL-15Rα cells were

chosen for in vivo evaluation, using a NSG mice model injected with CD19+ Raji cells. NK-92 cells
and NK-92-CAR cells do not proliferate and do not permanently engraft in NSG mice (45,46). For
this reason, we have used a systemic injection scheme of NK cells. Treatment with parental NK-92
cells had little effect, resulting in extensive growth of disseminated lymphoma in several organs of
the animals. In contrast, tumor development was largely controlled by the engineered NK-92-
CAR.19-IL-15/IL-15Rα cells, demonstrating that these cells also have a higher in vivo specific
antitumor activity. In contrast, NK-92 cells expressing 15Rα-sushi/IL-15 used to treat an animal
model of pancreatic adenocarcinoma were not able to eradicate the tumor (38). However, these cells
showed an enhanced cytotoxicity and prolonged the survival time of tumor-bearing mice, compared
to NK-92 cells without cytokine expression (38).

In conclusion, we described here a novel immunotherapy strategy using engineered NK-92-

CAR.19 cells that express IL-15 linked to its receptor IL-15Rα, with strong cytotoxic ability, and
capable of expanding independently of IL-2 (Figure 9). These features favor in vitro large-scale NK
expansion for clinical applications with lower costs. Our data highlighted the critical nature of IL-15
signaling as a mechanism that can enhance in vitro NK cell expansion along with target-specific
cytotoxic activity of NK-CAR cells. The results of our in vivo studies demonstrated the enhanced
potential of NK-92/CAR-19-IL-15/IL-15Rα cells compared to their parental cells and other variant,
which may further promote the clinical application of CAR-NK cells. Although we have chosen the
CD19 as target for this proof-of-concept study, other targets may be used with this innovative vector
strategy, thereby extending the CAR-NK-based cell platform to treat other cancer types.
Figure legends

Figure 1. Generation of NK-92-CAR.19 cells. (A) Lentiviral vectors encoding different CARs
under the control of the spleen focus-forming virus promoter (SFFV). The CAR sequences are
followed by a self-cleaving peptide (T2A) and IL-15 or IL-15/IL-15Rα (IL-15 and IL-15Rα fused by
a flexible linker). (B) Enrichment of CAR+ populations after positive selection of CAR-NK-92 cells
performed in two steps and evaluation of CAR expression after 30 days of culture and (C) Illustrative
dot plots of enriched NK-92-CAR.19 cell populations by flow cytometry.

Figure 2. NK-92-CAR.19 cells can specifically eliminate CD19+ B cell lines. NK-92, NK-92-
CAR.19, NK-92-CAR.19-IL-15 and NK-92-CAR.19-IL-15/IL-15R⍺

cells were co-cultivated with (A) Raji, (B)

+ SD are shown.

Figure 3. NK-92-CAR.19-IL-15/IL15R⍺ cells produce high amount of anti-tumor pro-

inflammatory cytokines. NK-92, CAR.19, CAR.19-IL-15 and CAR.19-IL-15R⍺. NK cells (2.5 ×
105

) were incubated for 5 h with Raji lymphoma cells at an E/T ratio of 10:1. Supernatants were collected, and the

Figure 4. Metascape functional analysis of transcriptome profiles of NK-92-CAR.19 cells after

coculture with a target B cell line. A) Metascape enrichment analysis of statistically enriched
ontology terms (GO/KEGG terms, canonical pathways, and hallmark gene sets. B) The Circos plot
shows how genes from the different input gene lists after coculture setups overlap. On the outside,
the arc represents the identity of each gene list. On the inside, the orange color represents the genes
that appear in multiple lists, and the light orange color represents genes that are unique to that gene
list. Purple lines link the same genes shared by multiple gene lists. Blue lines link the genes that fall
into the same ontology term (the term has to be statistically significantly enriched). The greater the
number of purple links and the longer the dark orange arcs, the greater is the overlap among the input
gene lists. Blue links indicate the amount of functional overlap among the input gene lists. C) GO
enrichment analysis was applied to the network to assign biological "meanings" of sub-protein
networks. GO enrichment analysis was applied to each MCODE network to assign "meanings" to the
network component, where the top three best p-value terms were retained. MCODE components
were identified from the merged network. Each MCODE network is assigned a unique color. D)
Metascape enrichment analysis of all statistically enriched Transcription Factor-target interaction
networks.

Figure 5. IL-15 maintains low expression of immune checkpoint molecules in NK-92-CAR.19

cell variants. NK-92-CARs and indicated control cells were co-incubated with Raji cells at an E:T
ratio of 1:2 and repeatedly stimulated with fresh target cells were added again after 24 h and 48h.
Three days after initiation of the experiment, surface expression of PD-1, LAG-3 and TIM-3 was
measured by flow cytometry (n=3). One-way ANOVA statistical test, Tukey's multiple comparison
posttest. Mean values + SD are shown. ***P < 0.001; **P < 0.01; *P < 0.05; ns, P 0.05.

Figure 6. NK-92-CAR.19-IL-15/IL15R⍺ cells proliferate independently of exogenous IL-2.

Proliferation of parental and genetically modified NK-92 cells: NK-92-CAR.19, NK-92-CAR.19-
IL15 and NK-92-CAR.19-IL-15/IL15R⍺

in X-Vivo10 supplemented with 5% of human plasma. (A) Cells were cultured e

Figure 7. NK-92-CAR.19-IL15/IL15R⍺ cells eliminate B-cell lymphoma in vivo and prolong

animal survival. A) Schema of in vivo study demonstrating the antitumor activity of transduced NK
cells in a disseminated human B-cell malignancy xenogeneic NSG mice model. NSG mice were
intravenously injected with 2 × 104 Raji-Luc lymphoma cells. At days 0, 3, 7, 10 and 14, the animals
were treated by intravenous injection of 7 × 106 NK-92 cells (NK-92: n=5, CAR.19: n=5, CAR.19-
IL15: n=5, CAR.19 IL15/IL15RA: n=5) (B) Lymphoma development was monitored by in vivo
bioluminescence imaging. Images were taken on days 8, 15 and 21, (C) Bioluminescence intensity
and (D) Bioluminescent quantification of tumor growth shown in individual mice within each group
at day 29. Statistics determined with the XXX *p

Figure 8. Proposed model of action of engineered NK-92/CAR-19-IL-15/IL-15Rα cells. In the

absence of exogenous IL-2: A) NK-92-CAR.19 cells do not proliferate; B) NK-92-CAR.19-IL-15
cells poorly proliferate till day 12, as they secrete soluble IL-15, which may binds to the IL-15Rα
and subsequently interacts with cells expressing IL-2/IL-15Rβ–γc receptors (such as other NK cells)
and activate them; C) NK-92-CAR.19-IL-15/IL-15R
cells intensively proliferate, as they express the IL-15/IL15R complexes on their cellular
cells expressing IL-2/IL-15Rβ–γc receptor. The NK-92-CAR.19-IL-15
/IL-15R cells can expand
independently of IL-2 and have strong cytotoxic ability. Of note, IL-15
signaling activates the PI3K/AKT pathway in all three NK-92-CAR.19 cells, which culminates in
cellular activation, survival and proliferation.
 Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.

Authors’ Contributions
RNS performed the experiments and drafted the original manuscript and figures; JE performed the
experiments and drafted the original manuscript and figures; JTCA helped with some experiments
and drafted the original manuscript and figures; MCT helped with some experiments and drafted the
original manuscript; DMCF helped with in vivo assays; LEB helped with in vivo assays; KCRM
drafted and edited the manuscript; MLF helped with analysis of the RNAseq data; KS drafted and
edited the original manuscript, RC analyzed the data and edited the manuscript; DTC edited the
original manuscript, TT analyzed the data, drafted the original manuscript and figures and VPC
analyzed the data, drafted the original manuscript and figures.

Funding

This study was financially supported by the São Paulo Research Foundation - FAPESP (grant
#2020/08279-8; grant #2019/25309-0; grant#2013/08135-2; grant #2008/578773; NPOP-Nutec grant
# 2020/07055-9); and by the National Council for Scientific and Technological Development - CNPq
(grant #573754-2008-0; grant #442484/2020-8). This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

Acknowledgments

The authors thank Patricia Vianna Bonini Palma and Camila Cristina de Oliveira Menezes Bonaldo
(Flow Cytometry Laboratory of the Regional Blood Center of Ribeirão Preto) for performing the
flow cytometry analysis. The authors also thank Fernanda T. Udinal for the English language review
and Sandra Navarro Bresciani for the illustration and graphic design.

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