Re-Targeting of Primary NK Cell Subsets by Chimeric Antigen Receptors is Dependent upon their Intrinsic Functional Potential
Vincent Yi Sheng Oei1,2, Marta Siernicka3,4, Agnieszka Graczyk-Jarzynka3, Hanna Julie Hoel1, Weiwen Yang1, Daniel Palacios1,2, Hilde Almåsbak1, Malgorzata Bajor3, Dennis Clement1,2, Ludwig Brandt5, Björn Önfelt5,6, Jodie Goodridge1,2, Magdalena Winiarska3, Radoslaw Zagozdzon3,7, Johanna Olweus1,2, Jon-Amund Kyte1,8, Karl-Johan Malmberg1,2,9
1Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Norway. 2The KG Jebsen Centre for Cancer Immunotherapy, Institute of Clinical Medicine, University of Oslo, Norway, 3Department of Immunology, Centre for Biostructure Research, Medical University of Warsaw, Warsaw, Poland. 4Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland. 5Science for Life Laboratory, Department of Applied Physics, KTH - Royal Institute of Technology, Solna, Sweden; 6Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. 7Department of Clinical Immunology, Transplantation Institute, Medical University of Warsaw, Warsaw, Poland.
8Department of Oncology, Oslo University Hospital; 9Centre for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.
Running title: NK Subsets’ Intrinsic Potential Affect CAR Re-Targeting
Keywords: CAR, Natural Killer Cell, NK cell Education, Differentiation, Killer cell Immunoglobulin-like Receptors
Conflict of interest: KJ Malmberg serves on the Scientific Advisory Board of Fate Therapeutics. The remaining authors have no conflicts to declare.
Corresponding Author: Karl-Johan Malmberg, Ullernchausseen 70, 0372 OSLO, +47- 45390926, [email protected]
Abstract: 250 Words; Word count (excluding Abstract): 5479; Figures: 7, Supplementary Figures 3.
Abstract
Natural Killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR into primary NK cells and investigated how the intrinsic potential of discrete NK cell subsets affect re-targeting efficiency. After screening five 2nd- and 3rd-generation anti- CD19 CAR constructs with different signaling domains and spacer regions, a 3rd-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after three days of IL-15 stimulation prior to transfection, consistently achieving over 80% expression. CAR- engineered NK cells acquired increased degranulation towards CD19+ targets, combined with maintained intrinsic levels of degranulation to CD19- K562 cells. The response of discrete redirected NK cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIRs) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained partially sensitive to inhibition through KIR depending on the level of HLA class I expression on the target cells. Adaptive NK cells, expressing NKG2C, CD57 and self-HLA specific KIR(s), displayed superior ability to kill CD19+, HLA low or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK cell repertoire diversity to optimize the overall efficacy of CAR-NK cell based cancer immunotherapy.
Introduction
Chimeric antigen receptor (CAR)-redirected T cells have seen success in CD19-positive hematological malignancies, leading to cure of acute lymphoid leukemia (ALL) and lymphoma of B cell origin (1-5). Despite decades of studies, it is only recently that the relative importance of distinct T cell subsets has been considered in T-cell based CAR strategies (6).
There is experimental evidence that CAR modified CD8+ memory stem cells provide superior anti-tumor responses in xenograft models compared to conventional CD8+ T cells (7).
Likewise, central memory T cells display persistence and the ability to repopulate functional memory niches (8,9). Selective engineering of specific cell populations has also been tested clinically with promising results (10,11). It is feasible that engineering of defined subsets may provide more uniform outcomes and more predictable safety profiles. Paralleling this development, attempts have also been made to study other cell types for redirection (12) including natural killer (NK) cells targeting hematological (13,14) and solid tumor cells (15,16). CAR transfected NK cells showed significant cytotoxicity in vitro and in vivo (17).
Expanded allogeneic NK cells were used with minimal reported severe adverse side effects attributed to NK cells. Furthermore, expanded NK cells and NK cell lines transduced with anti-CD19 CAR have reached the clinic and are currently tested in several Phase 2 trials [NCT01974479, NCT02892695, NCT03056339]. Still, the potential of NK-CAR immunotherapy remains largely untapped.
Most previous studies have used feeder-cell expanded NK cells or the NK-92 cell line as a template for CAR engineering (5,18). Thus, it remains unknown how the extensive repertoire diversity within the NK cell compartment influence the functional potential of CAR redirected NK cells. Notably, NK cells are tightly controlled by integration of signals from activating and inhibiting receptors with a constitutive dominance of the latter (19). Major inhibitory receptors include the killer cell immunoglobulin-like receptor (KIRs) and CD94/NKG2A. KIRs bind to polymorphic residues on groups of HLA class I alleles and
NKG2A binds to HLA-E, a HLA class Ib molecule. Together, these receptor families establish the functional capacity of NK cells in a process termed NK cell education (20,21).
NK cells that lack self-HLA specific (self-specific) inhibitory receptors are not eliminated but rendered hypo-responsive and therefore remain tolerant to normal tissues. Like T cells, NK cells also undergo a continuous differentiation from naïve to terminally differentiated stages (22). NK cell differentiation is associated with a transcriptionally regulated functional maturation involving increased expression of effector molecules and a shift from cytokine responsiveness to receptor-mediated triggering of effector function (23).
Here, we set out to study the feasibility of CAR engineering of short-term stimulated primary NK cells and to explore whether CAR-engineering is influenced by the intrinsic heterogeneity in functional potential in the NK cell repertoire determined by NK cell education and states of NK cell differentiation.
Materials and Methods
Cells and culture conditions.
Human K562, Nalm6, 721.221 wildtype (221.wt), Bjab, Ros-50, and murine P815 cell lines were cultured in complete medium of RPMI 1640 with glutamine (Sigma) supplemented with 10% (20% for Bjab and Ros-50) heat-inactivated FBS (Sigma) and 50µg/ml gentamycin (Sigma). 221.C1/C2 transfectants were maintained with Geneticin® (ThermoFisher Scientific), and 221 cells transduced to overexpress HLA-E presenting signal peptide of HLA- A (221.AEH) were maintained in Hygromycin B (ThermoFisher Scientific). All cell lines were tested at least quarterly for mycoplasma infection using the MycoAlert mycoplasma kit (Lonza).
Human subjects and culture of primary NK cells.
Buffy coats from healthy donors were purchased without identifier from blood bank (Ullevål Hospital, Oslo). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (LymphoprepTM; Axis-Shield, and SepMateTM; Stemcell Technologies). CD19+-depleted PBMC or NK cells magnetically purified by negative selection (Miltenyi Biotec) were obtained from freshly isolated PBMC and cultured at 37°C in 5% CO2 in CellGro® SCGM (CellGenix GmbH) supplemented with 5% human serum (Trina Biotechnologies) and 20ng/ml of IL-15 (Miltenyi Biotec) for at least 72 hours prior to mRNA transfection.
HLA genotyping.
Genomic DNA was isolated from 200µl of buffy coat using the DNeasy blood and tissue kit (Qiagen). KIR ligands were determined using the KIR HLA ligand kit (Olerup SSP) for detecting the HLA-Bw4, -C1, and -C2 motifs.
Antibodies and flow cytometry.
The following conjugated Abs were used: anti-Granzyme B (2C5/F5), anti-CD3 (UHCT1), anti-CD14 (M5E2), anti-CD19 (HIB19), anti-CD107a (H4A3), and anti-DNAM1 (Dx11) from BD Biosciences; anti-KIR2DL1/S1 (EB6), anti-KIRDL2/S2/L3 (GL183), anti-NKG2A (z199), and anti-CD56 (N901) from Beckman Coulter; anti-DNAM1(Tx25), anti- KIR3DL1(Dx9), anti-CD57(HCD57), and anti-IFNg(B27) from BioLegend; anti- KIR2DL1(143211), anti-KIR2DL3(180701) from R&D Systems; and anti- KIR2DL1(REA284), anti-KIR2DL1/S1(PB6), anti-NKp30(AF29-4D12), anti- NKG2C(REA205) from Miltenyi Biotec. Streptavidin Qdot605 (Invitrogen) was used to detect biotinylated mAbs. A Live/Dead Fixable Aqua Dead Cell Stain (Invitrogen) was used to exclude dead cells from the analyses. Anti-CD19 CAR was detected using a goat anti- human Immunoglobulin (Ig)G1 Fcg antibody (Jackson Immuno Research Europe Ltd). Anti- HLA-ABC (W6/32), from BD Biosciences was used to detect expression of HLA class I in cell lines and PBMC. Data were acquired by FACSDiva software on BD LSRII equipped with a 488-nm laser, a 633-nm laser, and a 405-nm laser. Ultra-Comp® beads (eBiosciences) were stained with each of the fluorochrome-conjugated Abs separately and used as compensation controls. Acquired data were analyzed in FlowJo 10.3 (Tree Star).
Vectors construction and in vitro mRNA synthesis.
Both of the anti-CD19 CAR constructs – FMC63-IgGFc-CD28-OX40-CD3ζ and FMC63- CD8STK-41BB-CD3ζ (kindly provided by Dr. Martin Pule, University College London) comprise an anti-CD19 single-chain fragment variable fragment (scFv) derived from the FMC63 mouse hybridoma. The first construct additionally comprises an IgG1-CH2CH3 spacer region and costimulatory domains from CD28 and OX40, while the second consists of CD8 spacer region and 4-1BB costimulatory domain. This CAR was originally described in ref. (24) and cloned into a retroviral SFG vector by M. Pule and colleagues. For the purpose
of this study both constructs were recloned from the retrovirus vector SFG into the mRNA expression vector pCIpA102 as described in ref. (25). In vitro mRNA synthesis was performed as previously described (26). Anti-Reverse Cap Analog (Trilink Biotechnologies Inc.) was used to cap the RNA. The mRNA was assessed by agarose gel electrophoresis and quantified with Nanodrop (Thermo Fisher Scientific).
CAR mRNA electroporation.
Previously described transfection protocol was adapted for the optimized CAR expression in NK cells (26). Cytokine stimulated primary NK cells or B cell-depleted PBMC were washed and resuspended in serum-free SCGM medium at 10–50 × 106 cells/ml. mRNA was mixed to a final volume of 400 μl of the cell suspension at 100 μg/mL, and electroporated in a 4-mm gap cuvette at 500 V and 2 ms using a BTX 830 Square Wave Electroporator (BTX Technologies Inc.). Transfected cells were immediately transferred to pre-warmed SCGM medium supplemented with 5% human serum and 20ng/ml of IL-15 then cultured overnight (minimum 15 hours).
NK cell expansion.
NK cells isolated from PBMC were co-cultured with 221.AEH cell lines at NK:feeder ratio of 10:1 for 14 days in RPMI with 10% heat-inactivated FBS (Sigma) and 10ng of IL-15 (Miltenyi Biotec). The medium was replaced every 48 hours and feeder cells were replenished on day 7 of culture. Donors with at least 10% adaptive NK cells (NKG2C+, CD57+) among NK cells prior to culture were selected for the expansion to ensure retention of the subset at the end of culture. NK cells after 14 days of expansion were transfected with CAR and tested as per short term cultured NK cells described above.
Functional flow cytometry assays
NK cells after expansion and electroporation procedures were seeded with 2:1 E:T ratio in U-bottom 96-well plates in RPMI full medium and incubated for 4 h at 37°C and 5% CO2. The assay was performed within 24 hours post-transfection. For functional assays anti- CD107a antibody was added together with monensin (GolgiStopTM) and brefeldin A (GolgiPlugTM) at the start of the assay. At the end of the assay cells were stained for surface markers to identify the NK cells subsets. After surface staining, the cells were fixed and permeabilized with BD CytofixTM and CytoPermTM for intracellular staining of IFN-γ according to manufacturer’s instructions from BD Biosciences.
Killing assay
Purified NK cells were stimulated with IL-15 (10 ng/ml) for 3 days, electroporated with HA21 mRNA and rested overnight in the presence of IL-15. The next day NK cells were stained with CellTrace™ Far Red dye - CTFR (Thermo Fisher), washed and incubated with Nalm-6 in V-bottom 96-well plates at various E:T ratio in the RPMI full medium for 2 h at 37°C and 5% CO2 in the presence of FITC-DEVD-FMK (Abcam) that allows for detection of activated caspase 3 in living cells. At the end of the assay cells were washed and stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher) for 20 min, washed and resuspended in 200 µl of staining buffer. Cells were analyzed using BD LSR II and BD High Throughput Sampler (BD Biosciences). From every well 100 µl of cell suspension was aspirated and analyzed. Cytotoxicity of NK cells was calculated based on the number of live target cells (active caspase 3 and dead cell stain negative) within the population of CTFR negative cells.
Microwell assay and imaging
NK cells were stained with 0.6 μM CellTrace Calcein Red-Orange AM and 221 target cells with 1 μM CellTrace™ Calcein Green AM and 2.5 μM CellTrace™ Far Red (Thermo Fisher)
at 37°C for 10-20 min. The stained cells were seeded onto a silicon-glass microchip divided into two separate compartments containing wells with dimensions of 50×50×300 μm3.
Imaging of the microchip was performed using a Zeiss LSM 710 microscope equipped with an environmental chamber kept at 37°C and 5% CO2. Images were acquired using a 10×
objective every 10 min for 6 h. Excitation was provided by a 488nm Ar-Laser (0,3%) , 561nm DPSS-561-10 laser diode (1,0 %) and 633nm HeNe-laser (1,1%) and emissions were collected with maximal pinhole diameter. The Zen 2011 (Zeiss) software package was used for collecting data. Image processing and analysis was conducted using a customized script in MATLAB (Simulink) (27).
CAR expression time-course.
For experiments requiring CAR surface staining on NK cells in the presence of target cells, NK cells were purified, stimulated with IL-15 (10 ng/ml) for 3 days, electroporated with HA21 mRNA and rested overnight in CellGro® SCGM (CellGenix GmbH) medium supplemented with 5% FBS and 10 ng/ml IL-15. On the next day CAR NK cells were seeded in the U- bottom 96-well plate either alone or together with CD19+ cells (Raji and Nalm-6) or CD19- control mouse cell line P815 at 1:1 E:T ratio in the RPMI full medium and incubated at 37°C and 5% CO2. CAR staining was performed at different time-points 0-24h (time 0h indicates the initial CAR expression on the NK cells prior to stimulation with target cells). Two additional rounds of target cells were added to NK cells (at time 4h and 12h). At each time point NK cells were stained with anti-CAR antibody (goat anti-human Fc antibody), anti- CD3, anti-CD14, anti-CD19 and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher) for 20 min, washed and resuspended in 200 µl of staining buffer. Cells were analyzed using BD LSR II and BD High Throughput Sampler (BD Biosciences).
Statistical analysis.
Statistical analyses were performed with GraphPad Prism software 6.0 and Microsoft Excel with statistical plug-in package from Real Statistics. For comparisons of matched groups, Wilcoxon matched test was used. A Mann-Whitney U test was used for comparisons of unpaired groups. A p-value < 0.05 was considered statistically significant. * <0.05, ** <0.01,
*** <0.001, **** <0.0001.
Results
Optimizing conditions for electroporation of CAR into primary human NK cells
Transfection of resting or short-term stimulated human NK cells has shown varying transfection efficiencies (28). Therefore, we first performed time-kinetics experiments to determine the ideal conditions for robust re-direction of primary NK cells (Fig. 1A). Freshly isolated NK cells were either directly transfected or stimulated with IL-15 for up to 5 days before electroporation with anti-CD19 CAR mRNA. Transfection efficiency improved both in magnitude and reliability with increased length of stimulation reaching 80-90% CAR+ NK cells after three days of prior stimulation (Fig. 1A). Therefore, in subsequent experiments, NK cells were stimulated with IL-15 for 72 hours before transfection to ensure robust CAR expression.
To determine the stability of CAR expression after transfection, we labelled NK cells with cell-trace violet immediately after isolation on day 0 of culture, transfected NK cells on day 3 and followed the expression of CAR over 4 days (until day 7 of culture). Notably, CAR transfection efficiency was similar in dividing and non-dividing cells (Fig. 1B-C), consistently reaching 80-90% within 24 hours post-transfection. Thereafter, CAR expression declined, particularly in dividing cells. CAR expression also declined precipitously in non-dividing cells 48 hours post-transfection, becoming undetectable 4 days after transfection (Fig. 1B-C). Thus, both cell proliferation rate and receptor turnover contributed to diminished CAR expression 48 hours after electroporation, defining the expression kinetics of CAR-redirected primary NK cells.
The anti-CD19 CAR construct selected in this study (HA21) consist of an anti-CD19 single-chain fragment variable (scFv) fragment bearing only extracellular CH3 domain, CD28 with its transmembrane domain, and intracellular OX40 and CD3z signaling domain (Fig. 1D) (26). It lacked Fc-binding CH2 domain to avoid any possibility of cis-binding of NK cells through CD16, permitting only trans-activation through CD19 binding scFv region. The
activating signal is mediated via CD3z, an activating adaptor protein also used by various NK activating receptors: CD16, NKp44 and NKp30. Almasbak et al. previously established 9 variants of anti-CD19 CAR with different lengths of hinge-spacers and intracellular signaling motifs for engineering of T cells (26). Therefore, the selected construct was compared to relevant constructs in terms of hinge-spacer length and signaling motifs shown in Fig. 1D to investigate the effects of these structural modifications on CAR expression and transfection efficacy. Expression intensities of the different constructs were similar (Fig. 1E), at >80%
(Fig. 1F), without altering the NK cell repertoire (Fig. 1G). No preferential cell losses were observed. As expected, HA22, having only scFv without any hinge-spacer (Fig. 1D), was undetectable by anti-human IgG1 Fcg, exhibiting only background staining of anti-human IgG1 Fcg.
Functionality of NK-cells transfected with alternative CAR constructs
Next, we evaluated functional responses of the transfected NK cells. We first tested HA21 transfected primary NK cells against various CD19-positive and negative cell lines (Fig. 2A- B). K562 and 221 are sensitive to natural cytotoxicity by NK cells due to their low HLA expression but only 221 express CD19. Nalm6 is a CD19+ pre-B cell leukemia cell line with intact HLA-C1 and HLA-C2 expression, ligands for KIR2DL2/3 and KIR2DL1, respectively (data not shown). Compared to mock-transfected cells, CAR (HA21) re-directed NK cells showed significant CD19-specific responses to both Nalm6 and 221 cells with near two-fold increase of degranulation (p<0.01). Simultaneously, CAR-modified NK cells did not show increased degranulation against CD19- K562 cells nor any significant response to murine cell line P815 (Fig. 2B) as compared to mock-transfected cells. Thus, CAR-mRNA transfected NK cells exhibited target-specific redirection response, retaining natural cytotoxicity against class I deficient CD19- target cells without auto-activation.
Functional responses of cytotoxic lymphocytes depend on their ability to form immune synapses and generate an activating signal within the cell. The signaling motifs and length of the spacer on the CAR are known to affect the functionality of redirected T cells, also when using the present mRNA platform (25). Therefore, we determined if intracellular signaling motifs and length of the spacer could result in any difference in functional response by comparing HA21 with other tested constructs (Fig. 2C-D). Redirected NK cells showed similar degranulation and cytokine release responses to P815 and K562 (Fig. 2C-D) as compared to mock transfected cells. This again confirmed that redirection did not modify functional response nor cause auto-activation in NK cells even with HA20 that has a full spacer that could potentially bind to CD16. However, HA21 and HA22 redirected NK cells consistently showed better responses to CD19+ targets 221 and Nalm6 compared to NK cells modified with other constructs, suggesting that the removal of CH2 region may increase CAR potency. There was no significant difference in degranulation or cytokine release between HA21 and HA22 transfected NK cells (Fig.2C-D). Thus, the removal of CH3 domain did not appear to confer further functional advantage in vitro. Since HA21 could be readily detected with anti-Ig monoclonal antibodies and provided consistent NK redirection, this construct was selected and validated against a classical second-generation CAR based on the CD8 hinge region and with 4-1BB and CD3z as signaling motifs (Fig.2E-F and Supplementary Fig.1).
Importantly, HA21 and 41BB/z CAR-engineered primary NK cells showed similar functional activity in all conditions tested (Fig. 2E-F).
CAR expression kinetics experiments revealed relatively stable CAR expression during the first 48 hours after transfection (Fig. 1B-C). However, it remained unclear whether this expression would be lost upon interaction with target cells, thereby compromising the serial killing potential of NK cells. Therefore, we studied the CAR expression after 24 hour- incubation of NK cells with target cells. Following the first exposure to CD19+ targets, CAR expression was rapidly reduced to approximately 30% of initial levels and remained so beyond
12 hours despite additional rounds of target cell exposure. The CAR expression decline within this time frame was dependent upon presence of the CD19 antigen, since exposure of NK cells to CD19- P815 cells had no effect on the CAR expression (Fig 2G). Despite this rapid downregulation of CAR expression, CAR-engineered NK cells displayed consistently higher killing potential in FACS-based killing assays (Supplementary Fig. 2A) and contributed to a higher extent to serial killing in a recently described micro-well read out (Supplementary Figure 2B and Supplementary video 1) (27).
Redirected NK cells respond to CD19+ targets according to the functional diversity established by differentiation
The functional capacity of NK cells is established through a transcriptionally regulated differentiation program in combination with functional tuning through inhibitory and activating receptors during NK cell education (29). We set out to study whether differentiation states influenced the ability of NK cells to respond to CAR-mediated redirection by stratifying NK cell responses based on expression of inhibitory receptors (NKG2A and KIRs), and markers of terminal differentiation (CD57 and NKG2C) (29-31) (Fig. 3A). Terminally differentiated NK cells typically express CD57 (22), display epigenetic alterations in signaling components (23,32,33) and are more proficient in performing antibody-dependent cellular cytotoxicity (ADCC)(30).
CAR-redirection with HA21 or the 41BB/z had no effect on NK cell function against CD19 negative, HLA class I deficient K562 cells, establishing the intrinsic functional diversity of the six different NK cell subsets (Fig. 3B-C left panel). Redirected NK cells across all subsets showed significantly increased degranulation and even greater IFN-g production against CD19+ targets relative to mock transfected NK cells (Fig. 3B-C middle and right panels). Notably, the response hierarchy among the different NK cell subsets largely followed a similar trend as mock transfected cells, suggesting that intrinsic functional capacity
established through NK cell differentiation constrained the response of CAR transfected NK cells. Specifically, the least differentiated subset (KIR-NKG2A-CD57-) had the weakest response. Intriguingly, adaptive NK cells (NKG2C+CD57+KIR+NKG2A-), showed the greatest response to CAR engagement where most CAR+ cells degranulated and produced IFNg towards CD19+ targets. Thus, the state of NK cell differentiation determines the quality of CAR-redirected NK cell responses.
Redirected NK cells harness the functional capacity established by education in its response to CD19+ targets
To evaluate the effect of NK cell education on the functional response of redirected NK cells, we monitored CAR-induced responses in NK cells expressing various combinations of self- and non-self-specific inhibitory KIR (2DL3+ NK cells in C1/C1 donors and 2DL1+ in C2/C2 donors) and/or NKG2A (Fig. 4A). For both mock and CAR transfected NK cells, subsets expressing self-specific KIR exhibited stronger degranulation responses than NK cells expressing a non-self-specific KIR against K562, 221.wt cells and Nalm-6 cells. Thus, the functional imprints of NK cell subsets achieved through education were retained despite short- term IL-15 stimulation and CAR transfection (Fig. 4B). Similar results were observed when IFN-g production was assessed (Supplementary Fig. 2). While both educated and uneducated CAR transfected NK cells exhibited significantly greater response to CD19+ target cells, the response was always higher in the educated self-KIR expressing subsets. Furthermore, educated subsets generally showed a greater increase in response upon CAR transfection.
Therefore, the intrinsic functional capacity of a given NK cell subset influence the effector response in CAR-redirected NK cells.
CAR-redirected NK cells overcome NKG2A inhibition
The net outcome of NK cell target interactions depends on both intrinsic functional potential of the cell and the input through activating and inhibitory receptors at the immune synapse.
Therefore, we next examined whether CAR redirection is sufficient to overcome NKG2A inhibition and boost NKG2C stimulation in the context of HLA-CD19+ 221.AEH cells transduced to overexpress HLA-E presenting signal peptide of HLA-A (34) and stratified the functional response by gating on NKG2A and NKG2C single-positive NK cells (Fig. 5A). As expected, mock transfected NKG2C+ NK cells (Fig. 5B right graph, in blue) showed greater response to targets transduced to express HLA-E compared to 221.wt cells while NKG2A+ NK cells were less responsive to HLA-E+ target cells (Fig. 5B left graph). However, upon CAR redirection, the NKG2A+ NK cells responded significantly better to both wildtype and HLA-E transduced CD19+ targets as compared to mock-transfected cells and lost their susceptibility to HLA-E/NKG2A-mediated inhibition (Fig. 5B left graph, in red). However, CAR-redirected NKG2C+ NK cells displayed a significantly better response when stimulated by a combination of the CD19 antigen and HLA-E (Fig. 5B right graph, 221.AEH in red) compared to either of the ligands alone (Fig. 5B right graph, 221.wt in red and 221.AEH in blue, respectively). Thus, CAR re-direction can overcome NKG2A-mediated inhibition and, to some degree, further boost the response of NKG2C+ NK cells to HLA-E expressing target cells. Among the donors studied thus far, only one had a pre-existing adaptive NK cell subset, with high levels of NKG2C. Therefore, to test the potential additive effects of CAR-redirected stimulation and NKG2C/HLA-E interactions we expanded adaptive NKG2C+ NK cells from CMV+ donors by co-culture with 221.AEH for two weeks as previously described (35). Mock transfected adaptive NK cells responded strongly and specifically to stimulation by HLA-E expressing target cells (Fig.5C). Notably, this response was further boosted by CAR- redirection yielding superior overall responses of this particular subset against HLA-E expressing targets. The NK cells did not respond to murine P815 cell lines and both conventional and adaptive NK cells responded similarly to NK cell targets K562 and 221.wt according to their CD19 expression.
CAR-redirected NK cells remain sensitive to KIR inhibition
Although CAR-redirection overcame NKG2A-mediated inhibition, a stronger KIR-HLA inhibition could still influence CAR-redirected NK cells response. Therefore, we tested the degranulation response of CAR-redirected single KIR, NKG2A- NK cells from HLA-C heterozygous donors who express both HLA-C1 and C2 against HLA-Cw3(C1) or Cw4(C2) transduced 221 cells (Fig. 6A-B). Despite a similar overall increase in degranulation of CAR- redirected single KIR+ NK cells (Fig. 4B and 6B), we found that interactions between the KIR and their cognate HLA ligand significantly dampened the CAR-redirected NK cell response (Fig. 6B).
These results prompted us to explore the net effects of education and ligand inhibition on the efficiency of CAR-redirected primary NK cells against tumor cell lines with physiological expression of HLA-C. To this end, we tested the responses of CAR-redirected NK cells from C1/C2 donors against two CD19+, HLA-C2 homozygous cell lines Bjab and ROS-50. While ROS-50 cells expressed very low levels of total HLA class I, Bjab cells expressed relatively similar levels of HLA class I to that of normal B cells derived from PBMCs (Fig. 7A). The difference in HLA class I expression correlated with the degree of inhibition of mock and CAR-redirected 2DL3 and 2DL1 single positive NK cells (Fig. 7B).
Thus, in line with the minimal impact of low HLA class I expression on CAR-NK cell recognition of 222.wt cells in Fig. 4C, we observed similar degranulation of both 2DL3+ and 2DL1+ NK cells against ROS-50 (Fig. 7B Right panel). In contrast, both mock and CAR transfected 2DL1+ NK cells were inhibited by Bjab cells (Fig. 7B left panel, in blue). Together, these data show that redirected self-KIR+ NK cells respond strongly to its redirected target but remain sensitive to inhibition by physiological or supra-physiological levels of cognate HLA class I.
Discussion
NK cells posit themselves as an attractive alternative source of allogenic effector cells for CAR engineering. Here we show the feasibility of mRNA electroporation for CAR engineering of short-term activated NK cells. By probing the functional responses of NK cell subsets at the single cell level we determined the impact of subset diversity on the functional potency and specificity of CAR-engineered primary NK cells. We found that CAR signaling tapped into the intrinsic functional potential of any given subset determined by their state of differentiation and education through self-MHC interactions. Thus, CAR-triggered functional responses reached a maximum in adaptive NK cells, which are both terminally differentiated and educated through expression of self-specific inhibitory KIR. While CAR signaling efficiently overrode NKG2A-mediated inhibition, CAR-NK cells remained sensitive to inhibition by KIR binding to cognate MHC class I on target cells. These results suggest that CAR-NK cell therapy strategies may reach their full potential if one also consider the functional diversification of human NK cells as well as donor and recipient HLA genotypes.
We focused our analysis on short term-cultured NK cells transfected with mRNA. In a therapeutic setting, such CAR-NK cells will likely only provide anti-tumor effects for a short period of time, limited by the transient expression of the construct and the rate of rejection by the host immune system (18). Indeed, most CAR-redirected cells lost their CAR expression within 72 hours after transfection, and even faster following the interaction with target cells.
These data point to a limitation in the durability of the effector response of mRNA transfected NK cells. However, this also means that toxicity can be reliably managed based upon kinetics of NK cell turnover and CAR expression without suicide genes or attempts to control their persistence beyond their usual lifespan. In particular, the mRNA strategy may offer a safer and faster way for clinical testing of CARs against new targets. Transiently expressed CARs may also be useful in settings were a limited treatment period is desirable, e.g. for avoiding long term toxicity or eliminating minimal residual disease up front of bone marrow transplant.
Furthermore, repeated injection of RNA CAR electroporated T cells was shown to mediated regression of large vascularized flank mesothelioma tumors in a pre-clinical model based on transfer into NOD/scid/γc(−/−) mice (36).
Current one donor-one product strategies may be useful for early clinical translation of new CAR constructs for new clinical indications. However full-scale clinical implementation of CAR therapy will most likely require off-the-shelf cellular products which are transferable across institutions. Thus, the finding that subsets and receptor signaling matters is crucial to the development of master cell banks of NK cells for CAR engineering using not only mRNA but also more stable expression systems such as lentiviruses and transposon technologies (13,37-39). A key to develop off-the-shelf CAR therapy is the use of third party allogeneic effector cells. In this context, transfer across HLA barriers is a major challenge for T cell-based CAR therapy due to the risk for off-target toxicity caused by the endogenous TCR. Although there are several strategies to knock down the TCR in T cells, NK cells may be a superior allogeneic cell source for CAR therapy. In fact, transfer of NK cells across HLA barriers have shown limited toxicity and may instead contribute to the antitumor response (40). Our data suggest that it may be particularly attractive to selectively expand adaptive NK cells or reprogram/differentiate such cells from either NK cell precursors or inducible pluripotent stem cells (iPSCs) as potent off-the-shelf NK cell products that can be further enhanced with CAR engineering (41).
NK cell education is a process where NK cells achieve functional capacity through interactions between inhibitory receptors on NK cells and conserved residues on MHC class I expressed by cells of both stromal and hematopoietic origin (29). The molecular mechanism behind NK cell education remains elusive but likely operates by lowering the threshold for activation through activating surface receptors. We set out to explore the impact of education on the potency of CAR engineered NK cells. Like ADCC, CAR engineering led to significant responses in NK cells expressing non-self-specific KIR as their only inhibitory receptor.
However, educated NK cells remained superior in all functional read-outs tested suggesting that CAR signaling taps into the intrinsic potential of the cell.
We also noticed that more differentiated NK cells responded better to CAR stimulation, especially terminally differentiated adaptive NK cells. NKG2C+CD57+ adaptive NK cells typically express self-specific KIR and are therefore both educated and terminally differentiated (35). Adaptive NK cells are gaining increasing recognition as an attractive NK cell subset for cancer immunotherapy due to its robust cytotoxicity, cytokine release and potential for long-term persistence (42). We found that CAR-engineered adaptive NK cells responded stronger than all other subsets, demonstrating that the differentiation-driven functional potential serve a good template for CAR signaling.
It has been shown that adaptive NK cells undergo an epigenetic reprogramming associated with a specific reconfiguration of adaptor molecules including Syk, Eat-2 and FceR1g (32). Genetic engineering on CAR signaling motif posit itself as a possible approach to restore these activating molecules’ expression to specifically improve cytotoxicity and cytokine release responses of adaptive NK cells against its redirected target. FceR1g, together with CD3z used in this study are adaptor proteins for NK cell activating receptors CD16, NKp46 and NKp30. Creating a CAR with both would mimic these activating receptors on NK cells (43). It has also been proposed that DAP12, an adaptor for NKG2C and activating KIRs, is superior over CD3z at inducing NK cells to respond (44) and can be applied to further increase cytotoxicity of NK cells. HA27, without the OX40 motif, did not exhibit a similar functional response as HA21 despite the presence of known activating motifs in NK cells, including CD28 and CD3z. Therefore, the presence of OX40 may have a role in facilitating the activation of NK cells either as a spacer between the two activating motifs or interacting with intracellular proteins to induce a signaling cascade. Validating the finding that CAR signaling tap into the potential of discrete NK cell subset, we observed identical response hierarchies using a classical second-generation T cell CAR, based on 4-1BB and CD3z.
Next, we determined whether CAR-mediated activation modulate the NK cells’
response to its target and act in synergy with major activating receptors. We found that CAR redirection significantly increased the degranulation response of NKG2C+ NK cells against HLA-E expressing targets over and above its already high degranulation response. Thus, CAR signaling moieties not only override inhibitory signaling through NKG2A, they boost NKG2C+ adaptive NK cells response. The additive nature of NKG2C+ NK cells with CAR redirection further support their utility against tumors that overexpress HLA-E (45).
While CAR-redirected adaptive NK cells are shown to respond robustly to CAR targets, they are also known to respond robustly to KIR inhibition. Inhibitory KIR induce strong inhibitory signal through multiple tyrosine-based inhibition motifs (ITIMs) to achieve tolerance and prevent cytotoxicity against normal cells (19). Similarly, CD94/NKG2A binding to HLA-E also induces an inhibitory signal via ITIM. While CAR redirection overcame NKG2A inhibition, KIR-mediated inhibition was still observed in assays with 221 HLA-C1/2 transfectants expressing supra-physiological levels of HLA-C. Assays with tumor cell lines confirmed that KIR-mediated inhibition also influenced the recognition of targets with physiological levels of HLA class I. Therefore, while CAR redirection is sufficient to globally lower the threshold for NK cell responses, the function of CAR-engineered NK cells can still be negatively regulated by strong inhibitory signals. Nevertheless, the fact that the threshold for CAR NK cell inhibition was dependent on the level of HLA-C, and even overcome in the case of NKG2A/HLA-E interactions, open up for the possibility that alternative CAR designs may be less sensitive to self-recognition and pave the way for autologous NK-CAR strategies.
The finding that KIR inhibition under some circumstances downtuned the function of CAR-engineered NK cells suggest this as a tool to dampen CAR-driven off-target toxicity under specific circumstances. One possibility is to CAR-redirect KIR matched NK cells to treat tumors with downregulated HLA class I, where KIR can be used to attenuate CAR-NK cells against normal tissues. Conversely, one can envisage combinations of anti-KIR check
point therapy similar to what has been proposed for PD-1-mediated inhibition of CAR-T cell therapy (46). In fact, a relatively large fraction of mature differentiated T cells express KIRs (47). Thus, KIR inhibition may also be relevant in the context of CAR T cell therapy.
CAR-redirection enhanced the functionality of hypo-responsive KIR-NKG2A- NK cells. Although the response was inferior to that of adaptive NK cells, the lack of inhibitory receptors may compensate to make this subset superior in HLA-matched conditions where tumor cells express high levels of HLA class I or in autologous settings. Given that NKG2A- inhibition was overridden by CAR transfection, another attractive subset for CAR engineering may be NKG2A+KIR- NK cells that have greater intrinsic functional potential compared to KIR-NKG2A- NK cells. In fact, there is data suggesting that this NK cell subset provide clinical benefit on its own in the context of stem cell transplantation (48). Notably, however, uneducated NK cells can still be inhibited via their KIR (49). Thus, the use of uneducated NK cells for CAR therapy across HLA barriers may lead to a functional limitation by lack of education and KIR-mediated inhibition (if the tumor express ligands for the non-educating KIR).
Taken together, our study show that CAR engineering of primary NK cells is feasible.
We found that CAR-signaling can overcome the hypo-responsiveness of uneducated NK cells and mediate significant killing of target cells. However, educated NK cells still responded more strongly than uneducated NK cells, revealing that the signaling motifs of the CAR construct tap into the intrinsic potential of the NK cell subset. Redirected CAR-NK cells remain partially sensitive to KIR inhibition, suggesting that one may further optimize CAR- NK therapy by carefully considering donor and recipient HLA genotypes. If these in vitro findings can be verified in therapeutic in vivo models, CAR-NK cells may be an attractive alternative to CAR T-cell strategies, in particular in allogeneic settings.
Conflict of interest statement:
KJ Malmberg serves on the Scientific Advisory Board of Fate Therapeutics. The remaining authors have no conflicts to declare. All relationships have been reviewed and managed by Oslo University Hospital and Karolinska Institute in accordance with its conflict of interest policy.Authors' Contributions
Conception and design: V.Y.S. Oei, and K-J. M
Development of methodology: V.Y.S. Oei, H. Almåsbak, Björn Önfelt, J-A Kyte and K-J.
Malmberg
Acquisition of data (mRNA production, electroporation of cells, flow cytometry and imaging.): V.Y.S. Oei, H.J. Hoel, W. Yang, H. Almåsbak, M. Siernicka, A. Graczyk-Jarzynka, Malgorzata Bajor, Merete Wiiger, Jodie Goodridge, Ludwig Brandt, Dennis Clement and Daniel Palacios.
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V.Y.S. Oei, M. Siernicka, A. Graczyk-Jarzynka and K-J Malmberg
Writing of the manuscript:
V.Y.S. Oei, and K-J Malmberg
Review, and/or revision of the manuscript: H.J. Hoel, W. Yang, H. Almåsbak, M. Siernicka, A. Graczyk-Jarzynka, M. Winiarska, R. Zagozdzon, J. Olweus, J-A. Kyte.
Grant Support
This work was supported by grants from the Swedish Research Council, the Swedish Children’s Cancer Society, the Swedish Cancer Society, the Karolinska Institutet, the Swedish Foundation for Strategic Research, the Norwegian Cancer Society, the Norwegian Research Council, the South-Eastern Norway Regional Health Authority, and the European Commission Horizon 2020 Program 692180-STREAMH2020-TWINN-2015, Grant from
National Science Center, Poland 2014/13/N/NZ6/02081 (MS) and Stiftelsen Kristian Gerhard Jebsen.
Figure Legends
Figure 1. Optimization of primary NK cell redirection by CAR. (A) Histogram showing CAR (empty) expression against background in mock (filled) transfected NK cells after various durations of cytokine (IL-15) stimulation. (B) Representative dot-plot overlays illustrating CAR expression (red) against background from mock (blue) transfected NK cells over the next 4 days post transfection and different generations of division in culture. (C) Summary graphs of (B) from 4 donors illustrating CAR expression over 4 days post transfection and various generations of division. (D) CAR constructs used in the study. (E) Representative histograms showing CAR expression of different constructs. (F) Summary graph from 4 donors illustrating CAR expression of different constructs in primary NK cells.
(G) Representative diagram from 1 of 3 donors illustrating NK cell KIR and NKG2A repertoire 24 hours after CAR transfection.
Figure 2. Functional analysis of CAR-redirected primary NK cells. (A) Representative gating strategy and contour plots to illustrate functional analysis of bulk primary NK cells.
(B) Summary graphs showing degranulation response against CD19- (P815 and K562) and CD19+ (221.wt and Nalm6) targets in NK cells transfected with HA21 CAR construct.
Statistical significance between mock and CAR transfected cells indicated by black asterisk with underline (n=12-14). Summary graphs showing NK cell (C) degranulation response and (D) IFNg positive NK cells against CD19- (P815 and K562) and CD19+ (221.wt and Nalm6) targets across different CAR constructs (n=15). Summary graphs showing (E) degranulation response and (F) IFN-γ positive NK cells against CD19- (K562) and CD19+ (221.wt and Nalm6) targets in NK cells transfected with either HA21 or 41BB/z CAR construct (n=12).
Statistical significance between mock and CAR transfected cells indicated by black asterisk with underline. (G) Graph showing CAR expression following repeated target or control cells interactions. An arrow indicates when the new pool of target cells was added (n=4)
Figure 3. CAR-redirected NK cells’ response to various cell lines according to their differentiation status. (A) Representative gating strategy and contour plots to identify NK cells at various differentiation states and their functional response (CD107a and IFNg).
Summary graphs showing (B) NK cells degranulation response and (C) IFNg release against CD19- HLA-I deficient (K562) CD19+ HLA-I low (221) and CD19+ HLA-I+ (Nalm6) according to selected stages of NK cell differentiation. Statistical significance between mock and CAR transfected cells (all subsets combined) as indicated in the legend.
Figure 4. Redirected NK cells harness the functional capacity established by education in its response to CD19+ targets. (A) Representative gating strategy to identify single KIR+ educated NK cells and their functional response (CD107a). (B) Summary graphs showing degranulation response of mock or CAR transfected 2DL3+ (red) and 2DL1+ (blue) NK cells from HLA-C1 or C2 homozygous donors, respectively, against K562, 221 cells and Nalm6 cells. Statistical significance between mock and CAR transfected cells indicated by blue and red asterisk for 2DL1 and 2DL3 NK cells, respectively. Statistical significance between single 2DL1 and 2DL3 expressing NK cells (without underlines) indicated by black asterisk. Data are aggregated from three experiments (n=4-15).
Figure 5. CAR-redirected NK cells overcome NKG2A inhibition and boost NKG2C activation of adaptive NK cells. (A) Representative contour plots illustrating gating strategy to identify NKG2A or NKG2C single receptor expressing NK cells and their response against 221wt (red) or 221.AEH (blue) targets. Representative contour plots of degranulation of NK cells from selected subsets against the two targets. Numbers represent percentage of the population CD107a positive. (B) Summary graphs showing CAR-redirected NKG2A (left) NKG2C (right) single positive NK cells’ degranulation response against 221.wt or 221.AEH
cells. Statistical significance between mock and CAR transfected cells indicated by black asterisk without underline. Statistical significance against 221.wt and 221.AEH cells (with underlines) indicated by black asterisk. (C) Representative graph from one donor illustrating degranulation response from conventional or adaptive NK cells with mock or CAR transfection against 221.wt and 221.AEH cells as well as K562 (CD19- NK cell target) and P815 (mouse cell line).
Figure 6. CAR-redirected NK cells remain sensitive to KIR-mediated inhibition. From HLA-C1C2 heterozygous donors, (A) representative contour plots illustrating gating strategy to identify 2DL1- or 2DL3-expressing NK cells and their response against 221.C1 (red) or 221.C2 (blue) targets. Numbers represent percentage of the population CD107a positive. (B) Summary graphs showing mock or CAR transfected KIR 2DL3 (C1, left) 2DL1 (C2, right) single positive NK cells’ degranulation response against 221.C1 or 221.C2 targets.
Figure 7. NK cells exhibit KIR mediated inhibition to cell line expressing similar levels of HLA-I molecules as normal PBMCs. (A) Histogram illustrating HLA-ABC expression from Bjab (center) and ROS-50 (right) against normal B cells (left) derived from PBMC. (B) Graphs illustrating mock or CAR transfected, single KIR expressing CAR-redirected 2DL3 (C1, left) or 2DL1 (C2, right) NK cells’ response to Bjab and ROS50 cell lines. Donors were all HLA-C1C2 heterozygous and the target are both HLA-C2 homozygous.
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