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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Cytotherapy. 2020 Jun 11;22(8):450–457. doi: 10.1016/j.jcyt.2020.05.001

Analysis of ex vivo expanded and activated clinical grade human NK cells after cryopreservation

Sudarshawn N Damodharan 1,*, Kirsti L Walker 1,*, Matthew H Forsberg 1, Kimberly A McDowell 1, Myriam N Bouchlaka 1, Diana A Drier 2, Paul M Sondel 1,3,4, Kenneth B DeSantes 1,4, Christian M Capitini 1,4
PMCID: PMC7387178  NIHMSID: NIHMS1595469  PMID: 32536506

Abstract

Background:

Several methods to expand and activate (EA) NK cells ex vivo have been developed for the treatment of relapsed or refractory cancers. Infusion of fresh NK cells is generally preferred to the infusion of cryopreserved/thawed (C/T) NK cells because of concern that cryopreservation diminishes NK cell activity. However, there has been little head-to-head comparison of the functionality of fresh versus C/T NK cell products

Methods:

We evaluated activity of fresh and C/T EA NK cells generated by IL-15, IL-2 and CD137L expansion.

Results:

Analysis of C/T NK cell products demonstrated decreased recovery of viable CD56+ cells but the proportion of NK cells in the C/T EA NK cell product did not decrease when compared to fresh EA NK cell product. Fresh and C/T EA NK cells demonstrated increased granzyme B compared to NK cells pre-expansion, but only fresh EA NK cells showed increased NKG2D. When compared to fresh EA NK cells, cytotoxic ability of C/T EA NK cells was reduced, but C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody independent- and antibody dependent mechanisms within 4 hours post-thaw. Fresh EA NK cells generated high levels of IFNγ, which was abrogated by JAK1/JAK2 inhibition with ruxolitinib, but C/T EA NK cells showed lower IFNγ unaffected by JAK1/JAK2 inhibition.

Discussion:

Usage of C/T EA NK cells may be an option to provide serial “boost” NK cell infusions from a single apheresis to maximize NK cell persistence and potentially improve NK-induced responses to refractory cancer.

Keywords: ADCC, CD137L, Cryopreserved, IL-2, IL-15, Natural killer cells, Ruxolitinib

Introduction

Natural killer (NK) cells are a cytotoxic subset of the innate immune system that account for approximately 5-20% of lymphocytes in human peripheral blood, and play a vital role in the elimination of virally infected and malignant cells1,2. NK cells provide a rapid immune response against stressed or transformed cells without needing activation or recognition of major histocompatibility complex (MHC) molecules on their target cells3.

Since NK cells are not thought to cause graft-versus-host-disease (GVHD), a T cell mediated complication of bone marrow transplantation, investigators have explored infusing allogeneic NK cells into patients, with or without interleukin-2 (IL-2), as a means of inducing anti-tumor effects without causing GVHD. Remissions have been observed in hematologic malignancies, including myeloid leukemias4–12 and multiple myeloma,13 as well as in solid tumors, such as non-small cell lung carcinoma14, neuroblastoma15, and pediatric sarcomas16. The infusion of a tumor-specific monoclonal antibody along with the NK cells may augment their cytotoxic potential17.

One obstacle to the therapeutic use of allogeneic NK cells is their low frequency within the circulation thereby limiting the number of cells that can be collected from the donor. Consequently, over the past decade, several methods to expand and activate (EA) NK cells ex vivo have been developed for adoptive immunotherapy of cancer. Expanding and activating NK cells ex vivo should provide sufficient numbers of effector cells to attack tumors in vivo2,18,19.

Irradiated artificial antigen presenting cells (aAPCs) can present membrane bound (mb) gamma (c) cytokines like IL-15 or IL-21 that stimulate NK expansion with or without co-stimulatory molecules (e.g. CD137L/4-1BBL, MICA, etc.)20,21 that drive NK cell activation. This method of NK cell expansion has led to increased cytotoxicity pre-clinically, but results in the clinic have have been variable. EA allogeneic NK cells have shown responses in pancreatic cancer22, multiple myeloma 23,24 and acute myeloid leukemia (AML)25. Other studies treating pediatric sarcomas26 , breast and ovarian cancer27 , gastric cancer28 , AML29 , lymphoma and solid tumors30,31 did not show durable benefits or complete remissions.

One limitation of these approaches is the inability to administer multiple NK cell infusions without performing repeat apheresis and expansion, which is a time consuming and expensive process. However, because NK cell activation and expansion methodologies can yield a high number of cells, one could envision infusing a fresh aliquot at the end of the expansion, and then cryopreserving the remaining aliquots for future infusions. Although it is widely considered “better” to use fresh NK cells over cryopreserved/thawed (C/T) cellular products based on observations with IL-2 activated NK cells24, head-to-head analyses of C/T versus fresh EA NK cells are lacking.

In this report, donor peripheral blood mononuclear cells (PBMCs) were cultured with an irradiated aAPC genetically modified to express the co-stimulatory molecules CD137L and mbIL-15 (K562-CD137L-mbIL15) + IL-2 to generate high numbers of EA NK cells as a means of performing qualification runs (QR) for a clinical trial using both fresh and C/T products for children with refractory or relapsed neuroblastoma (NCT03209869).

Material and Methods

Tumor cell lines

Human M21 melanoma cells (gifted by Dr. Ralph Reisfeld, La Jolla, CA), human M21-GFP+ melanoma cells (gifted by Krishanu Saha, University of Wisconsin), human CHLA-20 neuroblastoma cells (Children’s Oncology Group cell line repository), human K562 chronic myeloid leukemia cells (ATCC, Manassas, VA), and K562-CD137L-mbIL15 transfected cells [Provided by Dr. Dario Campana, Singapore, to Waisman Biomanufacturing Facility (WBF), Madison, WI] were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), L-Glutamine (2mM), penicillin/streptomycin (100μ/ml) at 37°C, 5% CO2. The working cell bank of K562-mbIL15-41BBL had to be counted and have ≥ 70% viability, undergo STR testing, be positive for GFP, CD137L and IL-15 by flow cytometry, be sterile, mycoplasma negative, endotoxin negative, and negative for human pathogenic viruses. To address contamination, the final NK cell product had to be negative for GFP (≤ 0.1%). After expansion, the K562-41BBL-mbIL15 cells were irradiated at 100 Gy for 15min using a Gammacell 1000 Cesium 137 irradiator, confirmed as replication incompetent by EdU analysis by flow cytometry, and frozen down at a concentration of 0.5 -1 × 107 cells/mL in freeze medium RPMI 1640 + 30% FBS + 20% DMSO usually 200mL in 750 mL CryoStore bags (OriGen Biomedical, Austin, TX).

NK cell expansion

All cellular expansions occurred at the WBF under current good manufacturing practice (GMP) quality system guidelines. Fresh leukopacks were commercially obtained (AllCells, Alameda, CA and Key Biologies, Memphis, TN) from paid human apheresis donors, shipped at 1-10° C, and processed within 24 hours of delivery. PBMCs were isolated from the donor apheresis products by lymphoprep (STEMCELL Technologies, Cambridge, MA) density gradient centrifugation. NK cells were enumerated utilizing flow analysis of CD56+CD3− cell population. A 1:10 co-culture of NK cells: thawed irradiated K562-CD137L-mbIL15 cells were transferred into a WAVE Bioreactor (GE Healthcare, Little Chalfont, UK) in X-VTVO 10 media (Lonza, Basel, Switzerland) supplemented with 10% human AB serum (Corning, Corning, NY)32, 1x Glutamax, 100 IU/mL of recombinant human IL-2 (National Cancer Institute Biological Resources Branch Preclinical Repository, Frederick, MD) at 37° C, 5% CO2 for 12-days. Additional media was supplemented on days 4 and 8.

NK cell isolation, cryopreservation and thawing procedures

On day 12 of the co-culture, cells were harvested, concentrated and CD3+ cell-depleted utilizing the CliniMACS CD3-reagent system and depletion tubing set (Miltenyi Biotec, Birgesch Gladbach, Germany). The final cell product was divided into four equivalent cell doses; one dose of fresh EA NK cells in 60% Plasmalyte, 40% HSA solution and 3 doses of cryopreserved EA NK cells in 50% Plasmalyte, 40% human AB serum, and 10% DMSO. The cryopreserved EA NK cell product was formatted in CryoStore Bags (Origen cat# CS250N), cryopreserved in a Controlled Rate Freezer (Custom Biogenics System, Bruce Township, MI) to reach −180C (3°C per min cooling rate) and then transferred to vapor phase liquid nitrogen for storage. CryoStore bags of frozen EA NK cells were thawed in a 37°C water bath for 4-5 minutes. Thawed NK cells were gradually diluted with 10% Dextran 40 and then with 5% HSA such that both volumes were equal to ½ of the total volume of the cryopreserved product. The cells and diluent were allowed to equilibrate and the component transfer pack was further diluted with 10% Dextran 40.

The diluted cells were centrifuged (2200 rpm, 15 minutes, 10°C) and resuspended in a final NK cell formulation medium that matched the fresh NK cells. Cells were used immediately upon thaw for analysis. Quality Control (QC) testing of the final product included cell count, cell viability, sterility, endotoxin, mycoplasma, NK cell and residual T cell content, and assessment of residual irradiated K562-CD137L-mbIL15 cells. This testing was performed on aliquots of the cell pool prior to dispensing into the final product CryoStore bag at the WBF.

Flow cytometry

EA NK cells at day +12 of culture were counted and 1 x 106 cells were stained at 4°C for 20 minutes with anti-human antibodies including Ghost Red-780, CD3-FITC, CD16-PE-Cy5, CD56-BV510, CD45-BV570, NKG2D-PE-Cy7, NKp30-AF647, NKp44-PE, and/or NKp46-PE-Cy7 (Biolegend, San Diego, CA). Separate tubes were incubated with PMA/ionomycin for 4 hours, washed, and then analyzed for CD3-FITC, CD56-BV421, CD45-BV510, and gamma interferon (IFNγ)-AF647 or Granzyme B-AF647. Samples were run on the MACSQuant Analyzer (Miltenyi Biotec), MQD files were converted to FCS files using MACSQuantify™ Software (Miltenyi Biotec) and analyzed using FlowJo (FlowJo, Inc, Ashland, OR).

Cytotoxicity assays

Four hour cytotoxicity was evaluated using a standard 51Cr release assay. Human K562 erythroleukemia, M21 melanoma, and CHLA-20 neuroblastoma cells were used as target cell lines. Target cell lines were incubated in 51Cr (PerkinElmer, Waltham, MA) for 2 hours prior to plating. Effector cells were all from the same qualification run: 1) PBMCs prior to expansion, 2) EA NK cell product after 12 days of expansion/activation (“fresh”), and 3) EA NK cell product after 12 days of expansion/activation followed by cryopreservation and thaw (“C/T”). Effector cells were serial diluted from a starting Effector: Target (E: T) cell ratio of 50:1 in media alone, media with 100 IU/ml IL-2 (to stimulate antibody independent cytotoxicity) or 100 ng/mL anti-GD2 hu14.18K322A (provided by St. Jude Children’s Research Hospital and the Children’s GMP, LLC, Memphis, TN) to evaluate antibody dependent cytotoxicity of GD2 positive tumor cells and incubated at 37°C for 4 hours. Supernatant was then harvested, allowed to dry overnight at room temperature and the amount of 51Cr release was analyzed on the Cobra II autogamma counter (PerkinElmer, Waltham, MA). Percent lysis was determined using the equation [(sample 51Cr release - spontaneous 51Cr release)/(max 51Cr release - spontaneous 51Cr release)] x 100 where spontaneous release is determined by incubating 51Cr labeled target cells in media alone and max release is determined by incubating 51Cr labeled target cells in 2% Cetrimide (Sigma-Aldrich, St. Louis, MO).

Twenty four cytotoxicity was assessed using an Incucyte Live Cell Analysis System (Essen Bioscience, Ann Arbor, MI) according to manufacturer’s instructions. C/T EA NK cells immediately post-thaw and 24 hours post-thaw in 100 IU/mL IL-2 were serially diluted 2-fold from 5 x 105 cells to 1 × 104 cells in triplicate, and 500ng of anti-GD2 dinutuximab (National Cancer Institute Biological Resources Branch Preclinical Repository) was added to each well of a 96-well plate. GFP+M21 melanoma cells were added to each well at 1 × 104 cells/well with or without 100 IU/mL IL-2 to generate effector to target ratios of 50:1 to 1:1. Cells were labeled with Annexin V Red Reagent to quantify early apoptosis. Plates were then placed within the Incucyte at 37°C for 24 hours. Images were taken hourly whereby green object count (GFP+ cells), red object count (Annexin V+ cells) and overlap object counts (GFP+Annexin V+ cells) were enumerated by Incucyte Base Software.

ELISA and JAK1/JAK2 inhibition

Human IFNγ levels in supernatants from Day +12 EA fresh or cryopreserved NK cells immediately after thaw, were quantified using Legend Max ELISA pre-coated human IFNγ plates. ELISA was performed according to manufacturer’s instructions (BioLegend, Inc., cat#: 430107). Select wells were treated with 1.25uM Ruxolitinib for 6 hours (Chemgood). IFNγ cytokine concentration was extrapolated relative to a standard curve created by serial dilution of human IFNγ standards run in parallel. ELISA plate absorbance was analyzed at 450 nm on a VERSAmax Tunable Plate Reader (Molecular Devices, Sunnyvale, CA) and data were collected using SOFTmax PRO software (Molecular Devices, Sunnyvale, CA).

Statistical analysis

Statistics were performed using GraphPad Prism version 6.0 for the Macintosh OS (GraphPad Software, San Diego, CA). Data were expressed as mean ± SEM. For analysis of three or more groups, a non-parametric ANOVA test was performed with the Bonferroni or Sidak’s multiple comparisons post-test. Analysis of differences between two normally distributed test groups was performed using a two-sided Mann Whitney test. A p-value less than 0.05 was considered statistically significant.

Results

A single expansion of PBMCs from a standard leukapheresis with aAPCs that express mbIL-15 and CD137L in combination with IL-2 should generate sufficient numbers of highly activated NK cells allowing for infusion of a fresh EA NK product, and cryopreservation of several EA NK aliquots at the same dose for potential future infusion. The average fold expansion of EA NK cells was approximately 85-fold (Table 1) while T cell expansion was a minimal 1.6-fold and stayed well under 5% of the cellular product (Table 1 and Figure 1), which was comparable to prior reports from other groups19,33. When comparing the cell subset composition of fresh EA NK cell products to cryopreserved EA NK cell products via flow cytometry, the percentage of NK cells in the product did not decrease after cryopreservation and thaw (Table 2). Cryopreserved aliquots from QR1 were available for analysis, and showed a predominance of CD56+CD3− cells. QR2 and QR3 compared both fresh and C/T products. They show similar percentage and predominance of CD56+CD3− cells in both fresh and C/T products (Figure 1B). Despite the high percentage of NK cells, cell recovery was impacted by decreased viability after C/T (Table 3).

Table 1:

NK cell and T cell numbers over 12-day PBMC expansion.

QR1 QR2 QR3 QR4 QR5
Day NK cells T cells NK cells T cells NK cells T cells NK cells T cells NK cells T cells
0 0.31 1.6 0.3 2.5 0.25 4.0 0.32 0.77 0.18 1.8
3 0.22 2.1 0.17 2.4 ND ND ND ND ND ND
4 ND ND ND ND ND ND 4.9 2.0 0.41 3.7
5 0.82 2.6 ND ND 0.61 2.2 ND ND ND ND
6 ND ND 1.8 1.3 ND ND ND ND ND ND
7 19 2.2 ND ND 6.2 1.8 ND ND ND ND
8 ND ND ND ND ND ND 29 1.2 10 1.4
10 54 1.9 14 3.1 7.9 1.8 ND ND ND ND
11 58 2.0 15 4.8 6.9 1.1 18 1.2 19 1.1
12 60 2.1 13 4.3 ND ND ND ND ND ND
Fold Increase 194 x 1.3 x 43 x 1.7 x 28 x 3.6 x 56 x 1.6 x 106 x 0x
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NK cells and T cells were enumerated by flow analysis of CD3−CD56+ (NK cells) and CD3+CD56− (T cells) periodically over a 12-day period, following seeding on Day 0, for 5 separate QRs. These values reflect T and NK cell content prior to CD3+ depletion on Day 12, and are shown as a factor of 109 cells. QR = qualification run. ND = not done.

Figure 1. NK cells are enriched from fresh and cryopreserved products after 10:1 NK:aAPC and IL-2 expansion.

Figure 1.

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Over the course of 12 days, (A) the number of both NK cells (CD3−CD56+) and T cells (CD3+CD56−) were enumerated every 1 to 3 days by flow cytometry. Data are pooled from 5 QRs. (B) After expansion and CD3 depletion, cryopreserved EA and fresh EA products contained ~90-95% NK cells with minimal other immune cells. Data are pooled from 2 QRs.

Table 2:

Cell subset analysis after CD3 depletion.

QR1- Frozen QR2- Fresh QR2- Frozen QR3- Fresh QR3- Frozen
CD56−CD3+ 0.02% 0.82% 0.89% <0.01% <0.01%
CD56+CD3− 99.40% 98.90% 98.80% 80.40% 87.00%
CD19+ 0.10% 0.09% 0.09% 4.10% 3.70%
CD20+ 0.10% 0.04% 0.05% 3.70% 3.40%
CD14+ 0.10% <0.1% <0.1% 0.20% 0.20%
CD33+ <0.1% 0.1% <0.1% 8.20% 3.20%
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NK (CD3−CD56+), T cell (CD3+CD56− percent), B cell (CD19, CD20) and monocyte/myeloid (CD14, CD33) presence were enumerated on Day +12 by flow cytometry after CD3 depletion.

Table 3:

NK cell recovery after cryopreservation and thawing.

QR 1 QR 2 QR 3
Day 11/12 NK cells 60 13 6.9
Viability post-C/T 92% 73% 74%
Absolute Recovery (NK cell count x % viability x % CD56+CD3−) 54.9 9.4 4.4
% Recovery 91% 72% 64%
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Absolute values are reported as a factor of 109 cells. C/T = cryopreserved/thawed, NK = natural killer, QR = qualification run.

The cytotoxicity of NK cells relies upon the interaction of target cell ligands with activating and inhibitory receptors on NK cells. NKG2D, and the natural cytotoxicity receptors (NCRs) NKp44, NKp46, and NKp30, are activating receptors that play a crucial role in NK cell mediated cytotoxicity. To assess the functionality of the EA NK cells, NKG2D surface receptor and NCR expression were enumerated pre- and post-EA, as well as after cryopreservation. The percentage of NKG2D+ cells were significantly increased in fresh, but a significant increase was not detected in cryopreserved, EA NK cells when compared to NK cells pre-EA (Figure 2A). The percentage of NKp46+, NKp44+ and NKp30+ cells were similar both pre- and post-EA and between fresh and C/T EA NK cells (Figure 2A). KIR expression remained unchanged pre- and post-EA and between fresh and C/T EA NK cells (Figure 2B).

Figure 2. NKG2D is significantly increased in fresh but not cryopreserved EA NK cells, while KIR expression is preserved.

Figure 2.

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Expression of NKG2D, NKp44, NKp46, and NKp30 receptors by flow cytometry on EA NK cells. Percent cells with NKG2D, NKp44, NKp46, and NKp30 receptors by flow cytometry on NK cells gated by flow cytometry within Day 0 PBMCs, and Day 12 fresh and C/T EA NK cells. Data pooled from 2 QRs. * = p < 0.05.

NK cells also play a major role in anti-tumor immunity through the secretion of cytokines and degranulation of enzymes such as IFN-γ and Granzyme B, respectively. Significant increases in the percentage of both IFN-γ and Granzyme B+cells were observed in fresh EA NK cells compared to pre-expansion (Figure 3A). However, C/T EA NK cells only showed increases in the percentage of Granzyme B+ cells (Figure 3B), whereas IFN-γ cells actually decreased (Figure 3C), after cryopreservation.

Figure 3. Granzyme B, but not IFN-γ, expression is increased in cryopreserved EA NK cells after expansion.

Figure 3.

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The percentage of NK cells expressing IFN-γ and Granzyme B in cryopreserved EA NK cells compared to fresh EA NK cells from the same donor on Day 12, and a separate PBMC donor pre-EA on Day 0. (A) Representative and (B) pooled data from 2-3 QRs on Granzyme B and (C) IFN-γ.

We next evaluated both antibody-independent and antibody-dependent cellular cytotoxicity (ADCC) comparing fresh and cryopreserved EA NK cells from the same donor to fresh unexpanded PBMCs from a separate control donor against K562 (GD2− erythroleukemia), M21 (GD2+ melanoma), and CHLA-20 (GD2+ neuroblastoma) lines. With all 3 targets, cytotoxicity by fresh EA NK cells was superior to C/T NK cells from the same donor, as shown by high cytotoxicity at low E:T ratios (Figure 4A–C) and potentially higher frequency of CD16+ cells (Figure 4D). But the frequency of CD16+ cells was not a major limiting factor overall since both fresh and C/T EA NK cells showed increased cytotoxicity as compared to fresh unexpanded PBMCs from a separate donor (Figure 4A–C). C/T EA NK cell products are able to mediate antibody-independent cytotoxicity in the presence of IL-2 (Figure 4A) and demonstrate further augmented killing of GD2+ targets when anti-GD2 antibody is included (Figure 4B–C). However these results are seen when C/T EA NK cells are used immediately post-thaw in a 4 hour cytotoxicity assay. When C/T EA NK cells are used immediately post-thaw in a 24 hour cytotoxicity assay, ADCC against M21 melanoma is only seen in the first 4 hours post-thaw, then tumor outgrowth occurs (Figure 4E). For C/T EA NK cells, viability decreases from 72.1% immediately post-thaw to 33.6% 24 hours post-thaw in IL-2. When used in a cytotoxicity assay after 24 hours, ADCC against M21 melanoma is not observed (Figure 4F).

Figure 4. Cytotoxicity by fresh and C/T EA NK cells from the same donor showed increased activity compared to unexpanded PBMCs.

Figure 4.

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(A) Both fresh and C/T EA NK cell products are able to mediate antibody-independent cytotoxicity of K562 targets with or without 100U/mL IL-2. Both fresh and C/T EA NK cell products are able to mediate ADCC using IL-2 and an anti-GD2 monoclonal antibody against (B) melanoma and (C) neuroblastoma. (D) The percentage of CD16-expressing EA NK cells was enumerated by flow cytometry using unexpanded fresh NK cells, fresh EA NK cells and C/T NK cells. Cytotoxicity by anti-GD2 mediated ADCC against GFP+ M21 melanoma cells was assessed by enumerating green object fluorescent counts after exposure to C/T EA NK cells (E) immediately post-thaw and (F) 24 hours post-thaw using 24 hour live cell imaging.

IL-2 and IL-15 each bind the gamma (c) receptor (CD132), which signals through JAK1. The affinity of IL2 or IL15 binding is greatly influenced through interaction of the respective IL-2 (CD25) or IL-15 receptor alpha (CD215). However it is unclear what the effect of blocking JAK1 would be on IL-2/mbIL-15/CD137L EA fresh NK cells, and whether similar effects would be observed on C/T EA NK cells. Fresh EA NK cells and C/T EA NK cells were treated with ruxolitinib and analyzed for IFNγ production. Although C/T EA NK cells show a lower percentage of IFNγ cells (Figure 3C), when the number of IFNγ+ cells are equalized, C/T EA NK cells generate far less IFNγ than EA fresh NK cells (Figure 5). As seen with other methods of generating fresh EA NK cells, IFNγ production by fresh EA NK cells was completely abrogated by JAK1/JAK2 inhibition, however no effect on IFNγ production was observed when ruxolitinib was added to C/T EA NK cells (Figure 5).

Figure 5: Impact of ruxolitinib treatment on IFNγ production by fresh and C/T EA NKs.

Figure 5:

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Fresh and C/T EA NKs were plated at 1 × 106 cells/0.5mL in a 12 well plate and exposed to vehicle or 1.25uM ruxolitinib and then IFNγ was measured by ELISA. Data is pooled from 2 separate experiments. N.S. = not significant. **** = p < 0.0001

Discussion

NK cells possess the unique ability to recognize malignant cells without the need for activation by tumor-specific antigens, and thus play an important role in immunosurveillance, but are present in the peripheral blood in numbers that are too low to allow for effective adoptive transfer from a healthy allogeneic donor34. The numbers of effector cells that can be generated with present methodologies of NK cell expansion should allow cancer patients to receive multiple NK cell infusions, including an initial fresh product and subsequent infusions from aliquots of C/T NK cells derived from the initial expansion. This hypothesis will be tested in a pilot trial utilizing haploidentical EA NK cells in conjunction with an anti-GD2 immunocytokine to treat children with relapsed or refractory neuroblastoma (NCT03209869). The goal of this present report was to characterize the fresh EA NK cells, and evaluate fresh versus C/T cell products generated during QRs as a lead up to opening this clinical trial.

Infusion of NK cells that have been EA ex vivo should provide large numbers of highly active effector cells to attack tumors in vivo2,18,19. The intent of one donor apheresis in the pilot trial is to generate enough NK cells to give one fresh infusion at 1 x 107 cells/kg and 3 C/T infusions at 1 x 107 cells/kg each, all to one patient. All qualification runs were able to meet these thresholds for children up to 70kg. In this study, using IL-2/mbIL-15/CD137L aAPCs yielded 6.9-58 × 109 (28-194 fold increase) NK cells, with only 1.1-4.3 × 109 (0-3.6 fold increase) T cells, consistent with a NK product with >95% purity prior to CD3 depletion. While higher yields have been reported using other EA methods, one notable difference from other trials with EA NK cells is the dose of IL-2 used here was 100U/mL18,23. Some studies with feeder cells used IL-2 as low as 10U/mL20,21,24, whereas others used up to 500U/mL24,30.

Both autologous and allogeneic fresh EA NK cells have been utilized to treat a variety of hematologic malignancies and solid tumors, with mixed success. The advantage of using allogeneic NK cells is that the cells are derived from a healthy donor, and thus should function better than a heavily pre-treated patient’s cells, and can potentially exploit HLA-mismatches and/or KIR-KIR ligand mismatches to enhance alloreactivity against the tumor35. However, adoptive cell transfer of fresh EA NK cells from HLA-matched donors26 and mis-matched donors36 have been shown to cause GVHD. Because NK cell expansion protocols typically also stimulate T cell proliferation given the use of IL-2, IL-15, and IL-21, it is important that CD3+ T cells are depleted to prevent the risk of GVHD.

Unexpanded NK cells historically showed diminished cytotoxicity in vitro after C/T37–43, except during ADCC44, but have been shown to have better cytotoxicity in vitro up to 24 hours after C/T as long as IL-2 is present20,45,46. In contrast, NK cells that were E/A with irradiated CD3-depleted PBMC feeder cells supplemented with IL-2 and OKT3 showed high cytotoxicity in vitro up to 96 hours after C/T without IL-247,48. In vivo, E/A NK cells show high47,49 or decreased48 cytotoxicity against humor tumor xenografts after C/T, especially if mediating ADCC49. The only studies that analyzed C/T NK cells E/A from feeder cells in the context of a GMP workflow, like in our study, found poor cytotoxicity against K562 cells immediately post-thaw which improved after 24 hours of IL-220,34,50. One study showed equivalent cytotoxicity to fresh EA NK cells51. Our study adds comparisons of both fresh and C/T EA NK cell cytotoxicity to unexpanded NK cells, which importantly show that while C/T EA NK cells have worse cytotoxicity than fresh EA NK cells, C/T EA NK cells are still superior to using pre-expanded fresh PBMCs. We also show for the first time retention of ADCC against 2 different solid tumors that can be maintained up to 4 hours post-thaw, and differential responses between fresh and C/T EA NK cells to JAK1/2 inhibition. The only clinical data using haploidentical C/T EA NK cells involved 8 patients with multiple myeloma who were given fresh or C/T EA NK cells, where interestingly only recipients of fresh EA NK products showed in vivo expansion but the one patient who had a partial response received C/T EA NK cells24. Of note, no published data exists for infusing both fresh and C/T NK cells that were EA from genetically modified feeder cells and infused into the same patient, and/or combined with an immunocytokine like our NCT03209869 trial.

Other investigators have used mbIL-2152 or IL-29, which share CD132 with IL-15, to provide signals that enhance NK cell expansion and activation. Head to head comparisons of EA methodologies in the context of a clinical trial are lacking. In this ex vivo study, IL-2 was combined with an aAPC expressing CD137L and mbIL-15 for expansion and activation. T cell concentrations were low in fresh and C/T EA NK cell products, showing only an average of 1.6-fold expansion after IL-2/mbIL-15/CD137L stimulation. The percentage of NK cells recovered from C/T EA aliquots were similar to the percentage of NK cells present in the fresh product at the end of incubation, however absolute recovery was impacted by decreased viability post-thaw.

A variety of assays have been used to measure the functional capacity of EA NK cells, such as quantifying cell surface NCR expression, cytotoxicity and cytokine secretion, but little data are available characterizing cryopreserved EA NK cell products. Increases in NKG2D+ cells were seen in fresh NK cell products and granzyme B+ cells were observed in fresh and C/T EA NK cell products compared to pre-expansion levels, and at levels comparable to fresh EA NK cell products, consistent with what has been demonstrated by others utilizing fresh EA NK cells19,33. Compared to pre-expanded cells, fresh and C/T EA NK cells showed enhanced antibody independent cytotoxicity of K562 leukemia cells as well as tumor cell lines that are typically resistant to antibody independent killing (M21 and CHLA-20). This is consistent with increased granzyme B on both fresh and C/T EA NK cells compared to pre-expansion levels. In addition, both C/T and fresh EA NK cells demonstrated increased ADCC of melanoma and neuroblastoma cells lines, compared to that of pre-expanded cells. Although killing capacity was somewhat reduced compared to fresh EA NK cells34, C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody independent- and antibody dependent mechanisms. These data demonstrate that IL-2/mbIL-15/CD137L-mediated expansion and activation enhances NK cell cytotoxicity of solid tumor cells, even after cryopreservation and thawing of the cell product.

EA NK cells secrete high levels of tumor necrosis factor-alpha (TNF-α) and IFN-γ, cytokines that play crucial roles in antitumor responses and immunoregulation. We observed that fresh EA NK cells have an increased percentage of IFN-γ+ cells compared to pre-expanded PBMCs, as has been observed by others with fresh EA NK cells19 but C/T EA NK cells demonstrate a lower percentage of IFN-γ+ cells than both fresh EA NK cells and pre-expanded PBMCs. When quantified by ELISA, C/T EA NK cells produce less IFN-γ than fresh EA NK cells. We next determined if ruxolitinib, which is being studied to treat acute GVHD53,54, would reduce IFNγ production by inhibiting JAK1/JAK2. Inhibition of JAK1 using ruxolitinib has recently been shown to attenuate IL-12/IL-15/IL-18 EA fresh NK cell function55, but it is unclear what the effect would be on IL-2/mbIL-15/CD137L EA fresh NK cells, and whether similar effects would be observed on C/T EA NK cells. To our surprise, C/T EA NK cells were not affected by JAK1/JAK2 inhibition, even though ruxolitinib abrogated IFN-γ production completely in fresh EA NK cells. One explanation is that ruxolitinib could have inhibited the effects of IL-2 and IL-15 completely in the fresh EA NK cells, and the results could indicate that IFNγ production in cryopreserved EA NK cells is solely driven by CD137L. Alternatively, it is conceivable that the IFNγ measured after thawing NK cells could have been released from cells that died during cryopreservation, and thus would not be regulated by JAK1/JAK2 inhibition post-thaw. Lastly, it is possible that the absolute number of IFNγ+ NK cells after cryopreservation is so low (Figure 3C) that the assay is unable to detect any measureable inhibition. Understanding the signaling pathways that drive EA NK cell activation will be helpful for future studies so that clinicians may potentially selectively abrogate toxicities like cytokine release syndrome and GVHD, but preserve cytotoxicity.

Taken together, our results show that EA NK cell recovery following C/T is decreased due to reduced viability post-thaw but the percentage of NK cells in the EA NK cell product does not decrease with C/T. Both fresh and C/T EA NK cell products demonstrate increased expression of NKG2D and granzyme B, and demonstrate superior antibody-independent cytotoxicity and ADCC compared to pre-expansion PBMCs. However fresh EA NK cells show superior cytotoxicity to C/T EA NK cells in vitro, and C/T EA NK cells produce a lower percentage of IFNγ+ cells and total amount of IFNγ than fresh EA NK cells. Surprisingly, IFNγ production was not abrogated by JAK1/JAK2 inhibition, which has potential implications for managing EA NK-associated toxicities. Given the limited persistence of NK cells in vivo, and relative inability to form memory, usage of C/T EA NK cells may be an option to provide serial “boost” infusions to augment anti-tumor responses in patients. While viability and granzyme B production is similar between the products, the reductions in cytotoxicity and gamma interferon production in C/T EA NK cells suggest there may be reduced potency. The additional benefit of administering multiple doses of a tumor-directed therapy, such as a monoclonal antibody or immunocytokine, with multiple infusions of EA NK cells could allow for more potent effects against solid tumors. Further data from combination immunotherapy trials utilizing EA NK cells from fresh and C/T products will inform the feasibility, safety and hopefully efficacy of this strategy for patients with chemo-refractory or relapsed cancers.

Supplementary Material

1

Acknowledgments

This work was supported by grants from the National Heart, Lung, and Blood Institute/National Institutes of Health (NHLBI/NIH) T32 HL07899 (K.L.W.), American Association for Immunologists Careers in Immunology Fellowship (M.N.B.), St. Baldrick’s-Stand Up To Cancer Pediatric Dream Team Translational Research Grant SU2C-AACR-DT-27-17 (P.M.S., K.B.D., C.M.C.), NCI/NIH R35 CA197078 (P.M.S.), NHLBI/NIH PACT HHSN268201000010C, Solving Kids Cancer, Vince Lombardi Cancer Foundation (K.B.D.), NCI/NIH R01 CA215461, American Cancer Society Research Scholar Grant RSG-18-104-01-LIB, Hyundai Hope on Wheels and the MACC Fund (C.M.C). We would like to thank the UWCCC flow cytometry core facility, who is supported in part through NCI/NIH P30 CA014520. Stand Up To Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. The contents of this article do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. None of these funding sources had any input in the study design, analysis, manuscript preparation or decision to submit for publication.

Abbreviations

aAPCs

artificial antigen presenting cells

ADCC

antibody-dependent cellular cytotoxicity

AML

acute myelogenous leukemia

C/T

cryopreserved/thawed

EA

expanded and activated

FBS

fetal bovine serum

GFP

green fluorescent protein

GMP

good manufacturing practice

GVHD

graft-versus-host-disease

IFNγ

gamma interferon

IL

interleukin

JAK

Janus Kinase

MHC

major histocompatibility complex

NCR

natural cytotoxicity receptor

NK

natural killer

PBMCs

peripheral blood mononuclear cells

QR

qualification run

STR

short tandem repeats

TNFα

tumor necrosis factor alpha

WBF

Waisman Biomanufacturing Facility

Footnotes

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Disclosure of Interest

C.M.C reports honorarium from Nektar Therapeutics. This company had no input in the study design, analysis, manuscript preparation or decision to submit for publication. No other relevant conflicts of interest are reported.

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Supplementary Materials

1
NIHMS1595469-supplement-1.pdf (147KB, pdf)

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