Promising treatment for neuroblastoma: CD276-CAR NK-92

There is an increasing understanding of the biology of neuroblastoma (NB) - the most frequently encountered solid extracranial tumor in pediatrics. Despite this and ongoing improvements in the standard of care and treatment, outcomes for children with NB remain poor.

The high mortality rates and frequent relapse of high-risk NB patients require the development of new therapeutic approaches, including chimeric antigen receptor (CAR)-modified immune cells.

CAR T cells have recently demonstrated excellent potential in clinical response targeting CD19 and CD22 in hematological malignancies.

However, targeting solid cancers is an ongoing challenge, and the production of autologous CAR T-cell products requires a highly sophisticated manufacturing process.

The widely recognized natural killer (NK)-92 cell line provides a favorable alternative when looking to produce 'off-the-shelf' CAR-modified effector cells. This article outlines a study that aims to demonstrate that the immune checkpoint molecule B7-H3 (CD276) is aberrantly expressed on NB cells.

It should be noted that second-generation CD276-CAR-engineered (but not parental) NK-92 cells offer the capacity to facilitate the specific and long-term elimination of NB cells in vitro without affecting CD276-negative cancer cells.

CD276-CAR NK-92 cells were also found to display increased cytotoxicity in a three-dimensional NB spheroid model. This can recapitulate in vivo morphology, cell connectivity, gene expression, polarity, and tissue architecture, effectively bridging the gap between in vitro and in vivo models.

CD276-CAR NK-92 cells produced a wide range of NK effector molecules, plus a series of pro-inflammatory cytokines able to stimulate the host immune system.

It was noted that CD276-CAR surface expression and cytotoxic effector function were consistently stable for over six months, with data suggesting that CD276-CAR NK-92 may form a viable treatment option for patients exhibiting high-risk NB.

The pediatric tumor neuroblastoma (NB) is derived from sympathoadrenal progenitor cells within the neural crest. It remains the most frequently encountered extracranial solid neoplasm in childhood, accounting for 8%-10% of pediatric malignancies and over 15% of childhood deaths from cancer.1,2

This tumor entity is categorized by extreme clinical heterogeneity, ranging from spontaneous differentiation to cases of aggressive metastatic disease, resulting in patient survival below 50%, even when employing intensive multimodal therapy.3

Current treatment options for high-risk NB include induction chemotherapy, surgical resection, radiotherapy, and high-dose chemotherapy.

These techniques are frequently followed by autologous stem cell transplantation and immunotherapy using monoclonal antibodies (mAb), which target the cell-surface disialoganglioside GD2 in combination with retinoic acid and cytokines.4,5

Significant challenges remain, however, including drug resistance, disease recurrence, and late effects - particularly in patients with high-risk NB. There is an urgent need for novel targeted therapies that are able to minimize toxicities while improving cure rates.

Chimeric antigen receptor (CAR)-modified lymphocytes may represent an immunotherapeutic approach that holds great promise. This approach involves the genetic modification of immune cells, prompting these to express synthetic recombinant receptors which induce lymphocyte activation when binding with an antigen.6

CAR T cells have achieved significant clinical recognition due to their notable response rates in patients receiving autologous CD19-redirected T cells for the treatment of refractory or relapsed B-cell malignancies.7,8,9

The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recently approved two autologous CD19 CAR T cell products for use with patients presenting acute lymphoblastic leukemia and specific types of refractory or relapsed large B-cell lymphoma.

CAR T cells have the potential to induce severe side effects, however; for example, cytokine release syndrome (CRS) and neurotoxicity. The manufacturing process is also complex and expensive because this treatment must be produced on an individual patient basis.10

Autologous CAR-T cells are manufactured from patients' peripheral blood lymphocytes. This means that their transduction efficiency, yield, T-cell subtype distribution, and activation state can be significantly varied, impacting overall product quality and composition.

Contemporary CAR-based immunotherapeutic strategies tend to focus on T cells as effector cells.

However, natural killer (NK) cells have become an increasingly appealing option due to their robust antitumor activity and improved safety in an allogeneic setting, factors that could overcome a number of barriers to effective autologous CAR T cell therapies.

Most of the CAR NK cell studies thus far were conducted using NK-92 cells - an IL-2 dependent, constantly growing human NK cell line with phenotypic and functional characteristics of activated NK cells, other than the expression of FcγRIII (CD16).11

NK-92 cells have demonstrated persistent antitumor activity against a range of hematological malignancies and solid tumors when investigated in pre-clinical studies.12,13 The safety of irradiated NK-92 cells was also noted in phase I clinical trials, with a number of patients undergoing long-lasting treatment responses.14,15,16

This range of characteristics makes NK-92 cells an attractive option for CAR-engineering, offering good potential in the development of standardized 'off-the-shelf' therapeutics for adoptive cancer immunotherapy.17

CAR T cells have demonstrated excellent effectiveness in the treatment of hematological malignancies, but these have displayed limited activity in solid tumors.18

One notable barrier to successful immunotherapy for solid tumors lies in the lack of genuine tumor-specific antigens - an issue that leads to the targeting of tumor-associated antigens overexpressed on tumors and shared with healthy tissues, potentially increasing the risk of severe on-target off-tumor toxicity.19

These barriers are further complicated due to antigen expression typically being heterogeneous within a tumor and the majority of antigens not being broadly expressed across different tumor types.

CD276 (B7-H3) is part of the B7 family of immune checkpoint molecules - important regulators of the adaptive immune response and key factors in human cancer.20,21,22,23,24

It would seem that CD276 mRNA is ubiquitously expressed in a diverse array of tissues and cell types, but immunohistochemical (IHC) analysis has shown that the expression of CD276 protein is either weak or entirely absent in normal cells.25,26

CD276 protein is typically overexpressed in most solid human tumors, however. These tumors include prostate cancer, pancreatic cancer, non-small-cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, and within the tumor vasculature.27

It is possible that this disparity is the result of reduced levels of microRNA-29 – a factor that is inversely related to the upregulation of CD276 expression in malignant cells.28

The overexpression of CD276 is a factor in tumor immune evasion, also resulting in an increased risk of metastasis.

It also regularly correlates with rapid cancer progression and adverse clinical outcomes in a number of malignancies; for example, prostate cancer, pancreatic adenocarcinoma, ovarian cancer, lung cancer, and renal carcinoma.29,30,31,32,33

A recent article published by Zhang et al. gave a detailed analysis of the transcriptional profiles of five Gene Expression Omnibus datasets containing clinically annotated NB cases. This analysis demonstrated that high CD276 expression was linked to poor overall survival.25,34,35,36

Both primary and metastatic tumors may see CD276 expressed on various cell types, including differentiated tumor cells, cancer-associated fibroblasts, tumor-initiating or cancer stem cells, and cells of the tumor vasculature.27,37,38,39

When evaluated against other immune checkpoints, it has been noted that CD276 regulates innate and adaptive immunity as well as promotes cancer progression and metastasis via a range of non-immunologic functions.

These varied characteristics and its tendency towards differential expression in tumors versus healthy tissues make CD276 a key target for novel immunotherapeutic strategies.40

An evaluation was conducted around CD276-specific mAb and antibody-drug conjugates in xenograft mouse models and phase I clinical trials. This study highlighted specific antitumor activity and a promising safety profile.41,42,43,44

There is also evidence to suggest that CAR T cells targeting CD276 may mediate significant antitumor effects in pre-clinical studies of different solid tumors, including NB.45,46,47,48,49,50,51

The study presented here aims to explore the benefits of CD276-CAR-engineered NK-92 cells, evaluating their potential as an 'off-the-shelf' cellular therapeutic able to recognize and target NB cells.

Materials and methods

Cell lines and culturing conditions

A number of neuroblastoma cell lines were commercially acquired from the European Collection of Authenticated Cell Cultures. These included LAN-1, SH-SY5Y, SK-N-AS, SK-N-BE, and IMR-32.

Both the NB cell line Kelly and the acute myeloid leukemia (AML) cell line KG-1a were commercially acquired from Cell Line Services.

The NB cell line LS was established by Rudolph et al. This was provided by Prof. Handgretinger, University Hospital Tuebingen, Children's Hospital, Germany.52

The tumor cell lines were all maintained in Roswell Park Memorial Institute (RPMI) 1640 medium that had been supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific), 2 mmol/L L-glutamine, and 1 mmol/L sodium pyruvate (Biochrom) – a combination termed 'RPMI complete medium'.

Lenti-X 293T cells (Clontech) have been cultivated in DMEM high glucose (4.5 g/L) medium (Thermo Fisher Scientific), which contained GlutaMAX supplemented with 10% FBS and 1 mmol/L sodium pyruvate (Thermo Fisher Scientific).

NK-92 cells were commercially acquired from the American Type Culture Collection.

These were maintained at a concentration of 105 cells/mL in Minimal Essential Medium (MEM) Alpha Medium containing GlutaMAX (Thermo Fisher Scientific) supplemented with 20% FBS and 100 U/mL IL-2 (PROLEUKIN, Aldesleukin, Chiron) – a mixture termed 'NK-92 complete medium'.

Primary NK cells were successfully isolated from healthy donors by utilizing the EasySep human NK cell isolation kit (STEMCELL Technologies).

These were maintained in RPMI 1640 medium (Thermo Fisher Scientific) and appropriately supplemented with 10% pooled AB-serum from healthy donors (Transfusion Medicine, University Hospital Tuebingen, Germany).

All instances of media included one antibiotic-antimycotic solution (Thermo Fisher Scientific) comprised of 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B.

The complete range of cells and cell lines was maintained at 37 °C in a humidified 5% CO2 atmosphere. These were routinely tested to look for potential mycoplasma contamination.

Flow cytometry

Cells were stained at 4 °C with the specified antibodies. This was done in flow cytometry buffer containing PBS (Sigma-Aldrich) that had been supplemented with 2% FBS and 0.5 mol/L EDTA (Sigma-Aldrich) for a total of 15 minutes.

Live cells were gated based on forward and side scatter. This was done using a BD FACSCanto II flow cytometer, with CD276-CAR expression on NK-92 cells determined via CD34 marker gene expression.

Immunohistochemistry

Immunohistochemistry for B7-H3 was performed on formalin-fixed, paraffin-embedded tissue sections.

An automated immunostainer (BenchMark ULTRA; Ventana Medical Systems) was used to perform staining, with operation in line with the manufacturer's protocol. A murine anti-human-CD276 (6A1) mAb (Abcam) was employed as the primary antibody.

An independent evaluation of cytoplasmic B7-H3 expression was conducted by two investigators. This was measured as the percentage of stained tumor cells versus total tumor cells, resulting in the semiquantitative scoring of cytoplasmic staining intensity (IHC grade): 0 (negative), 1 (weak), 2 (moderate), or 3 (strong).33

Generation of lentiviral vectors

Creative Biolabs supplied a 2nd generation lentiviral vector plasmid which was used to encode the CD276-specific CAR (CD276-CAR) construct.

The CD276-CAR was created by conjugating a B7-H3 clone m851-derived scFv on a 2nd generation CAR backbone. This incorporated an EF1α promotor, a CD28 transmembrane domain, a CD8 hinge domain, a cytoplasmic CD28 co-stimulatory, and a number of CD3-ζ signaling domains.

Truncated CD34 (tCD34) is co-expressed after a T2A site to facilitate detection and enrichment. Transfer plasmid encoding a green fluorescent protein (GFP)-luciferase construct was provided by Dr. Irmela Jeremias, Helmholtz Center, Munich, Germany.

Lentiviral particles (LVP) were generated in a Lenti-X™ 293T (Clontech) following lipofection (Lipofectamine 3000, Thermo Fisher) of a VSV-G envelope plasmid, a 2nd generation packaging plasmid, and the specified transfer plasmid.

Supernatants containing LVP were concentrated using Lenti-X concentrator (TaKaRa) prior to storage at −80 °C until use.

Lentiviral transduction

NK-92 cells were seeded using a concentration of 1.25 × 106 cells/mL of MEM Alpha Medium and supplemented with a combination of 8 ng/µL of protamine sulfate (Sigma-Aldrich) and 2.5 µmol/L of BX-795 (Cayman Chemical Company). These were then transduced with CD276-CAR LVP for a total of 16 hours.

Transduced cells were then cultivated using NK-92 complete medium, with transduction efficiency ascertained via flow cytometric analysis of the CD34 surface expression. Next, CD276-CAR NK-92 cells were single-cell sorted and screened to ensure the highest CAR expression.

Neuroblastoma cells were seeded using concentrations of 5.0 × 104 or 1.25 × 105 cells/ mL in RPMI medium. This was done without supplements, and these cells were then transduced with GFP-luciferase LVP for a total of 16 hours.

These cells were maintained in RPMI complete medium, with transduction efficiency determined via flow cytometry.

Calcein-release cytotoxicity assay

Target cells were labeled using 10 µmol/L of calcein acetoxymethyl (Calcein AM) (Thermo Fisher Scientific) in RPMI medium supplemented with 2% FBS. Its concentration was 106 cells/ml.

CD276-CAR NK-92 cells were washed prior to being resuspended in RPMI medium supplemented with 2% FBS. These were then co-incubated with target cells for a total of 2 hours. It was possible to determine CD276-CAR-specific cytotoxicity via the fluorescence measurement capabilities of the Spark multimode microplate reader (Tecan).

Real-time label-free live cell analysis

Neuroblastoma cell lines were modified to reach a concentration of 105 cells/mL in the RPMI complete medium. These were then seeded in E-Plate 96 VIEW (OLS) micro-well plates.

Effector CAR NK-92 cells were modified to reach an E:T ratio of 5:1 in NK-92 complete medium without IL-2. These were then co-incubated with the target cells.

The xCELLigence real-time cell analysis (RTCA) system enabled cells to be efficiently monitored for a total of 72 hours. The RTCA 2.0 software was used to calculate NB cell viability, enabling CAR-mediated cytotoxicity to be subsequently ascertained.

Neuroblastoma three-dimensional spheroid cytotoxicity assay

GFP-positive NB cells were modified to reach a concentration of 5.0 × 103 cells/mL. These were then seeded in a 96-well low-attachment U-bottom plate (Nexcelom).

Spheroids were grown for a total of 72 hours prior to being co-incubated with CD276-CAR NK-92 cells. Fluorescence was determined using the Celigo S imaging cytometer (Nexcelom) at the specified time points over a 96 hour period (at least).

The average integrated fluorescence intensity of NB spheroids was used to calculate CAR-mediated cytotoxicity.

Quantification of cytokine release

Both effector and target cells were co-incubated in equal amounts of target cell complete medium and NK-92 complete medium without IL-2. An E:T ratio of 5:1 was used, and co-incubation was done at 37 °C for a total of 6 hours.

Supernatants were acquired and stored at −80 °C until these were required.

It was possible to achieve CD276-CAR NK-92 maximum degranulation by employing a cell activation cocktail (BioLegend) which included phorbol 12-myristate 13-acetate (PMA)/ionomycin.

The Bio-Plex Pro human cytokine 17-plex assay (Bio-Rad) was utilized to determine cytokine release. The LEGEND MAX human granulysin and granzyme B ELISA kits (Biolegend) and the human perforin ELISA kit (Thermo Fisher Scientific) were also employed for this purpose.

Data analysis

Statistical analyses were conducted using the GraphPad Prism 8 software package (GraphPad Software Inc), while flow cytometry data was analyzed via the FlowJo software package, V10.0.8 (FlowJo LLC).

Results

B7-H3 is aberrantly expressed on NB cells

Analysis was done on the CD276 expression on NB cell lines and patient tumor samples to ascertain if B7-H3 (CD276) could be a suitable target antigen for CAR-based immunotherapy of NB.

Figure 1. Immunostaining of neuroblastoma tissue for CD276 expression. Formalin-fixed, paraffin-embedded neuroblastoma tissue sections of five patients with aggressive and/or recurrent disease were stained for CD276 expression using immunohistochemistry and evaluated by two independent investigators for positivity of staining and staining intensity using an immunohistochemical grading score. Image Credit: Nexcelom Bioscience LLC

Formalin-fixed, paraffin-embedded NB tissue sections of patients with recurrent and/or aggressive disease were stained for CD276 expression. This was done using immunohistochemistry, revealing that 4 of 5 samples displayed either moderate or high cytoplasmic and membrane expression of CD276 (Figure 1).

Figure 2. CD276 and NK ligand expression profile analysis of neuroblastoma (NB) cell lines. NB cell lines LAN-1, Kelly, and LS were analyzed for B7-H3 (CD276) surface expression (A) and characterized using a panel of known NK cell ligands (B) via flow cytometry. Image Credit: Nexcelom Bioscience LLC

Flow cytometry was used to screen established high-grade NB cell lines (LAN-1, Kelly, and LS) for CD276 surface expression. It was observed that all NB cell lines expressed significant levels of CD276, highlighting the antigen's suitability for targeted, CAR-based NB therapy (Figure 2A).

Source: Nexcelom Bioscience LLC

Age (y) Gender Clinical stage IHC grade Stained tumor cells (%)
Patient 1 3 M NB IV relapsed 1 70
Patient 2 5 F NB III 3 90
Patient 3 7 F NB IV relapsed 3 100
Patient 4 5 F NB IV relapsed 2 80
Patient 5 6 M NB IV relapsed 2 80

Additional characterization of the NB cell lines was done via flow cytometry to screen for expression of known NK cell ligands (Figure 2B).

All three of the cell lines exhibited similar NK ligand expression profiles, each expressing human leukocyte antigen E (HLA-E) - a significant ligand for the inhibitory receptor complex CD94/NKG2A on NK cells.

LAN-1, Kelly, and LS cells were also noted to share a comparatively low expression of major histocompatibility complex (MHC) class I chain-related proteins A and B. This protein functions as an activating signal for NK cells via the natural-killer group 2, member D (NKG2D or CD314) receptor on NK-92 cells.

All three cell lines were also found to express Nectin-2 (CD112) and PVR (CD155) - two receptors that are widely recognized targets to DNAM-1 (CD226), triggering an activating signaling cascade. The immune checkpoint receptor TIGIT can also trigger an inhibitory NK response.

It was noted that the inhibitory NK ligand HLA-ABC was entirely absent on Kelly cells, while LAN-1 cells exhibited average expression. HLA-ABC expression was found to be elevated on LS cells, however.

Lentiviral transduction of NK-92 cells results in surface expression of CD276-specific cars

CAR constructs with an anti-CD276 scFv derived from clone "m851" w subcloned into a 2nd generation lentivirus vector. This CD276-CAR is generally encoded under the control of the human elongation factor 1 alpha promoter. It is, therefore, co-expressed with truncated CD34 as a marker gene (Figure 3A).

Figure 3. Schematic representation of CD276-CAR plasmid and generation of CD276-CAR NK-92 cells. Schematic representation of the lentiviral transfer plasmid encoding the CD276-CAR construct which was purchased from Creative Biolabs (A). The second-generation CAR comprises a CD28 co-stimulatory domain and a CD34 tag sequence. CD34 marker gene expression was measured via flow cytometry on untransduced (grey, filled) and transduced NK-92 cells before (black line) and after (grey line) single-cell sort (B) and monitored for 6 mo (C). The average proliferation rate of parental NK-92 and sorted CD276-CAR NK-92 was measured over the same time frame and calculated as mean ± SD (D), n = 3, ns: P = .6012. CAR, chimeric antigen receptor. Image Credit: Nexcelom Bioscience LLC

It was necessary to produce VSV-G pseudotyped lentiviral particles to transduce the human NK cell line NK-92. Flow cytometry of CD34 marker gene surface expression was employed in the determination of transduction efficiency.

Cells were then single-cell-sorted, with a total of eight NK-92 clones screened for CAR expression and proliferation. The NK-92 cell clone exhibiting the highest CD276-CAR expression was selected for additional experiments and investigation.

Cell number, viability, and CAR expression were monitored regularly to investigate the potential for transduction of NK-92 cells with the CD276 CAR construct to adversely affect proliferation.

Neither the proliferation nor viability (>90% at all times; data not shown) of CD276-CAR NK-92 cells were found to decrease in comparison to parental NK-92 cells. CAR expression levels were also found to maintain stability for over 6 months (Figure 3B, Figure 3C, Figure 3D).

CD276-CAR NK-92 cells express different receptor profiles compared to primary NK cells

Flow cytometry was used to screen cells for the expression of a range of activating and inhibitory NK cell receptors, immune checkpoint molecules, and chemokine receptors.

This was done to determine if CAR transduction had any impact on NK-92 receptor expression profile and to compare NK-92 cells with primary NK cells from healthy donors,

When evaluated against primary NK cells, NK-92 and CD276-CAR NK-92 cells displayed a somewhat lower expression of DNAM-1 (CD226). The expression of NKG2D (CD314) was similar to that of isolated NK cells. It should be noted that these are two of the main activating NK receptors.

Parental and CD276-CAR-transduced NK-92 cells showed a high expression of CD94 and NKG2A (CD159a). These two membrane receptors form an immune checkpoint complex recognizing the human HLA-E peptide, allowing them to inhibit NK effector function.

Characterization of CD276-CAR NK-92 cells. CD276-CAR-transduced and parental NK-92 cells were characterized for the expression of NK receptors (A), immune checkpoint molecules (B) and chemokine receptors (C) via flow cytometry. Results were compared to flow cytometric analysis of isolated NK cells from three healthy donors. Median fluorescence intensity ± SD was calculated using FlowJo software, n = 3. CAR, chimeric antigen receptor.

Figure 4. Characterization of CD276-CAR NK-92 cells. CD276-CAR-transduced and parental NK-92 cells were characterized for the expression of NK receptors (A), immune checkpoint molecules (B), and chemokine receptors (C) via flow cytometry. Results were compared to flow cytometric analysis of isolated NK cells from three healthy donors. Median fluorescence intensity ± SD was calculated using FlowJo software, n = 3. CAR, chimeric antigen receptor. Image Credit: Nexcelom Bioscience LLC

It was also noted that NKp80 – a key receptor expressed on all activated NK cells -was only found to be present on the isolated NK cells (Figure 4A).

Isolated NK and NK-92 cells were found to exhibit a comparable pattern of immune checkpoint receptor expression (Figure 4B). PD-1 was not highly expressed – this is the receptor for PD-L1 and functions as both the first- and second-line target for immune checkpoint inhibition-based cancer therapies.

However, both NK-92 cells and isolated NK cells did exhibit significant expression of CD96 (TACTILE) and TIGIT, two noteworthy emerging targets for immune checkpoint inhibition.

Flow cytometric analysis revealed that high expression of three of the most vital chemokine receptors - CD183 (CXCR3), CD184 (CXCR4), and CD197 (CCR7) – was present in both the NK-92 and primary NK cells. CD181 (CXCR1) expression was entirely absent in the NK-92 cell line, however (Figure 4C).

CD276-CAR NK-92 cells specifically lyse CD276+ NB cell lines in vitro

NK-92 cells were co-incubated with calcein-labeled NB cells at various effector-to-target (E:T) ratios to ascertain if CD276-CAR NK-92 cells offered the potential to specifically lyse NB cells via CAR signaling.

CD276-CAR NK-92-mediated tumor cell lysis. CD276-CAR NK-92 cells as well as untransduced control NK-92 cells were co-incubated with calcein-labeled neuroblastoma cell lines LAN-1, Kelly and LS as well as the CD276-negative acute myeloid leukemia cell line KG1a for 2 h. Specific tumor cell lysis is shown as mean ± SD, n = 3 (A). CD276-CAR and parental NK-92 cells were  irradiated with 10 Gy and used as effector cells in a CRA with neuroblastoma (NB) cell lines LAN-1, Kelly, and LS at an E:T ratio of 5:1 after indicated time points. Specific tumor cell lysis is shown as mean ± SD, n = 3 (B). Irradiated and non-irradiated CD276-CAR NK-92 cells as well as untransduced NK-92 cells were co-incubated with unlabeled NB cell lines and constantly monitored over time using the xCELLigence RTCA system. NK-mediated tumor cell lysis is portrayed as decrease in the dimensionless “cell index”, n = 3 (C). The release of cytokines by CD276-CAR NK-92 and parental NK-92 cells in the presence or absence of NB cells was measured using the Bio-Plex Pro human cytokine 17-plex assay and is shown as a heatmap (D). *P < .1; **P < .01; ***P < .001; ****P < .0001; ns = P ≥ .1. CAR, chimeric antigen receptor.

Figure 5. CD276-CAR NK-92-mediated tumor cell lysis. CD276-CAR NK-92 cells as well as untransduced control NK-92 cells were co-incubated with calcein-labeled neuroblastoma cell lines LAN-1, Kelly, and LS as well as the CD276-negative acute myeloid leukemia cell line KG1a for 2 h. Specific tumor cell lysis is shown as mean ± SD, n = 3 (A). CD276-CAR and parental NK-92 cells were irradiated with 10 Gy and used as effector cells in a CRA with neuroblastoma (NB) cell lines LAN-1, Kelly, and LS at an E:T ratio of 5:1 after indicated time points. Specific tumor cell lysis is shown as mean ± SD, n = 3 (B). Irradiated and non-irradiated CD276-CAR NK-92 cells as well as untransduced NK-92 cells were co-incubated with unlabeled NB cell lines and constantly monitored over time using the xCELLigence RTCA system. NK-mediated tumor cell lysis is portrayed as a decrease in the dimensionless “cell index”, n = 3 (C). The release of cytokines by CD276-CAR NK-92 and parental NK-92 cells in the presence or absence of NB cells was measured using the Bio-Plex Pro human cytokine 17-plex assay and is shown as a heatmap (D). *P < .1; **P < .01; ***P < .001; ****P < .0001; ns = P ≥ .1. CAR, chimeric antigen receptor. Image Credit: Nexcelom Bioscience LLC

When evaluated against parental NK-92 cells, specific lysis of NB cells was found to increase up to 80% after 2 hours. This was even the case at low E:T ratios (Figure 5A).

It was also noted that CD276-CAR NK-92 cells did not exhibit unwanted off-target effects. This was likely due to the fact that the cells did not induce any CAR-specific lysis of the CD276-negative control cell line, KG1a.

Since irradiation of NK-92 cells is necessary for all active clinical trials, it was deemed important to determine whether irradiation impacted cytotoxic efficacy. Parental NK-92 and CD276-CAR NK-92 cells were irradiated at 10 Gy. These were employed as effector cells in a standard Calcein-release assay (CRA).

It was noted that irradiation immediately prior to utilization had very little impact on CAR-mediated lysis of NB cell lines. CD276-CAR NK-92 cells were found to steadily lose effector function and undergo apoptosis over time, however (Figure 5B).

An examination of NK-92-mediated cytotoxicity of the NB cell lines was conducted over an extended time period. As the xCELLigence RTCA system is able to monitor label-free adherent cells in real-time, it was possible to accurately determine the start and end of cytotoxicity.

To achieve this, NB cells were co-incubated with CD276-CAR NK-92 or parental NK-92 cells, with electrical impedance measured at 5-minute intervals for more than 36 hours (Figure 5C).

Electrical impedance is illustrated as a dimension-less 'cell index' that can be understood as being directly proportional to the number of adherent cells at certain time points in comparison with those at the beginning of the experiment.

All three NB cell lines displayed a noteworthy decrease in cell index, occurring less than 2 hours following the addition of CD276-CAR NK-92 cells. There were no instances of lysis detected when parental NK-92 cells were used.

CD276-CAR NK-92 cells were found to eliminate a sufficient number of NB cells to prevent regrowth entirely. The use of irradiated CD276-CAR NK-92 cells did not hinder cytotoxic ability while inhibiting tumor regrowth.

The analysis was performed on the NK-92 cytokine secretion for a range of cytokines, including NK cell effector molecules. This was done using the Bio-Plex Pro human cytokine 17-plex assay (Figure 5D).

It was noted that, following co-incubation with NB cells, CD276-CAR NK-92 cells sharply increased secretion of a number of cytokines, including IL-2 (10- to 36-fold) and IL-10 (5- to 19-fold).

A notable increase in the secretion of the pro-inflammatory molecules IFN-γ (21- to 82-fold) and TNF-α (38- to 85-fold) was also detected. NK effector function was primarily linked to perforin release (10-fold increase) rather than granzyme B or granulysin secretion (1.3- and 2-fold increase, respectively).

CD276-CAR NK-92 cells successfully target NB spheroids

Most immunotherapy in vitro studies are focused on the investigation of tumor cell monolayer culture systems, typically overlooking the three-dimensional (3D) tumor structure. This oversight leads to limited translation potential to the in vivo situation.

To help address this gap in the literature, 3D multicellular NB spheroids were employed to evaluate infiltration and intratumoral cytotoxicity of CD276-CAR NK-92 cells.

The Celigo S imaging cytometer was used to monitor 3D cultures of the GFP-transduced NB cell lines Kelly, LAN-1 and LS, with a focus on spheroid growth and fluorescence intensity.

CD276-CAR NK-92-mediated lysis of 3D neuroblastoma spheroids. GFP-transduced neuroblastoma cell lines LAN-1, Kelly, and LS were grown as 3D spheroids and subsequently co-incubated with CD276-CAR NK-92 or parental NK-92 cells for 96 h in at least three individual experiments. Representative fluorescence images show LAN-1 spheroids (A). Integrated fluorescence intensity of neuroblastoma (NB) spheroids (LAN-1, Kelly, LS) was measured regularly using the Celigo S Imaging Cytometer (Nexcelom), representative fluorescence pictures of the NB spheroids are shown after co-incubation of 96 h (B, C). NK-92 and CD276-CAR NK-92 cells were irradiated prior to co-incubation and added to tumor spheroids (Kelly) at indicated E:T ratios and compared to non-irradiated effector cells (D). NB spheroids (Kelly) were treated with irradiated CD276-CAR as well as parental NK-92 cells at an E:T ratio of 5:1. Fresh, irradiated effector cells were added twice every 48 h (E). Integrated fluorescence intensity was compared to untreated control spheroids and is shown as mean ± SD, n = 3. CAR, chimeric antigen receptor.

Figure 6. CD276-CAR NK-92-mediated lysis of 3D neuroblastoma spheroids. GFP-transduced neuroblastoma cell lines LAN-1, Kelly, and LS were grown as 3D spheroids and subsequently co-incubated with CD276-CAR NK-92 or parental NK-92 cells for 96 h in at least three individual experiments. Representative fluorescence images show LAN-1 spheroids (A). Integrated fluorescence intensity of neuroblastoma (NB) spheroids (LAN-1, Kelly, LS) was measured regularly using the Celigo S Imaging Cytometer (Nexcelom), representative fluorescence pictures of the NB spheroids are shown after co-incubation of 96 h (B, C). NK-92 and CD276-CAR NK-92 cells were irradiated prior to co-incubation and added to tumor spheroids (Kelly) at indicated E:T ratios and compared to non-irradiated effector cells (D). NB spheroids (Kelly) were treated with irradiated CD276-CAR as well as parental NK-92 cells at an E:T ratio of 5:1. Fresh, irradiated effector cells were added twice every 48 h (E). Integrated fluorescence intensity was compared to untreated control spheroids and is shown as mean ± SD, n = 3. CAR, chimeric antigen receptor. Image Credit: Nexcelom Bioscience LLC

NB spheroids were co-incubated with CD276-NK-92 or parental NK-92 cells before being monitored for over 96 hours (Figure 6A). This was done following 4 days of culture.

Parental NK-92 cells did appear to have a notable effect on spheroid growth, but CD276-CAR NK-92 cells were observed to eradicate LAN-1 spheroids entirely after less than 48 hours (Figure 6B and Figure 6C).

Spheroids established from the cell line Kelly were also found to be almost fully eliminated after 72 hours. Levels of CAR-mediated cytotoxicity were not enough to fully lyse spheroids of the cell line LS over the specified time period, however.

CD276-CAR NK-92 cells were noted to specifically target and eliminate NB cells in a 3D spheroid model. Additional investigation was conducted into whether irradiation of CD276-CAR NK-92 cells would adversely affect cytotoxic effect.

It was determined that the utilization of irradiated effector cells would lead to the integrated fluorescence intensity of NB spheroids (Kelly cell line) decreasing to approximately 60% within 24 hours, maintaining stability for an additional 120 hours.

It was also noted that by increasing the effector to target ratio to 15:1, irradiated CD276-CAR NK-92 cells fully lysed the NB spheroid within a similar length of time to the non-irradiated CD276-CAR NK-92 cells at an E:T ratio of 5:1 (Figure 6D).

This finding prompted an additional investigation into whether the sequential addition of irradiated CD276-CAR effector NK-92 cells at low E:T ratios offered the potential to increase cytotoxic effector function.

This involved neuroblastoma spheroids using the Kelly cell line being co-incubated with CD276-CAR and parental NK-92 that had been previously irradiated before addition to the tumor cells. Effector cells were added every 48 hours.

The introduction of fresh CD276-CAR NK-92 cells was found to increase CAR-mediated cytotoxic effector function, therefore expanding the therapeutic window (Figure 6E).

Conclusions

A number of challenges remain in the treatment of patients with refractory or relapsed NB. The Children's Oncology Group's protocol for standard therapy of high-risk NB features the use of a combination of immunotherapy with mAb targeting disialoganglioside (GD2), immunostimulatory molecules and retinoic acid.4,53,54,55

A total of 12 active clinical trials for CAR T are currently ongoing for the cell-based therapy of NB. Only one of these trials does not employ GD2 as its primary target antigen, while GD2-CAR T cells have demonstrated highly promising results in the treatment of NB.56,57

It should, however, be noted that expression levels of GD2 are highly varied within NB. Low expression of GD2 prior to immunotherapy has also been linked to higher relapse rates in patients under GD2-targeted therapy.58

Besides cost-efficiency, one of the primary barriers to the widespread adoption of CAR T cell therapies is the prominence of severe side effects resulting from on-target, off-tumor toxicity.

Pre-clinical studies of NB mouse models have highlighted the potential or fatal neurotoxic events following treatment with GD2-specific CAR T cells.59,60

Due to these risks, an ideal candidate for CAR T-based therapy must be clearly expressed on the surface of the tumor and scarcely on normal tissue to minimize toxicity. There is, therefore, a significant need for new options for targeted immunotherapy of NB.

CD276 (B7-H3) – a member of the B7 superfamily - is a vital immune checkpoint molecule. It was recently recognized as a prognostic marker for a number of tumor types and is emerging as a promising immunotherapeutic target structure.40,61,62

CD276 has a tendency to be aberrantly expressed in a range of solid cancers, including NB.63,64 Its high expression across various tumor types and its restricted expression in normal tissue have led to CD276 becoming an appealing pan-cancer target for immunotherapy.

A number of active clinical trials are currently ongoing, aiming to assess the efficacy of omburtamab - a radioactive, 131 iodine-labeled anti-CD276 mAb which was awarded the FDA's breakthrough therapy designation in 2017 for its potential in the treatment of NB-associated CNS metastases.

The ongoing pivotal phase II trial for refractory or relapsed high-risk NB was anticipated to enter the biologics license application process in 2020.65

CD276 has been investigated and assessed as a potential immunotherapy target for a range of solid tumor entities in pre-clinical studies.46

CD276-CAR T cell therapies have demonstrated excellent efficacy in pre-clinical models looking at a range of malignancies, including brain tumors, pancreatic adenocarcinoma, pediatric solid tumors, liver cancer, colon cancer, cervical cancer, ovarian cancer, breast cancer, and melanoma.26,45,46,47,48,66,67

There are currently a limited number of clinical trials that are focused on CD276 as a target. Many of these are still in their early stages, and most notably, there is no active trial being undertaken specifically for NB.

The study outlined in this article introduced the 2nd generation CD276-targeting CAR to the widely recognized, FDA-approved NK cell line NK-92.

As NB cells typically exhibit a low level of MHC class I expression, the utilization of NK cells is an attractive option for immunotherapy.68,69

Enhanced NK isolation and expansion protocols are able to compensate for low cell quantities of autologous NK cells, while allogeneic NK cells are able to provide graft versus tumor effect. These are also effective at preventing graft versus host disease.70,71,72

Despite these advances, genetic modification of primary NK cells remains problematic, with poor CAR functionality and low transfection efficiencies.73,74,75

These issues can be addressed by employing the NK-92 cell line, however. This cell line provides a consistent, flexible platform for CAR-engineered immune cell production.

Both CD276-CAR expression and the viability of NK-92 were found to be stable for more than six months with no observable loss of cytotoxic function. CD276-CAR NK-92 cells were found to exhibit NK signaling that was independent of MHC expression. They also exhibited the expression of other inhibitory ligands on NB cells.

Initial in vivo studies and clinical trials with CAR- engineered NK-92 cells have highlighted a reduced tendency to induce severe side effects; for example, CRS and neurotoxicity.76,77

The study outlined here highlights that a key factor in CRS – IL-6 - was not produced by CD276-CAR NK-92 cells following co-incubation with NB cells.

These experiments have also highlighted that the secretion of pro-inflammatory cytokines, such as IFN-γ and MIP-1b, may further stimulate the endogenous immune system, enhancing antitumor activity.17

It is vital that CAR-immune cell therapies are effective in solid tumors, particularly in the metastatic setting. A key factor in their ability to do so lies in their ability to target and infiltrate tumors.78,79

Data presented within this article clearly highlights that CD276-CAR NK-92 cells can efficiently infiltrate and lyse NB spheroids which imitate the features of a solid tumor.

Further investigation into this potential for CD276-CAR NK-92 cells will be comprised of pre-clinical in vivo models for NB and the development of strategies to protect CAR-engineered NK-92 cells from the immunosuppressive conditions present in the tumor microenvironment.

The use of an NK cell line – for example, NK-92 - offers a number of distinct advantages over primary NK cells. These cells offer the potential to address a number of barriers to the clinical translation of donor-derived NK cells, including variable transduction efficiency and limited expansion potential.80

NK-92 cells exhibit potent ex vivo expansion to high cell numbers when generated under good manufacturing practice. They also boast an excellent safety profile and ease of genetic modification; factors that help make this cell line an optimum choice of platform for targeting tumors with CARs.81,82

The most notable disadvantage of NK-92 cells lies in their origin from a non-Hodgkin lymphoma patient, meaning that γ-irradiation of NK-92 cells must be performed prior to transfusion into a recipient.

Pre-clinical titration experiments have shown that an irradiation dose of 10 Gy is adequate in properly inhibiting proliferation of both unmodified NK-92 cells and CAR-engineered NK-92 cells.

In xenograft models, this approach has not negatively impacted the cells' in vitro cytotoxicity for at least 48 hours or limited their in vivo antitumor activity.77,83,84,85

It is also possible to freeze autologous CAR T-cell products after transduction, and these will then expand in vivo upon injection of comparatively small cell amounts. NK cells - particularly NK cell lines – must be expanded to a therapeutic dose prior to infusion, however.

It should also be noted that irradiation will prevent any in vivo expansion, meaning that repeated therapeutic dosages are required for ongoing control of cancer growth.

It is important to adjust the manufacturing process to account for these factors.

In an effort to modify the existing strategy for unmodified NK-92 cells, Burger et al. (2019) employed a qualified master cell bank of CAR-engineered NK-92 cells which acted as a reliable source for the subsequent production of patient doses destined for use in clinical trials.77,86

To bridge the cell proliferation's lag phase following thawing and to ensure a therapeutic dose of CAR NK-92 cells is available for a patient within less than one week, it was necessary to develop a process reliant on a maintenance culture of CAR NK-92 cells.

Therapeutic dosages can be expanded from this maintenance culture in batch culture within 5-6 days upon seeding the cells at an initial density of 5 × 104 cells/mL in 2 L of medium. Gas permeable cell culture bags are used to accommodate this culture.

After around 3.5 doublings (the doubling time of CAR NK 92 cells is 32-36 hours), the culture is able to yield a total cell number of 1 × 109. This number can be expanded in some instances, though this depends on factors such as the number of patients undergoing simultaneous treatment or the individual cell dose per patient.

A recent phase I clinical trial employed CD33- specific CAR NK-92 cells for the treatment of AML. No dose-limiting toxicities were observed during this study, even after several intravenous infusions of up to 5 × 109 irradiated cells per dose.76

The study presented here was able to generate CAR-engineered NK-92 cells with the capacity to eliminate NB cell lines in both monolayer cultures and 3D models in vitro.

The cells achieved this by targeting the pan-cancer antigen CD276, offering excellent potential as an 'off-the-shelf' treatment option for high-risk neuroblastoma and a range of other solid tumor entities.

Clinical implications

Adoptive immunotherapy with CAR-engineered immune cells has been found to be highly effective in hematologic malignancies. A number of clinical CAR NK-92 trials are currently ongoing.

The study presented here demonstrated the potential benefits of utilizing CAR-engineered NK-92 cells to target CD276 in neuroblastoma.

CD276-CAR NK-92 cells also offer strong potential for use in targeting a wider range of B7-H3-positive solid cancers. The data presented here suggested that further research into this area would be highly beneficial, including in vivo models and later clinical trials.

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Acknowledgments

Produced from materials originally authored by Stefan Grote, Kenneth Chun-Ho Chan, Caroline Baden, Martin Ebinger, Rupert Handgretinger and Sabine Schleicher from The Department of Hematology/Oncology, Children's University Hospital Tuebingen; and Hans Bösmüller, Mihály Sulyok, Leonie Frauenfeld from The Institute of Pathology, University Hospital Tuebingen.

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