Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)–grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.
Natural killer (NK) cells are innate lymphoid cells that are a promising cellular immunotherapy for the treatment of cancers; however, the optimal approach to enhancing NK cell effector function and recognition remains a key area of investigation (1–3). Brief activation of NK cells via combined IL-12, IL-15, and IL-18 receptors induces memory-like (ML) NK cell differentiation, and results in subsequent enhanced NK cell persistence in vivo, target recognition, and effector functions (4–6). These findings led to clinical testing of ML NK cells in a first-in-human NK cell study at Washington University, revealing safety and the ability of these cells to induce remissions in patients with hematologic malignancies (7, 8). Despite the fact that NK cell therapy has been translated to the clinic and mechanisms important for ML NK cell differentiation have been elucidated (8–12), several aspects of human ML NK cell biology still remain unclear, including the epigenetic mechanisms responsible for enhanced function and metabolic attributes (13, 14). Currently, advancing this ML NK cell–based therapy is limited by Good Manufacturing Practice (GMP)–grade recombinant cytokines, in particular recombinant human (rh)IL-18, to induce the ML NK cell differentiation program. Thus, a critical barrier in applying this approach in large-scale clinical trials, and combining with expansion approaches, is a lack of readily produced GMP-grade cytokine receptor–stimulating agents.
One approach to enhance cytokine signaling is to engineer fusion proteins providing new attributes, most commonly with the Fc portion of antibodies (Ab; ref. 15). For example, N-803 (previously ALT-803), represents a complex between an IL-15 variant and the IL-15Rα sushi domain fused to IgG Fc, yielding a soluble form of the physiologic trans-presentation cytokine–receptor complex with prolonged in vivo persistence (16). N-803 acts as an IL-15Rβγ super agonist in enhancing the activation, proliferation, cytokine secretion, and cytotoxicity of NK cells, resulting in potent killing of tumor and virus-infected cells (16–20). However, there remain challenges in large-scale GMP production of more complex immune-modulating agents that include proper protein folding and efficient purification for larger fusion proteins that include multiple protein domains.
Here, we addressed this challenge by developing a platform to express and purify soluble fusion proteins and protein complexes comprising cytokine, ligand, receptor, and single-chain antibody (scFv) domains. We found that the extracellular domain of human tissue factor (TF) could act as a fusion protein scaffold to allow high-level mammalian cell expression of difficult-to-produce proteins (i.e., IL-15). The TF fusions could also be readily purified by anti-TF Ab affinity chromatography. This protein expression strategy was scalable and could be used to generate large amounts of heteromeric fusion protein complexes (HFPC) under GMP conditions for cancer immunotherapy. The first HFPCs produced comprised IL-12, IL-15, and IL-18 (referred to as HCW9201) and IL-12, IL-15, IL-18, and a CD16 ligation domain (HCW9207). In this report, we evaluated HCW9201 and HCW9207 for their ability to activate NK cells through individual cytokine receptors, and in turn differentiate into ML NK cells. We also provide new data on the epigenetic changes in human ML NK cells and metabolic features of cytokine- and HCW9201-activated NK cells.
Materials and Methods
Reagents, mice, and cell lines
Recombinant human (rh) cytokines were obtained from the following: IL-12p70 (BioLegend), rhIL-18 (InvivoGen or R&D Systems), rhIL-15 (Miltenyi or NCI), and rhIL-2 (Proleukin, Clinigen). CHO-K1 cells (ATCC, CCL-61) have been validated for GMP production of recombinant proteins as outlined in (21). Daudi cells (ATCC, CCL-213; ref. 18), Raji cells (ATCC, CCL-86; ref. 18), K562 cells (ATCC, CCL-243; CBReGFP; ref. 7), and 32Dβ cells (ATCC, CRL 11346) transfected with pREP9 (Invitrogen) encoding human IL-15Rβ were cultured as described previously (22). All cell lines obtained from ATCC were cultured and expanded per ATCC recommendations, viably cryopreserved, and stored in liquid nitrogen. Once thawed, cultures were maintained for less than two months of continuous culture according to ATCC instructions. All cell lines were verified Mycoplasma free by the MycoAlert Plus Mycoplasma Detection Kit (Lonza; performed by the Washington University Tissue Culture Support Service) or ATCC Universal Mycoplasma Detection Kit (ATCC, 30-1012K). HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell lines were from InvivoGen and cultured as recommended and verified Mycoplasma free using the ATCC Universal Mycoplasma Detection Kit. Antibodies for flow cytometry and CyTOF are described in Supplementary Tables S1 and S2.
NSG mouse models
NOD-scid IL-2Rgammanull mice were obtained from Jackson Laboratory and maintained under specific pathogen–free conditions until 6–8 weeks of age. Trimethoprim and sulfamethoxazole (0.258 mg/mL, Hi Tech Pharmacal) were provided via drinking water on day of irradiation and maintained for 3 weeks of the study. For the tumor rejection model, nine normal human donors, (11–12 mice per group from three separate experiments) or NK cell persistence experiments (4 normal human donors performed in 3 separate experiments, 2 mice per group) were examined. All animal experiments were performed in accordance with our animal protocol approved by the Washington University Animal Studies Committee.
Production of HCW9201 and HCW9207
HCW9201 is a complex of two fusion proteins: one (IL-18/TF/IL-15) comprising IL-18 and IL-15 domains linked to the extracellular amino acid domain of human TF and another (IL-12/IL-15RαSu) comprising a single-chain form of IL-12 linked to the soluble domain of IL-15Rα (IL-15RαSu). HCW9207 is a similar two-protein IL-18/IL-15/IL-12 complex with the addition of an anti-human CD16 scFv linked to the IL-12/IL-15RαSu fusion protein (IL-12/IL-15RαSu/anti-CD16 scFv; ref. 23). The individual protein domains were fused without additional linker sequences (Fig. 1A). The corresponding coding DNA sequences were synthesized (Genewiz; ref. 24), cloned into pMSGV-1 modified expression vectors (25), and transfected into CHO.K1 cells. Coexpression of the two polypeptides in CHO cells allows for formation of the protein complex via high-affinity interactions between the IL-15 and IL-15RαSu domains and secretion of the complexes into the culture media. The HFPCs were then detected with product-specific ELISAs (24).
Production cell banks (HCW9201 and HCW9207) and a GMP-compliant master cell bank (HCW9201) were generated from stably transfected clonal cell lines following limited dilution cloning. Subsequent HFPC production was conducted using fed-batch methods with chemically defined media in scalable stir tank bioreactors (Sartorius). Briefly, 0.4 × 105 cells were seeded in a bioreactor at 80% of the final working volume of the bioreactor in EX-CELL Advanced CHO Fed-batch Medium (Sigma-Aldrich) supplemented with 4 mmol/L L-glutamine and 2 mmol/L GlutaMAX (Life Technologies). Culture pH was controlled at pH 7.0 ± 0.2 with a CO2/sodium carbonate cascade, dissolved oxygen was controlled at 40% using an O2/CO2 cascade, and temperature was maintained at 37°C. After 4 days in culture, the vessel temperature was dropped to 28°C, and 20% of the final culture volume of CHO CD EfficientFeed B AGT Nutrient Supplement (Life Technologies) was added to the bioreactor. The bioreactor was harvested on day 18 or when the culture viability dropped below 60%. The harvest was clarified using D0SP/X0SP depth filters (Millipore) at a 2:1 ratio (0.0054/0.0028 m2/L of harvest) followed by aseptic filtration using an Opticap XL 150 Capsule filter (Millipore; 0.0044 m2/L of harvest). HCW9201 and HCW9207 were purified from clarified bioreactor harvests using immunoaffinity chromatography with anti-TF Ab-conjugated Sepharose resin. Briefly, monoclonal anti-TF (designated as HCW9101) was immobilized on CNBr-activated Sepharose 4 Fast Flow (Cytiva) resin per the manufacturer's instructions. The clarified cell culture harvest was loaded onto the HCW9101 immunoaffinity column equilibrated with 4 column volumes of PBS, followed by wash with 5 column volumes of PBS and 5 column volumes of 0.1 M sodium citrate (pH 5.0). HCW9201 or HCW9207 bound to the immunoaffinity column was eluted with 0.1 M acetic acid (pH 2.9–3.0). The protein peak was collected and then neutralized to pH 7.5 with 1 M Tris base. The pH-adjusted sample was then buffer-exchanged into PBS using a 50-mL centrifugal device with a molecular weight cutoff of 30 kDa (Millipore). A GMP-compliant manufacturing process (scaled from 2 to 200 L) was developed for HCW9201 consisting of immunoaffinity chromatography, low pH viral inactivation/depth filtration, anion exchange chromatography, nanofiltration, and ultrafiltration/diafiltration steps using commercially scalable methods (Fig. 1E). The purified product was characterized and released using the quality tests shown in Supplementary Table S3.
High-performance liquid chromatography–size exclusion chromatograph
HCW9201 or HCW9207 sample was diluted to 0.5 mg/mL in PBS mobile phase (0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.0) and applied onto TSKgel G3000 SWxl, 7*8×300 mm analytical SEC column (Tosoh Bioscience) with a mobile phase flow rate of 0.8 mL/minute. Each injection was run for 20 minutes and detected by ultraviolet light at 280 nm. Molecular weight was calculated using Gel Filtration Standard (Bio-Rad) run under the same conditions.
Deglycosylation of HCW9201 or HCW9207 was performed with Protein Deglycosylation Mix II (New England Biolabs) following the manufacturer's instructions. Briefly, 100 μg of HCW9201 or HCW9207 was heated at 75°C for 10 minutes in 1× Mix Buffer 2, then mixed with 5 μL of Protein Deglycosylation Mix II enzyme in a total volume of 50 μL. The deglycosylation reaction was first incubated at room temperature for 30 minutes, then transferred to 37°C for 16 hours.
Polyacrylamide gel electrophoresis
HCW9201 or HCW9207 protein (deglycosylated or nondeglycosylated) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4% to 12% NuPage Bis-Tris gel (Thermo Fisher Scientific) under reducing conditions. After electrophoresis, the gel was stained with InstantBlue (Abcam) for 35 minutes, followed by destaining overnight in purified water.
N-terminal amino acid sequence analysis
HCW9201 protein was deglycosylated and resolved by reduced SDS-PAGE gel as described above. Protein bands on the SDS gel were then electroblotted onto a PVDF membrane (Immobilon PSQ, Millipore). The PVDF membrane was stained with InstantBlue (Abcam) for 35 minutes, followed by destaining overnight in purified water. Each protein band was cut from PVDF membrane and sent to Molecular Structure Facility, University of California, Davis (CA 95616) for N-terminal sequence analysis using Edman Sequencing method.
Biological activity on HEK cells (IL-15, IL-18, and IL-12)
IL-15 activity of the HFPCs was determined based on 32Dβ cell proliferation in a WST-1 assay per manufacturer's instructions (Fisher Scientific). Briefly, 32Dβ cells (2×104 cells/well) were incubated with increasing concentrations of HFPCs for 48 hours at 37°C. Cell proliferation reagent WST-1 (Roche, 11644807001) was added during the last 4 hours of cell growth according to the manufacturer's procedures. Conversion of WST-1 to the colored formazan dye by metabolically active cells was determined through absorbance measurements at 440 nm. IL-12 and IL-18 activities were determined using HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell lines as recommended by the manufacturer (InvivoGen). Briefly, HEK-Blue IL-12 or HEK-Blue IL-18 reporter cells (5×104 cells/well) were incubated with increasing concentrations of HFPCs for 24 hours at 37°C. Next day, 20 μL of supernatant from the induced HEK-Blue IL-12 or HEK-Blue IL-18 reporter cells was added to 180 μL of QUANTI-blue (InvivoGen, rep-qb1), a subtrade for SEAP (secreted embryonic alkaline phosphatase), followed by incubation for 3 hours at 37°C. The absorbance was measured at 620 nm to determine IL-12 or IL-18 activity based on SEAP activity to change QUANTI-blue to a purple-blue color. The EC50 was determined with the dose–response curve generated from the experimental data by nonlinear regression variable slope curve-fitting with Prism 8 software (GraphPad).
NK cell isolation and culture
Fifty-six healthy donor peripheral blood mononuclear cells (PBMC) were collected from anonymous, healthy platelet donors through the Mississippi Valley Regional Blood Center or One Blood, Orlando, FL. NK cells (≥95% CD56+CD3−) were isolated using RosetteSep (STEMCELL Technologies) as described (9). ML and control NK cells were generated as described previously (7), except the media used for activation, ML differentiation, phosphorylation, RNA sequencing (RNA-seq), CyTOF, and NSG experiments used NK media (Miltenyi) supplemented with 5% (v/v) heat-inactivated human AB pooled sera (MilliporeSigma), Complete NK Medium (CNKM). RPMI-1640 supplemented with 2 mmol/L L-glutamine, antibiotics (penicillin, 100 U/mL; streptomycin, 100 μg/mL; Thermo Fisher), and 10% (v/v) fetal bovine serum (Cytiva) was used for the methylation and metabolic studies. For activation, NK cells were incubated with individual cytokines: IL-12p70 (10 ng/mL), IL-15 (50 ng/mL), and IL-18 (50 ng/mL; 12/15/18; ref. 7), or HFPCs at 50 to 100 nmol/L at two million per mL in a low adherence cell culture tray (Costar). After 12 to 16 hours of activation, NK cells were washed three times in 0.5% (w/v) human serum albumin in HBSS to remove residual cytokines or HFPCs. Cells were resuspended to two million per mL and plated in the presence of rhIL-15 (1 ng/mL), with every other day media exchanges supplemented with fresh rhIL-15 (1 ng/mL) for 6 to 7 days in assay-specific media.
For labeling of NK cells, cells were suspended in FACS buffer: 0.5% (w/v) BSA, 2 mmol/L EDTA in PBS with no Ca2+ and Mg2+. All washes consisted of resuspension in the FACS buffer and pelleted at 660 × g for 4 minutes at ambient temperature. Cells were incubated with antibody cocktails to surface molecules as described in the specific assay for 30 minutes at ambient temperature in the dark and washed. For detection of intracellular molecules, cells were processed using Fix/Perm System (BD Biosciences) per the manufacturer recommendation, blocked with 7% (v/v) heat-inactivated goat serum in Perm/Wash buffer, and incubated with cytokine antibodies for 30 minutes at 4°C in the dark. Cells were washed twice and fixed until collection on a Navios, 3 laser 10 parameter flow cytometer (Beckman Coulter).
NK cell functional and cytotoxicity assays
For human NK cell activation status, a portion of activated or control NK cells were harvested and incubated with anti-CD107a and GolgiPlug/GolgiStop per manufacturer's recommendation (BD Biosciences) for 1 hour at 37°C. The cells were washed, placed in FACS buffer, and labeled with antibodies for CD45/CD3/CD56/CD16/CD25 for 30 minutes at ambient temperature. Cells were washed and then fixed/permeabilized per manufacturer's recommendation (BD Biosciences) at 4°C. Permeabilized cells were incubated in 7% (v/v) heat-inactivated goat serum (Sigma) to block nonspecific sites and then incubated with antibodies to IFNγ and TNF for 30 minutes at 4°C in the dark. Finally, cells were washed and immediately collected on a Navios3 laser 10-parameter flow cytometer (Beckman Coulter). For detection of intracellular cytokines produced in response to tumor targets or IL-12/IL-15, cells were incubated at a 5:1 effector:target ratio, and functional assays were performed as described (5), except Zombie Green (BioLegend) was used to detect viable cells in this assay. Data were acquired on Navios Flow Cytometer and analyzed on FlowJo v.10.6.
Cytotoxicity assays (4-hour 51Cr-release) were performed as described (26) with K562, Daudi, and Raji targets, or using K562-Luc2 (ATCC), and the One-Glo Ex Kit per manufacturer's recommendations (Promega) in 96-well white plates (Corning). Luminescence was detected on a Genios luminometer (Tecan). For antibody-dependent cellular cytotoxicity (ADCC), target cells were incubated with Rituxan (monoclonal anti-CD20 at 10 μg/mL; Roche) for 30 minutes at room temperature and washed to remove excess antibody prior to use. For 51Cr release assays, 4-hour lytic supernatants were collected per the manufacturer recommendation and resultant counts per minute (cpm) was assessed on a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter. Percent-specific lysis was calculated: [(cpmtest − cpmspontaneous)/(cpmmax − cpmspontaneous)]*100.
Assessments of intracellular signaling
Phospho-flow cytometry methods were previously described (27). Briefly, viably cryopreserved NK cells were thawed in complete NK media and allowed to rest at 37°C for 30 minutes. HFPC at 50 and 100 nmol/L or 12/15/18 (10/50/50 ng/mL, respectively) individual cytokines were used to activate NK cells at appropriate time intervals: 2 hours for STAT4, 1 hour for AKT and ERK, and 15 minutes for NFΚB-p65, STAT5, and p38-MAPK. Plate-bound anti-FcγRIIIa experiments were performed as previously described (10).
Proliferation of flow-sorted CD56bright and CD56dim NK cells was performed as described (5), with modifications. Freshly isolated human NK cells were purified by RosetteSep (STEMCELL Technologies), and labeled with CFSE according to the manufacturer's recommendations (Invitrogen). After washing, cells were stained with anti-CD45/CD3/CD56/CD16 for 30 minutes at ambient temperature in the dark, washed again, passed through a 70-μm nylon mesh, and then immediately were flow-sorted (Sony SY3200 Synergy, Sony Biotechnology) using a 70-μm tip into CD45+CD3−CD56+CD16-bright (br) or -dim populations to greater than 75% CD56brCD16dim or greater than 90% CD56dimCD16br purity. Five million sorted cells were seeded into one milliliter of NK media and activated with 12/15/18 (10/50/50 ng/mL) or 100 nmol/L HFPC or low-dose IL-15 (LD15) control, incubated for 16 hours, washed, and plated for ML differentiation in the presence of IL-15 (1 ng/mL) with 1:1 media exchanges every other day. On day 5 after activation, cells were harvested, washed, and labeled with anti-CD45/CD3/CD56/CD16 for 30 minutes, washed, and immediately acquired on Navios Flow Cytometer. Data were analyzed using FlowJo v.10.3 software with proliferation module to determine the percent divided cells in the CD56-bright and CD56-dim populations.
RNA-seq and analysis
NK cells from three normal human donors (1×106) from each step of activation to ML differentiation were centrifuged, lysed in TRIzol (Invitrogen), and immediately flash frozen and stored at −80°C. After all samples were collected, total RNA was isolated (Zymo). Samples passing RIN analysis (Agilent) were moved to high-quality cDNA preparation with a Clontech SMARTer ultra-low input kit according to the manufacturer's protocol. The samples were then indexed, pooled, and sequenced on an Illumina NovaSeq. Basecalls and demultiplexing were performed with Illumina's RTA version 1.9 and bcl2fastq2 software, with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.5.1a (28). Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.6-p5 (29). Isoform expression of known Ensembl transcripts was estimated with Salmon version 0.8.2 (30). Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 2.6.2 (31).
All gene counts were then imported into the R/Bioconductor package EdgeR (32), and TMM normalization size factors were calculated to adjust samples for differences in library size. Ribosomal genes and genes not expressed in at least three samples greater than one count-per-million were excluded from further analysis. The TMM size factors and the matrix of counts were then imported into the R/Bioconductor package Limma (33). Weighted likelihoods based on the observed mean-variance relationship of every gene and sample were then calculated for all samples with the voomWithQualityWeights (34) function using a model matrix that included every condition as well as patient-specific blocking factors. The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals. Differential expression analysis was then performed to analyze for differences between conditions, and the results were filtered for only those genes with Benjamini–Hochberg false-discovery rate–adjusted P values less than or equal to 0.05. For each contrast extracted with Limma, global perturbations in known Gene Ontology (GO) terms, MSigDb, and KEGG pathways were detected using the R/Bioconductor package GAGE (35) to test for changes in expression of the reported log 2 fold changes reported by Limma in each term versus the background log 2 fold changes of all genes found outside the respective term. The R/Bioconductor package heatmap3 (36) was used to display heat maps across groups of samples for each GO or MSigDb term with a Benjamini–Hochberg false-discovery rate–adjusted P value less than or equal to 0.05. Perturbed KEGG pathways where the observed log 2 fold changes of genes within the term were significantly perturbed in a single-direction versus background or in any direction compared with other genes within a given term with P values less than or equal to 0.05 were rendered as annotated KEGG graphs with the R/Bioconductor package Pathview (37). Data are available on Gene Expression Omnibus (GEO GSE172100).
For qRT-PCR of CISH, total RNA from six normal human donor NK cells, low dose (LD) 15 control, 12/15/18, and 100 nmol/L HCW9201 activations was isolated using DirectZol Micro RNA Prep (Zymo). 100 ng cDNA was prepared using Life Technologies High-Capacity cDNA Reverse Transcription Kit with random primers. CISH and ACTB control were amplified using the ABI primer sets Hs00367082_g1 and Hs01060665_g1, respectively, in triplicate, using the TaqMan Gene-expression system (Applied Biosystems) on an ABI7300 RT-PCR system (20 μL reaction). Fold change was calculated 2ΔΔCT.
Human NK cells (2 ×105) were seeded in Cell-Tak-coated Seahorse Bioanalyzer XFe96 (Agilent) culture plates in Seahorse XF RPMI medium, pH 7.4 supplemented with 2 mmol/L L-glutamine for the glycolysis stress test. For the mitochondrial stress test, cells were seeded in Seahorse XF RPMI medium, pH 7.4 supplemented with 10 mmol/L glucose and 2 mmol/L L-glutamine. Assays were performed following the manufacturer's instructions. The data were analyzed using Wave software (Agilent). Rapamycin (100 ng/mL) was added for inhibition studies, as indicated.
Pyrosequencing analysis of DNA methylation
The level of DNA methylation at the IFNG distal promoter conserved noncoding sequence 1 (CNS-1) enhancer region containing four characterized CpG sites [located at positions −4360, −4325, −4293, and −4278 relative to the transcription start site (TSS); ref. 38] was determined using pyrosequencing reactions performed at Johns Hopkins University Genetic Resources Core Facility (JHU GRCF). This DNA sequencing method relies on light detection based on a chain reaction when pyrophosphate is released during sequential addition of nucleotides during the synthesis of a complementary strand of DNA as described (39). Genomic DNA extraction: Primary human NK cells were purified from two healthy donor PBMCs as described (9). The purified NK cells were phenotyped, and treated with either individual cytokines (12/15/18) as described (7) or HFPCs (50–100 nmol/L). After 12 to 16 hours of activation, NK cells were washed three times and plated in rhIL-15 (1 ng/mL) with every other day media exchanges. Following treatment, NK cells (0.2–1.0 × 106) were collected and washed with 0.5 mL DPBS (pH 7.4). Wet cell pellets were then either stored at −20°C until processed directly for genomic DNA extraction using the QIAamp DNA Micro Kit (QIAGEN). The DNA concentration was determined using the μDrop with the Multiskan Sky microplate reader (Thermo Scientific). Bisulfite conversion of genomic DNA was performed using the EZ DNA Methylation-Direct Kit (ZYMO). Five hundred nanograms of genomic DNA determined using the μDrop Microplate Reader (Thermo) was used per manufacturer's recommendations. For DNA methylation controls, the human nonmethylated (UNMET) and methylated (MET) genomic DNA (ZYMO) were used. PCR amplification of dsPCR-CNS1 (247 bp) products from bisulfite-converted nDNA using the PyroMark PCR Kit (QIAGEN). For amplification of the IFNG CNS-1 region (4 kb upstream IFNG TSS) containing the four informative CpG sites, the PCR primers IFNG-CNS1F and IFNG-CNS1R-bio were used. Forty nanograms of bisulfite converted DNA was subjected to PCR amplification CFX96 thermal cycler (Bio-Rad; 15 min at 95°C, followed by 50 cycles of 35 sec at 95°C, 35 sec at 55°C, and 40 sec at 72°C, with a final extension of 5 min at 72°C). PCR products dsPCR-CNS1 (247 bp) were analyzed in a 1.2% TAE agarose gel and stained with SYBR Safe DNA Gel Stain per manufacturer's recommendation using a 1 kb Plus DNA Ladder (Thermo) as molecular weight marker. Pyrosequencing analysis of DNA methylation at the CpG sites within the IFNG CNS-1 region: dsPCR-CNS1 CpG PCR products were subjected to pyrosequencing analysis of DNA methylation using the DNA sequence primers C4399-CNS1F CNS1F (for analysis of CpG sites −4399, −4377, −4360, and −4325; DNA sequence: 5′-GGG GAT TTA GAA AAA T-3′) and C4293-CNS1F (for analysis of CpG sites −4293, −4278, and −4227; DNA sequence: 5′-TGT ATG ATG TTA GGA GTT T-3′). The pyrosequencing reactions were performed at JHU GRCF (https://grcf.jhmi.edu/dna-services/methlylation/analysis-via-pyrosequencing/). The pyrosequencing runs and profiles (determination of % DNA methylation) for each IFNG CNS-1 CpG sites (from −4399 to −4278) were provided by JHU GRCF. For analysis, the methylation percentages of the four IFNG CNS-1 informative CpG sites were averaged for each treatment. Unpaired Student t tests with two-tailed P value were used for statistical analysis of DNA methylation data using the GraphPad Software Prism 8 (Version 8.3.0).
Three normal human donor NK cells isolated by RosetteSep and activated as described earlier were used for both RNA-seq and CyTOF. Antibodies used for this study were either obtained by Fluidigm or conjugated in-house (Supplementary Table S2). Conjugations, titrations, surface and intracellular staining, and data acquisition and analysis were performed as described (8).
Ribbon model of HCW9201 heterodimeric cytokine fusion was created in PyMOL2. The crystal structures of IL-18 (PBD: 3WO2), TF (PBD: 1BOY), IL-15/IL-15RA (PBD: 2Z3R), and IL-12p70 (PBD: 1F45) were imported into the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. Relevant amino acids were added or deleted to accurately reflect the fusion protein sequence. Molecular domains were then aligned to minimize steric hindrance.
Bioluminescence imaging, survival, and persistence
Analysis was performed as described (7). Mice were irradiated at 125 cGy one day prior to leukemia or NK cell injection. For leukemia elimination model, one million K562 CBReGFP cells were introduced via tail vein, and tumor burden was monitored using an amiHT optical imaging system (1–60 sec exposure, bin 8, FOV 12 cm, open filter) by intraperitoneal injection with D-luciferin (150 mg/kg in PBS; Gold Biotechnology) after isoflurane anesthesia (2% vaporized in O2). Total photon flux (photons/sec) was measured from fixed regions of interest over the entire mouse using Aura Imaging Software v3.2. For leukemia challenge experiments, 3 to 5 million NK cells were administered retro-orbitally on day 3 after leukemia injection and supported with IL-2 (50,000 IU; Proleukin, Clingen) three times per week. Leukemia burden was assessed by weekly imaging and overall survival monitored until day 400.
For NK cell persistence in vivo, irradiated mice were injected with 2 to 5 million activated or control NK cells, through the tail vein and supported with IL-2 (50,000 IU) 3 times per week for one week. Blood and spleen were harvested one week post NK cell injection. Blood was obtained by cardiac puncture, and spleen single-cell suspensions were prepared by mechanical disruption and passage through 70-μm nylon mesh. RBCs were lysed, and splenocytes were stained with Zombie NIR (BioLegend) per manufacturer's recommendations, washed, and then stained with anti-murine CD45/anti-human CD45/CD3/CD56/hNKG2A staining for 30 minutes at 4°C. Cells were then washed and immediately collected on a Beckman Coulter Gallios, 3-laser, 10-parameter Flow Cytometer. Data were analyzed using FlowJo v10.7 software.
Statistical analyses were performed using GraphPad Software Prism 9. All ordinal data were tested for normal distribution (Shapiro–Wilk) and appropriate parametric/nonparametric tests used, as indicated in the figure legends.
HFPC production using a TF platform
To overcome the challenge with large-scale production of complex multimeric fusion proteins, a novel protein expression platform was developed using a truncated human TF domain as a fusion scaffold for expressing and purifying soluble fusion proteins and protein complexes comprising multiple functional domains. A pairing domain feature (e.g., IL-15 and soluble IL-15 receptor α) was further incorporated into the TF-based fusion concept to generate heteromeric, multifunctional protein complexes. To address the unmet need for GMP-grade reagents (7, 8) that stimulate NK cells (5), we designed and produced HCW9201 [IL-12p70, IL-18, and IL-15 trans-presented in the IL-15Rα sushi domain (IL-15RαSu)] and HCW9207 (IL-12p70, IL-18, IL-15/IL-15RαSu and an anti-CD16 scFv; Fig. 1A; Supplementary Fig. S1). After expression of the constructs in CHO-K1 cells and affinity purification of the soluble HFPCs, each polypeptide in the HFPC proteins was resolved by polyacrylamide gel electrophoresis (Fig. 1B and C). Bands for expected IL-12/IL-15RαSu or IL-12/IL-15RαSu/anti-CD16scFv, and IL-18/TF/IL-15 fusion proteins were clearly seen in HCW9201 and HCW9207 products, respectively. A truncated form of IL-18/TF/IL-15 was also detected in both HFPC preparations (Fig. 1B and C). N-terminal amino acid sequence analysis of this protein band indicated that IL-18 was cleaved between its C-terminal region and the N-terminal of TF. The partial degradation of the IL-18/TF/IL-15 fusion protein appeared to occur during the cell culture process. Other bands present in the purified protein lanes correspond to deglycosylation enzymes (Fig. 1C, lane B). High-performance liquid chromatography–size exclusion chromatograph (HPLC-SEC) analysis was conducted to evaluate product purity (Fig. 1D). The full-length and truncated forms of IL-18/TF/IL-15 copurified and were not resolvable by HPLC-SEC due to the small difference in their molecular masses (IL-18 is approximately 18 kDa). Although the presence of the cleavage product was noted in all batches of the fusion protein (including the GMP large-scale batch), the biological activity of IL-18 was ensured by quantitation on the HEK-IL-18–dependent reporter cell line (Supplementary Table S3). HPLC-SEC results indicated that both HCW9201 and HCW9207 existed as dimers, with a small percent of high molecular protein aggregates observed in HCW9201 and relatively higher percent of protein aggregates in HCW9207. Based on our experience, and as demonstrated for HCW9201, protein aggregates could be significantly reduced by process development. The advantage of this protein expression platform is the scalability and consistency of producing biologically active, multigram quantities of HFPC protein (Fig. 1E). Further characterization of identity, purity, and activity was completed for GMP-grade HCW9201 (detailed in Supplementary Table S3), and HCW9201 was qualified for ex vivo use in generating ML NK cells for human clinical trials. Thus, this unique protein manufacturing approach consistently produced the engineered HFPCs.
HCW9201 and HCW9207 induce specific signals downstream of individual cytokine receptors
We first performed experiments to confirm HCW9201 and HCW9207 induced signaling downstream of each individual cytokine receptor. Both HFPCs stimulated individual cytokine-dependent reporter cell lines with EC50 values defined for IL-12, IL-15, and IL-18 (Fig. 2A). There were differences in the activities using these cell-based assays, primarily with HCW9207 exhibiting less potent IL-15 biological activity compared with HCW9201 and showing similar IL-12 and IL-18 activities (within assay variation).
HCW9201 and HCW9207 signaling in primary human NK cells
NK cell responses via cytokine receptors depend on the signaling cascade and phosphorylation of multiple molecules (40, 41). To determine if signaling pathways normally activated in NK cells during cytokine stimulation were being triggered appropriately by HFPCs, we compared the median fluorescence intensity (MFI) fold change from baseline (resting) to activation with the combination of IL-12p70, IL-15, and IL-18 (12/15/18) or HFPCs in CD56bright and CD56dim subsets (ref. 42; representative flow histograms in Fig. 2B; Supplementary Fig. S2A; summary data in Fig. 2C; Supplementary Fig. S2B). IL-12R signaling through phosphorylated (p)STAT4 was equivalent in CD56bright NK for all stimulators, with minor differences in CD56dim NK cells suggesting that the EC50 value differences observed in Fig. 2A were not large enough to affect maximal signaling. For IL-15R signaling, pSTAT5, pAKT, and pERK were examined (27). The results demonstrated that both 12/15/18 and HCW9201 induced robust and equivalent STAT5 phosphorylation, whereas HCW9207 had reduced pSTAT5 in CD56dim NK cells. For IL-18R signaling, 12/15/18 and HCW9201 induced similar NFkB-p65 and p38 MAPK phosphorylation over baseline, but HCW9207 had substantially reduced activity. Cross-linking of CD16 concurrent with HCW9201 stimulation did not alter these signals, suggesting that the addition of ITAM-based signals did not result in the signaling change (Supplementary Fig. S2C), consistent with less effective IL-18R signaling by HCW9207. Collectively, these data confirmed that HCW9201 induced cytokine receptor signaling via IL-12R, IL-15R, and IL-18R, similar to 12/15/18, with reduced IL-18R and IL-15R signals observed with HCW9207 in primary NK cells.
We next compared the HFPCs to established ability of 12/15/18 to activate and induce proliferation of human NK cells (5). NK cells were labeled with CFSE and flow-sorted into CD56bright and CD56dim subsets (Fig. 3A) and incubated for 12 to 16 hours with LD (1 ng/mL) IL-15 (control), 12/15/18, HCW9201, or HCW9207, washed, and replated in IL-15 (1 ng/mL) to support survival for ML differentiation as modeled in Supplementary Fig. S3A. After 5 days, proliferation was quantified using CFSE dilution (Fig. 3B and C). CD56bright and CD56dim NK cells proliferated equivalently to 12/15/18 and HCW9201 or HCW9207 and significantly greater than IL-15 controls. Activation of human NK cells for 12 to 16 hours with IL-12, IL-15, and IL-18 synergistically increases expression of IFNγ and CD25, prototypic markers of combined cytokine activation (5, 43). Dose–response curves of HFPCs identified maximal activation of CD25 and IFNγ at ≥100 nmol/L (Supplementary Fig. S3B), and 50 and 100 nmol/L concentrations were then further tested (Fig. 3D and E). The expression of CD25 and IFNγ was equivalently increased over controls following activation with 12/15/18 and HCW9201. However, HCW9207 exhibited reduced CD25 and IFNγ expression, consistent with reduced IL-18 signaling (Fig. 2). In contrast, surface CD107a was induced with HCW9207 over HCW9201 or 12/15/18, which is consistent with CD16 signaling via ITAMs triggering degranulation (Supplementary Fig. S3C). Short-term cytokine activation also enhanced NK cell cytotoxicity, and HCW9201 and 12/15/18 had similar improvements in leukemia cell killing over IL-15 only controls (Fig. 3F).
Similar molecular programs are activated by 12/15/18 and HCW9201 and HCW9207
To define the molecular activation programs exhibited by 12/15/18 and HFPCs, bulk RNA-seq was performed after 12 to 16 hours of priming and after subsequent in vitro ML NK cell differentiation (Fig. 4A). Over three thousand genes were significantly differentially expressed (adjusted P < 0.05) when comparing baseline NK cells, LD15, and 12 to 16 hours of activation with 12/15/18, HCW9201, or HCW9207 (Supplementary Fig. S4A; Supplementary Table S4). The top 50 differentially expressed genes (P adj < 0.05) are shown in the heat map (Fig. 4B), with IFNG showing the highest induction over control. Linear regression analysis of the log fold change over control revealed greater similarity between 12/15/18 and HCW9201, compared with 12/15/18 and HCW9207 (Fig. 4C). There were some differentially expressed genes in 12/15/18 and HCW9201 that had reduced expression following HCW9207, including CSF2, EBI3, CD274, TNFSF4, CDKN1A, C15orf48, LGALS3, SOD2, and MFSD2A. When comparing the RNA-seq profiles of 12/15/18, HCW9201, and HCW9207-primed cells that were differentiated in vitro in IL-15, bulk RNA-seq revealed few transcript differences, consistent with a return to a more resting state (Supplementary Table S5) and likely partially confounded by an IL-15–induced molecular program (Fig. 4D; Supplementary Fig. S4B and S4C). Six genes (CCR5, CXCR6, IFNG, CCL5, FAM107B, and TCEAL9) were shared by all three activation methods (Supplementary Fig. S4C). Significant reduction in CISH transcript was evident in D6 ML NK cells induced by both HFPCs and validated by qRT-PCR (Supplementary Fig. S4D). Based on the activity assay comparisons (Figs. 2–4), HCW9201 was determined to be the lead HFPC to replace 12/15/18 and was the focus of the remainder of the study.
CNS-1 is equivalently demethylated in 12/15/18- and HCW9201-activated NK cells
The CNS-1 region of the IFNγ locus has been shown to be important in NK cell differentiation (44) and is regulated epigenetically by DNA methylation after activation with 12/15/18 in mice (11), leading to ML NK cell differentiation. To evaluate this mechanism in human NK cells and to compare both 12/15/18 and HCW9201 treatments, the methylation status of CNS-1 CpG sites (−4360, −4325, −4293, and −4278) was interrogated after overnight activation and 14 days of differentiation (Fig. 4E). These four CpG sites showed a significant reduction in DNA methylation in NK cells activated with 12/15/18 and HCW9201 (Fig. 4F), with the greatest reduction in DNA methylation observed at the CpG sites located at −4360 and −4325 (Supplementary Fig. S5). These data demonstrate that the IFNγ CNS-1 locus is demethylated in human ML NK cells after induction with 12/15/18 or HCW9201 agents.
12/15/18 and HCW9201 enhance cellular metabolism
Metabolism is an important factor in NK cell functional responses (45), and IL-2, IL-15, and IL-12 have been shown to affect glycolysis and oxidative phosphorylation in human NK cells (46), yet it is unknown how combined 12/15/18 activation affects measures of metabolism. To define the impact of 12/15/18 and HCW9201 on the metabolism of human NK cells, extracellular flux assays were performed. Both 12/15/18 and HCW9201 increased the rate of glycolysis in human NK cells to a significantly greater magnitude compared with resting or IL-15 controls (Fig. 5A and B). This included significantly greater glycolytic capacity. 12/15/18 and HCW9201 activation also increased the mitochondrial oxygen consumption rate (OCR) to similar extents (Fig. 5C and D) and showed significantly increased OCRs over IL-15 controls. The enhanced glycolysis, glycolytic reserve, glycolytic capacity, and basal respiration were inhibited by rapamycin (Fig. 5B and D; Supplementary Fig. S6), suggesting a role for mTOR signaling in these metabolic changes. Collectively, these data showed that signals via the IL-12, IL-15, and IL-18 receptors similarly enhanced key metabolic pathways of glycolysis and oxidative phosphorylation for energy production over resting and IL-15 control–stimulated NK cells. The metabolic fitness may be linked to enhanced proliferation and cytotoxic function of NK cells.
Mass cytometry reveals 12/15/18- or HCW9201-primed and ML NK cells are identical
Multidimensional analysis of 12/15/18-primed and resulting ML NK shows phenotypic differences from conventional NK cells, both in vitro and in vivo (7, 8). HCW9201- and 12/15/18-primed and ML NK cells were compared using a 37-parameter CyTOF panel (Fig. 6). Representative viSNE maps of 12/15/18- and HCW9201-activated human NK cells (Fig. 6A and B) revealed similar mapping patterns after the short-term activation (12–16 hours), as well as after ML NK cell differentiation (day 6). Unsupervised FlowSOM analysis defined six metaclusters (Fig. 6C), which did not differ in abundance between 12/15/18 and HCW9201 conditions (Fig. 6D). The median expression of all 37 markers was compared between baseline (resting) NK cells and HCW9201 and showed a lack of correlation after linear regression (Fig. 6E). In contrast, comparisons of HCW9201 and 12/15/18 after 12 to 16 hours or after subsequent ML NK cell differentiation (day 6) demonstrated a significant concordance. Collectively, these data demonstrate that incubation with 12/15/18 or HCW9201 results in a similar activation and ML NK cell multidimensional phenotype.
HCW9201- and 12/15/18-induced ML NK cells have potent function on restimulation in vitro and in vivo
Increased IFNγ expression and cytotoxic responses after challenge with the K562 leukemia are hallmarks of 12/15/18-induced ML NK cells (5). 12/15/18- and HCW9201-primed ML NK cells differentiated for 6 days were challenged with K562 cells, and IFNγ was measured after 5 hours. Both 12/15/18- and HCW9201-induced ML NK cells produced significantly more IFNγ than LD15 controls, with HCW9201-induced ML NK cells producing the greatest levels (Fig. 7A). ML NK cells differentiated after priming by 12/15/18 or HCW9201 had equivalently improved cytotoxicity against K562 targets compared with IL-15 controls (Fig. 7B). 12/15/18- and HCW9201-primed ML NK cells demonstrated similar improvements in killing of the resting NK-resistant Daudi and Raji tumor targets with and without anti-CD20 to simulate ADCC (Supplementary Fig. S7). To compare ML NK cells induced by HCW9201 and 12/15/18 in vivo, cells were injected into NSG mice engrafted with K562 leukemia and compared with IL-15 NK cell and no NK cell controls (Fig. 7C). Significantly enhanced survival was seen for mice treated with 12/15/18 (P = 0.012)- and HCW9201 (P = 0.037)-induced ML NK cells compared with the no NK cell control group (Fig. 7D). 12/15/18-induced ML NK cells resulted in improved survival compared with the IL-15–only control NK cell group (P = 0.048), with HCW9201-induced ML NK cell treatment trending toward survival improvement (P = 0.057). Using bioluminescence imaging (BLI), we found that leukemia was significantly and similarly reduced by both 12/15/18 (P = 0.02)- and HCW9201 (P = 0.0086)-primed NK cells compared with control NK cells (Fig. 7E and F; Supplementary Fig. S8A). To address in vivo persistence, we compared the in vivo NSG persistence of control NK cells to 12/15/18- and HCW9201-induced ML NK cells 7 days after transfer. There was a significant increase in human NK cells in the 12/15/18 and HCW9201 groups compared with control group (Fig. 7G and H), and increased persistence of NKG2A+ NK cells in the blood and spleen at D7 after injection (Supplementary Fig. S8B). These data overall indicate that HCW9201 recapitulates the ML NK cell induction with similar function to 12/15/18-induced ML NK cells.
In this study, we describe a new approach to construct multifunctional heteromeric fusion protein complexes using a TF scaffold platform. The extracellular domain of human TF was selected because it has a rigid elongated structure comprised mainly β-sheets with its N- and C-termini located at distal ends (>70 Å apart) of the polypeptide (47), permitting genetic fusions of other protein domains without anticipated steric interference. The extracellular domain of human TF does not interact with the cell membrane phospholipid bilayer and, as a result, does not exhibit procoagulant activity (48). Human TF is expressed at high levels by most cell types and is not expected to be immunogenic in humans. Consistent with these properties, we found that genetic fusion to the TF domain promoted increased production of difficult-to-express proteins, such as IL-15. The TF fusion proteins could be readily purified by affinity chromatography using an anti-TF and low pH elution conditions, similar to those used in protein A–based affinity purification of Abs. To generate multichain protein complexes, we also incorporated genetic fusions to the human IL-15 and IL-15RαSu domains. When coexpressed in CHO cells, the fusion proteins formed a soluble stable heterodimeric complex through high-affinity interactions between IL-15 and IL-15RαSu domains. This approach offers an alternative to immunoglobulin (Fc) and other engineered protein scaffolds, which typically require introduction of multiple mutations or other non-human sequences or complicated in vitro assemble/purification methods to generate bi- or multispecific complexes (49, 50). Using the TF scaffold platform, we constructed more than 30 fusion complexes comprising various cytokines, ligands, receptors, and scFvs. Our characterization of HCW9201 and HCW9207 described in this study verify high-level production and purification of HFPCs that retain the expected biological activities of their multiple cytokine and scFv domains. We also provide a scalable approach for generating large-scale GMP-grade HFPCs to support clinical applications.
HCW9201 and HCW9207 were designed to coordinately activate the IL-12, IL-15, and IL-18 receptors, with (HCW9207) or without (HCW9201) engagement of CD16. We hypothesized that HCW9201 and HCW9207 would result in changes to NK cell activation and differentiation into ML NK cells, similar to the 12/15/18 cytokine combination (5, 7). A comprehensive comparison of these HFPCs to recombinant human IL-12, IL-15, and IL-18 revealed similar cytokine receptor signaling, activation, molecular programs, and functional ML NK cell differentiation with HCW9201 and 12/15/18. However, HCW9207 exhibited reduced IL-18 and IL-15 signaling and had several differences in induced gene expression in primary NK cells compared with 12/15/18 and HCW9201. We also demonstrated that 12/15/18 and HCW9201 enhanced the metabolism of NK cells and resulted in reduced methylation of key CpG sites within the IFNγ CNS-1 regulatory region. Finally, HCW9201- and 12/15/18-primed NK cells exhibited similar enhanced cytotoxicity and ADCC against leukemia targets in vitro and equivalently controlled leukemia in NSG mice. Based on these results, HCW9201 was found to have essentially identical activity as the combination of individual IL-12, IL-15, and IL-18 cytokines in effectively generating ML NK cells, solving the barrier to GMP-grade reagents to produce ML NK cells for large/late-stage clinical trials.
ML NK cells were first reported following combined IL-12, IL-15, and IL-18 stimulation in mice (4), which was confirmed in human NK cells (5). Additional signals have been reported to contribute to ML NK cell induction, including ligation of the activating receptor CD16, which enhances ML NK cell functionality when combined with cytokines (5). The activity of CD16 and consequent ITAM signaling to promote ML NK cell responses was confirmed following activation with the high-affinity anti-CD16a/CD30 bispecific protein AFM13 (51). HCW9207 was designed to improve on individual 12/15/18 targeted with HCW9201. However, due to reduced cytokine signaling of HCW9207 in NK cells, HCW9201 was nominated as the lead HFPC with subsequent production of a large-scale GMP lot that is sufficient to support future clinical evaluation. Fusion of the anti-CD16 scFv domain did not provide additional activity to the HCW9201 complex; thus, further protein engineering is under way to test new approaches to coordinately engage the IL-12, IL-15, and IL-18 receptors and other activating and coactivating receptors to further improve on ML NK cell generation. We also explored new aspects of human ML NK cell biology, identifying that HCW9201 and 12/15/18 activation resulted in enhanced mTOR-dependent metabolism, which likely contributed to subsequent ML NK cell enhanced proliferation and effector functions. As previously reported following 5 days of IL-12/15/18 stimulation (52), we provide data demonstrating that similar to murine ML NK cells (11), HCW9201- and 12/15/18-induced human ML NK cells also had selective demethylation of key CpG regulatory sites within the IFNγ CNS-1 region, providing further evidence of epigenetic reprogramming of DNA methylation as one mechanism responsible for enhanced effector function. The mechanism of epigenetic regulation of ML NK cells is a nascent area in the field, and key questions include the relative contributions of DNA methylation and histone modifications. Finally, new HFPCs are being developed and tested via the TF-based platform to facilitate the controlled expansion and survival of ML NK cells.
We also examined the molecular program of NK cells after HCW9201, HCW9207, and 12/15/18 activation, and the subsequent ML NK cell differentiation. Coordinated ligation of the IL-12, IL-15, and IL-18 receptors resulted in changes in more than 3,000 mRNA transcripts, with a high concordance between HCW9201 and 12/15/18. Key hallmarks of NK cell activation were equivalent, including transcription of the IFNG and IL2RA (encoding CD25) genes. Although HCW9207 was also concordant with 12/15/18, the correlation was less prominent and differences were observed. The immune inhibitory checkpoint LAG3 was noted to be induced predominantly after 12/15/18, HCW9201, and HCW9207 activation, identifying a new potential checkpoint for NK cells following activation. The chemokine receptors CCR1 and CCR5 were also upregulated, indicating that ML NK cells may have an enhanced potential to traffic to sites of inflammation. In contrast to acute activation, following ML NK cell differentiation, few transcripts are differentially regulated between IL-15 only–treated control NK cells, and those primed with 12/15/18, HCW9201, or HCW9207. This may reflect a high “background” of IL-15-stimulated mRNA changes, which was evident when comparing to naïve NK cells. This also likely reflects molecular program heterogeneity because not all NK cells resulting from 12/15/18 receptor priming and differentiation are equal, and a subset of resulting cells may be ML with particularly enhanced antitumor function. Future studies will address this using single-cell RNA-seq. Despite this background, some genes were found to be altered in ML NK cells. One finding was that the suppressor of cytokine signaling CISH was downregulated at the transcript level in HCW9201- and HCW9207-induced NK cells. Because this negative regulator of cytokine signaling was previously implicated in limiting NK cell function (53), this may indicate a mechanism of enhanced ML NK cell functionality and persistence. Indeed, the enhanced metabolism and CISH could be linked, as CISH deletion in induced pluripotent stem cell–differentiated NK cells demonstrates improved metabolic activity dependent on mTOR signaling (54). Chemokine receptor and trafficking molecules were also identified, including SELL (L-selectin), CCR5, and CXCR6. Previous work has identified that CD62 L protein is increased on ML NK cells in vitro and in vivo (7, 8), suggesting that SELL transcription is one mechanism contributing to this phenotype. The gene encoding the serine protease granzyme K (GZMK) was also increased, suggesting an enhanced capacity for cell death and potentially triggering inflammation. CXCR6 was also more abundant in ML NK cells compared with controls, which has been implicated in other forms of NK cell memory (55). These hypothesis-generating findings will require further investigation to clearly define their role and importance for ML NK cell biology.
ML NK cells generated with signals via the IL-12, IL-15, and IL-18 receptors have been translated into the clinic as a cellular therapy for leukemia. These trials were preceded by a comprehensive preclinical evaluation of ML NK cells that established their cytokine responsiveness, unique aspects of their biology, and enhanced antileukemia activity in vitro and in vivo (5, 7, 9, 10). Here, we compared HCW9201 to 12/15/18 and found overlap with these preclinical attributes, supporting the use of HCW9201 as an approach to ligate cytokine receptors to generate ML NK cells for clinical use. In the first-in-human phase I study, ML NK cells were safe and did not cause inflammatory adverse events characteristic of chimeric antigen receptors (CAR) T cells, such as cytokine release syndrome and immune cell–associated neurotoxicity syndrome (ICANS). Complete remission was induced in nearly half of relapsed/refractory AML patients, identifying promising avenues of further clinical investigation in older, unfit patients and those who require additional therapy as a bridge to potentially curative hematopoietic cell transplantation (7, 8). Expanding on this, ML NK cells have also demonstrated prolonged persistence in vivo when used to augment allogeneic HCT (56, 57), and induce complete remission in the setting of relapsed AML after and allogeneic HCT (58). ML NK cells have robust CD16 expression and exhibit improved ADCC (10), indicating that they can be directed to targets using therapeutic mAbs or bi/trispecific NK cell engagers. ML NK cells can also be engineered with CAR and exhibit improved functional activity compared with conventional CAR NK cells (59). To explore these clinical opportunities in the clinic, HCW9201 now provides an ideal GMP agent that can be advanced from early-phase to late-phase clinical trials. Toward this end, GMP-grade HCW9201-primed NK cells have now been used as an NK cellular therapy, as part of a clinical trial (NCT01898793).
In summary, we report that HCW9201 is an HFPC that recapitulates the IL-12, IL-15, and IL-18 receptor signaling required to enhance NK cell function and results in ML NK cell differentiation equivalent to a combination of individual IL-12, IL-15, and IL-18 cytokines. New aspects of ML NK cells were revealed, including enhanced metabolism, and an improved molecular understanding of the human 12/15/18-induced ML NK cell. Using this novel TF-based platform, GMP-grade HCW9201 and other HFPCs can be generated as new tools for cancer immunotherapy, addressing a major barrier for advancing ML NK cell therapy to larger clinical trials.
N. Shrestha is an employee of and equity holder in HCW Biologics Inc. M.J. Dee is an employee of and equity holder in HCW Biologics Inc. P. Chaturvedi is an employee of and equity holder in HCW Biologics Inc. G.M. Leclerc is an employee of and equity holder in HCW Biologics Inc. X. Zhu is an employee of and equity holder in HCW Biologics Inc. C.M. Spanoudis is an employee of and equity holder in HCW Biologics Inc. V.L. Gallo is an employee of and equity holder in HCW Biologics Inc. C.A. Echeverri is an employee of and equity holder in HCW Biologics Inc. L.L. Ramirez is an equity holder in HCW Biologics Inc. L. You is an employee of and equity holder in HCW Biologics Inc. J.O. Egan is an employee of and equity holder in HCW Biologics Inc. P.R. Rhode is an employee of and equity holder in HCW Biologics Inc. J. Jiao is an employee of and equity holder in HCW Biologics Inc. G.J. Muniz is an employee of and equity holder in HCW Biologics Inc. E.K. Jeng is an employee of and equity holder in HCW Biologics Inc. R.P. Sullivan is an employee of and equity holder in Wugen, Inc. M.M. Berrien-Elliott reports personal fees and other support from Wugen, Inc. during the conduct of the study, as well as a patent for 017001-PRO1 licensed and with royalties paid from Wugen, Inc. H.C. Wong reports a patent for US2021/0070825 pending to Wugen, Inc., and is an employee of and equity holder in HCW Biologics Inc. T.A. Fehniger reports grants from HCW Biologics Inc. and NIH during the conduct of the study; grants, personal fees, and other support from Wugen, Inc., grants from ImmunityBio, Compass Therapeutics, and Affimed, personal fees from Kiadis, Nkarta, and Nektar, and other support from Indapta and OrcaBio outside the submitted work; a patent for 15/983,275 pending and licensed to Wugen, Inc., a patent for PCT/US2019/060005 pending and licensed to Wugen, Inc., and a patent for 62/963,971 pending and licensed to Wugen, Inc.; and equity interest in, consulting for, and royalty interest in Wugen, Inc. This includes intellectual property with T.A. Fehniger as a coinventor, licensed to Wugen, Inc. from Washington University, indirectly related to this work. No disclosures were reported by the other authors.
M.K. Becker-Hapak: Conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. N. Shrestha: Data curation, formal analysis, investigation, visualization, writing–review and editing. E. McClain: Data curation, formal analysis, validation, investigation, visualization, writing–review and editing. M.J. Dee: Formal analysis, investigation, visualization, writing–review and editing. P. Chaturvedi: Formal analysis, investigation, writing–review and editing. G.M. Leclerc: Formal analysis, investigation, writing–review and editing. L.I. Marsala: Formal analysis, investigation, writing–review and editing. M. Foster: Formal analysis, validation, investigation, visualization, writing–review and editing. T. Schappe: Formal analysis, investigation, writing–review and editing. J. Tran: Data curation, formal analysis, investigation, writing–review and editing. S. Desai: Formal analysis, investigation, writing–review and editing. C.C. Neal: Investigation, visualization, writing–review and editing. P. Pence: Data curation, investigation, writing–review and editing. P. Wong: Investigation, visualization, writing–review and editing. J.A. Wagner: Investigation, writing–review and editing. D.A. Russler-Germain: Data curation, investigation, visualization, writing–review and editing. X. Zhu: Investigation, writing–review and editing. C.M. Spanoudis: Investigation, writing–review and editing. V.L. Gallo: Formal analysis, investigation, writing–review and editing. C.A. Echeverri: Formal analysis, investigation, visualization, writing–review and editing. L.L. Ramirez: Formal analysis, investigation, visualization, writing–review and editing. L. You: Formal analysis, investigation, writing–review and editing. J.O. Egan: Formal analysis, visualization, project administration, writing–review and editing. P.R. Rhode: Formal analysis, visualization, project administration, writing–review and editing. J. Jiao: Formal analysis, investigation, writing–review and editing. G.J. Muniz: Conceptualization, supervision, investigation, visualization, project administration, writing–review and editing. E.K. Jeng: Formal analysis, investigation, visualization, methodology, project administration, writing–review and editing. C.A. Prendes: Conceptualization, supervision, investigation, project administration, writing–review and editing. R.P. Sullivan: Conceptualization, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. M.M. Berrien-Elliott: Data curation, formal analysis, investigation, methodology, writing–review and editing. H.C. Wong: Conceptualization, data curation, supervision, investigation, project administration, writing–review and editing. T.A. Fehniger: Conceptualization, data curation, supervision, funding acquisition, visualization, methodology, writing–original draft, project administration, writing–review and editing.
The authors thank Siteman Flow Cytometry for cell sorting assistance, the Siteman/ChiiPs Immunomonitoring Laboratory for CyTOF data acquisition, and GTAC@MGI for RNA-seq. This work was supported by the Siteman Cancer Center (P30CA091842) through use of multiple shared resources, including GTAC@MGI, Siteman Flow Cytometry, and the Immunomonitoring Laboratory. Experimental schemas were created with BioRender.com. This work was partially supported by HCW Biologics Inc. and NIHT32 HL007088 (J.A. Wagner). This work was also supported by the NIH: T32HL00708843 (J.A. Wagner and P. Wong), K12CA167540 (M.M. Berrien-Elliott), SPORE in Leukemia P50CA171063 (M.M. Berrien-Elliott and T.A. Fehniger), and R01CA205239 (T.A. Fehniger). This work was also supported by the NCI Cancer Center Support Grant P30CA91842 (Siteman Cancer Center).
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