Simultaneous Engagement of Tumor and Stroma Targeting Antibodies by Engineered NK-92 Cells Expressing CD64 Controls Prostate Cancer Growth

Metastatic castration-resistant prostate cancer (mCRPC) is uniformly lethal with 5-year survival rates remaining at 30%. Innovative treatments for patients with the disease are urgently needed, and efforts are being focused on developing immunotherapies (1). The FDA-approved autologous cell–based vaccine Sipuleucel-T, which stimulates T-cell response against the prostate tumor antigen prostatic acid phosphatase, improved median overall survival (OS) by 4.1 months, indicating that prostate cancer was a good candidate for cancer immunotherapy (2). Current immunotherapy clinical trials for mCRPC have focused on evaluating checkpoint inhibitors, however, they have had modest outcomes and none have been approved (3–5). The lack of immunotherapeutic efficacy in mCRPC is due, in part, to the immunosuppressive tumor microenvironment (TME) that promotes the survival of the cancer cells and progression of the disease (6). Cancer-associated fibroblasts are the major cell type in the TME that regulate growth and metastasis of epithelial cancers, including prostate cancer (7). Cancer-associated fibroblast paracrine signaling and mechanical stress contribute to tumor survival, inflammation, immunosuppression, therapeutic resistance, and drive tumor invasion (8, 9). Fibroblast activation protein alpha (FAP) is a transmembrane serine protease highly expressed by cancer-associated fibroblasts that contributes to extracellular matrix remodeling and protumor signal transduction (10). Immunotherapies that target malignant cells alone may not be enough to elicit an immune response capable of overcoming protumorigenic functions of the TME. In contrast, an immunotherapeutic approach that targets both the tumor stroma and malignant cells has been shown effective in multiple cancer models (11, 12).

Natural killer (NK) cells are innate, cytotoxic lymphocytes that detect and kill stressed, virus-infected, and transformed cells without prior sensitization (13). Infiltration of NK cells in prostate tumors corresponds with an increased OS among patients with mCRPC (14), which suggests the potential to exploit NK cells for the treatment of prostate cancer. A key antitumor effector function of human NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated through the IgG Fcγ receptor CD16A (FcγRIIIA; ref. 15). Several clinically successful antitumor mAbs mediate ADCC (16); however, attributes of CD16A, such as a low-to-moderate affinity for IgG and rapid downregulation by a disintegrin and metalloproteinase-17 (ADAM17) may limit the efficacy of mAb therapies (17).

CD64 (FcγR1), the sole functional high-affinity FcγR in humans, binds to IgG isotypes IgG1 and IgG3, like CD16A; however, CD64 binds monomeric IgG with an affinity ∼30-fold higher than CD16A (18). CD64 is expressed by myeloid cells including monocytes, macrophages, and neutrophils, but not lymphocytes, including NK cells (19). Unlike CD16A, CD64 expression is not downregulated by ADAM17 upon activation (20). CD16A noncovalently associates with the signaling adaptor FcRγ and CD3ζ as either homodimers or heterodimers (21, 22), whereas CD64 associates with FcRγ, because myeloid cells lack expression of CD3ζ (23). These immunoreceptor tyrosine-based activation motif (ITAM)–containing signaling adaptors trigger downstream signaling cascades for cell activation.

In this study, we stably expressed human CD64 in the NK-92MI human NK cell line that lacks expression of endogenous FcγRs and synthesizes IL2 to promote cell proliferation (24). These cells are referred to herein as NK-92MI^CD64 cells. Our analyses revealed that CD64 surface levels can be modulated by CD3ζ expression. Furthermore, we observed that CD64 induces ADCC and that this was facilitated through CD3ζ signaling. The antitumor activity of these genetically modified NK cells was tested against the tumor-associated calcium signal transducer 2 (TROP2)–expressing prostate cancer cell line, DU145, and the FAP-expressing prostate stromal cell line, hPrCSC-44. Effector-cell function was enhanced with the addition of TROP2- or FAP-targeted antibodies. Therapeutic efficacy of the combination therapy was also demonstrated in a prostate tumor stroma xenograft model. Overall, these data suggest the therapeutic potential of the engineered NK cells in combination with antibodies targeting both the tumor stroma and malignant cells, a strategy that could provide urgently needed immunotherapies for patients with mCRPC.

DU145 (#HTB-81) and HEK293T (#CRL-11268) cell lines were purchased from ATCC in 2019 and maintained in DMEM (Gibco, catalog no. 11965092) supplemented with 10% FBS (Gibco, catalog no. 16140071). hPrCSC-44 cells were obtained from Dr. W. Nathaniel Brennen (Johns Hopkins Medicine, Baltimore, MD) and maintained in RoosterNourish-MSC media (RoosterBio, catalog no. KT-001). Human NK-92MI (#CRL-2408) cells were purchased from ATCC in 2018 and maintained in MEM-|\alpha $| media without nucleosides (Gibco, catalog no. 12561056) supplemented with 12.5% FBS (Gibco, catalog no. 16140071), 12.5% Horse Serum (Gibco, catalog no. 26050088), and 0.1 nmol/L 2-mercaptoethanol (Sigma-Aldrich, catalog no. M6250). Jurkat (#TIB-152) cells were obtained from ATCC in 2009 and maintained in RPMI-1640 (Gibco, catalog no. 11875093) supplemented with 10% FBS (Gibco, catalog no. 16140071). Gryphon Ampho cells (293T cells stably expressing gag, pol, and env genes) were purchased from Allele Biotechnology in 2019 and were maintained DMEM (Gibco, catalog no. 11965092) supplemented with 10% FBS (Gibco, catalog no. 16140071). All cell experiments were performed within 4 months of thawing cell lines from frozen cell stocks. Cell lines were authenticated using short-tandem repeat analysis and routinely monitored for Mycoplasma contamination prior to our studies.

Fresh peripheral blood of healthy blood donors was purchased from the Innovative Blood Resources. Total RNA was isolated from two human peripheral blood leukocytes using TRizol total RNA isolation reagent (Thermo Fisher Scientific, catalog no. 10296010). Peripheral blood cDNA synthesized with the SuperScript First-Strand Synthesis System for RT-PCR Kit (Thermo Fisher Scientific, catalog no. 11904018) was used in RT-PCR for expression construct generation. Bam HI-flanked full-length CD64 cDNA (1,169 bps) was amplified using the forward primer 5′-CTC TAG ACT GCC GGA TCC GGA GAC AAC ATG TGG TTC TTG ACA ACT CT-3′ and the reverse primer 5′-TCG AAT TTA AAT GGA TCC CTA CGT GGC CCC CTG GGG CTC CTT-3′ (underlined nucleotides indicate Bam HI restriction sites). The RT-PCR reaction was performed with 2 μL of cDNA, 200 nmol/L of each primer, 200 μmol/L of dNTPs, 2.0 mmol/L of MgSO₄, and 1 U of Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific, catalog no. 11304011) in a 25 μL reaction volume. The ABI Veriti 96-well Thermal Cycler was used for the RT-PCR reaction starting with 94°C for 3 minutes, 35 cycles of denaturing at 94°C for 30 seconds, annealing at 56°C for 45 seconds, extension at 68°C for 1 minutes, and 30 seconds with a final extension at 72°C for 7 minutes. The In-Fusion HD Cloning Kit (Takara Bio USA, catalog no. 638917) was used to insert the CD64 cDNA into the bi-cistronic retroviral expression vector pBMN-I-GFP (Addgene Plasmid# 1736; deposited by Dr. Garry Nolan) at the Bam HI restriction enzyme site. Pseudo-retrovirus was generated as described previously (25). Briefly, approximately 24 hours before transfection, Gryphon Ampho cells were seeded in a 60-mm cell culture dish. Gryphon Ampho cells were incubated overnight at 37°C, 5% CO₂ to reach about 85% confluence. For transfection of Gryphon Ampho cells, the complexes of 5 μg CD64-pBMN-I-GFP plasmid DNA and 10 μL Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, catalog no. 11668019) were prepared according to the vendor's instructions. The DNA-Lipofectamine 2000 complexes were added to Gryphon Ampho cells in the dish and subsequently mixed with culture medium by rocking back and forth gently. The medium containing DNA-Lipofectamine 2000 complexes was removed from the culture dish and 5 mL of fresh culture medium was added after 12 to 16 hours of incubation. The cell culture medium containing pseudo-retrovirus particles was harvested after additional 48-hour incubation. The harvested cell culture supernatant was centrifuged at 2,500 RPM (1,282 × g) for 3 minutes at 4°C to remove cell debris and filtered through 0.45 μm filter before cell transduction.

For NK-92MI cell transduction, 1 mL of filtered cell culture supernatant containing virus particles was mixed with 4 mL of NK-92MI cell culture medium and 5 μg/mL polybrene (Sigma-Aldrich, catalog no. TR-1003) to form an infection cocktail. To infect NK-92MI cells, 5 × 10⁵ NK-92MI cells were mixed with 5 mL of the infection cocktail and seed into one well of a 6-well plate. The same volume of fresh medium was added 1 day after infection. Two days after infection, the transduced NK-92MI cells were stained with APC-conjugated anti-CD64 (BioLegend, catalog no. 305014) and examined by flow cytometric analysis to determine CD64 expression. The NK-92MI cells were sorted by flow cytometry for a homogeneous CD64 expression population.

Kpn I/Bam HI-flanked full-length CD64 cDNA was amplified using the forward primer 5′-TAA CGG GGT ACC GGA GAC AAC ATG TGG TTC TTG ACA ACT-3′ (underlined nucleotides indicate the Kpn I restriction site) and the reverse primer 5′-CCG GGA TCC CTA CGT GGC CCC CTG GGG CTC CTT-3′ (underlined nucleotides indicate the Bam HI restriction site). The human CD64 cDNA was cloned into the pcDNA3.1 expression vector (Thermo Fisher Scientific, catalog no. V79020). Bam HI/EcoR I-flanked full-length FcRγ chain cDNA (285 bps) was amplified with the sense (5′-CGC GGA TCC CAG CCC AAG ATG ATT CCA GCA GTG GTC-3′) (underlined nucleotides indicate the Bam HI restriction site) and anti-sense primer (5′-CCG GAA TTC CTA AAG CTA CTG TGG TGG TTT CTC ATG-3′ (the underlined nucleotides indicate the EcoR I restriction site). The human FcRγ cDNA was cloned into the pcDNA3.1 expression vector. Similarly, Kpn I/EcoR I-flanked full-length CD3ζ chain cDNA (546 bps) was amplified with the sense primer (5′-CGG GGT ACC TCT GAG GGA AAG GAC AAG ATG A-3′) (underlined nucleotides indicate the Kpn I restriction site) and antisense primer (5′-CCG GAA TTC GGT GAA ATC CCC TGC CTG TTA-3′ (underlined nucleotides indicate the EcoR I restriction site). The human CD3ζ cDNA was cloned into the pcDNA3.1 expression vector. Expression constructs were transfected into HEK-293T cells for transient expression analysis using the TransIT-293 transfection reagent (Mirus Bio, catalog no. MIR 2704). Successful transfection was confirmed via flow cytometric analysis.

Cells were lysed in SDS lysis buffer [1% SDS, 50 mmol/L Tris-Cl, 10 mmol/L ethylenediaminetetraacetic acid (EDTA), protease inhibitor cocktail (Millipore Sigma, catalog no. P8340), and phosphatase inhibitor cocktail 2 (Millipore Sigma, catalog no. P5726)] for Western blot analysis, or digitonin immunoprecipitation (IP) lysis buffer (1% digitonin, 20 mmol/L Tris-Cl, 135 mmol/L NaCl, protease, and phosphatase cocktail inhibitors) for IP. SDS lysates were sonicated and centrifuged at 12,000 × g to pellet insoluble material. Digitonin lysates were rocked gently for 30 minutes at 4°C and centrifuged at 10,000 × g to remove insoluble cell debris.

Protein concentrations were quantified using a Bicinchoninic acid assay (Pierce Biotechnology, catalog no. 23225). Fifty micrograms of protein [1X Laemmli sample buffer (Bio-Rad, catalog no. 161-0747), 0.1M DTT] was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Blots were blocked in Intercept TBS blocking buffer (LI-COR, catalog no. 927-60001) and incubated with primary antibodies and Quick Western IRDye 680RD detection reagent (LI-COR, catalog no. 926-69100) overnight at 4°C. Blots were visualized using an Odyssey infrared imager (LI-COR). Primary antibodies used were anti-CD64 (Abcam, catalog no. ab134073), anti-FcRγ subunit (EMD Millipore, catalog no. 06-727), anti-CD3ζ (Santa Cruz Biotechnology, catalog no. sc-1239), and anti–β-actin (Sigma-Aldrich, catalog no. A5316).

For IP, lysates were incubated for 1 hour with human serum IgG (Sigma-Aldrich, catalog no. I2511-10MG) at 4°C to block nonspecific interactions between CD64 and IP antibodies. CD64 or isotype matched control antibodies (Bethyl Laboratories, catalog no. catalog no. P120-101) were immobilized to Pierce Direct IP Kit immunoprecipitation beads (Pierce Biotechnology, catalog no. 26148) following the manufacturer's directions. Protein was eluted at 100°C using SDS elution buffer containing 0.1M DTT, and samples were resolved by SDS-PAGE for Western blot analysis as described above.

Single guide RNAs (sgRNA) were designed using Synthego's Knockout Guide Design tool (https://design.synthego.com/#). Alt-R CRISPR-Cas9 sgRNAs were purchased from IDT (Integrated DNA Technologies) and contain both tracrRNA and crRNA sequences; CD247 (CD3ζ) guide RNA sequence: AGCAGAGUUUGGGAUCCAGC. Cas9 recombinant protein was purchased from IDT (catalog no. 1081059). CRISPR/Cas9 ribonucleoprotein (RNP) complexes were assembled by mixing 100 pmol of guide RNA and 30 pmol of Cas9. Five million NK-92MI cells were resuspended in primary cell P3 buffer (Lonza, catalog no. PBP3-00675) with the Cas9 RNP complexes and nucleofected using the Amaxa 4D Nucleofector program CA-137. Knockout of CD3ζ was confirmed via Western blot analysis (anti-CD3ζ, Santa Cruz Biotechnology, catalog no. sc-1239), flow cytometry (anti-CD247, BioLegend, catalog no. 644106), and inference of CRISPR edits (ICE) analysis. For ICE analysis, DNA was isolated from NK-92MI cells using Qiagen's DNEasy Kit (Qiagen, catalog no. 69506) and PCR with the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, catalog no. 1725270) was used to amplify a 467 bp region that contained the gRNA cut site for CD3ζ (forward primer 5′-CCTTGTTCCTGAGAGTGAAG-3′, reverse primer 5′-CCAAATAAATCATGACACGGAG-3′). PCR products were submitted to UMN Genomics for Sanger sequencing with the sequencing primer 5′-GATGTGTTCTCGTCACCTT-3′. Sequence results were then uploaded to Synthego's ICE analysis tool to confirm gene ablation (https://ice.synthego.com/#).

An in-house murine naive single-chain variable fragment (scFv) antibody phage display library with a diversity of 1 × 10⁹ was used to identify clones against recombinant human FAP (R&D Systems, catalog no. 3715-SE-010). Anti-TROP2 was generated as described previously (26). The heavy and light chain variable domains of the anti-FAP (B12) and anti-TROP2 scFv sequences were cloned separately into the pFUSEss human IgG1 expression vectors pFUSEss-CHIg-hG1 (InvivoGen, catalog no. pfusess-hchg1) and pFUSE2ss-CLIg-hk (InvivoGen, catalog no. pfuse2ss-hclk). Anti-FAP light chain primers (forward primer 5′-AAAACGAATTCGGATATTGTGATCAGTCTCCATCCTCC -3′, reverse primer 5′-AAAACGTACGGCGTTTGATTTCCAGCTTGGTGC -3′) and heavy chain primers (forward primer 5′-AAAACGAATTCGGAAGTGATGCGAGTCTGGG-3′, reverse primer 5′- AAAACGTAGCCGAGGAGACTGTGAGAGTGGTGC-3′). Anti-TROP2 light chain primers (forward primer 5′-GCCGCGAATTCGGACATCCAGCTGACCCAAAGCCACAAGTTCATG-3′, reverse primer 5′-CCGCGCGTACGACGCTTCAGCTCCAGCTTGGTACCCGCGC-3′) and heavy chain primers (forward primer 5′- GCCGCGAATTCGCAAGTGCAACTGGGTGAAAGCGGTCCGG-3′, reverse primer 5′- GCCGCGCTAGCGCTGCTAACGGTCACGGTGGTACCTTGGCC-3′). The vectors were cotransfected into HEK293T cells using the calcium carbonate method. The cell supernatant was collected after 72 hours and purified using a 5 mL HiTrap Protein A HP column (GE Healthcare) following the manufacturer's guidelines. Eluted antibody was collected, and the purity was analyzed by reduced and nonreduced SDS-PAGE and visualized using GelCode Blue Safe Protein Stain (Thermo Fisher Scientific, catalog no. PI24596). Protein concentration was measured on the basis of absorbance at 280 nm using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific).

To measure antibody capture by CD64, NK-92MI^CD64 cells were incubated with the generated anti-TROP2 or anti-FAP (B12) (5 μg/mL) in serum-free MEM-α media (SFM) without nucleosides (Gibco, catalog no. 12561056) supplemented with 0.1 nmol/L 2-mercaptoethanol (Sigma-Aldrich, catalog no. M6250) for 2 hours at 37°C and 5% CO₂. Effector cells were washed once with SFM, and stained with APC-conjugated donkey anti-human IgG secondary mAb (1:200, catalog no. 709-136-14, Jackson Immunoresearch) for 30 minutes on ice. Cells were fixed with 1% paraformaldehyde, resuspended in FACS buffer, and analyzed on a BD LSR II flow cytometer. For IFNγ quantification, NK-92MI^CD64 cells were preincubated in SFM for 2 hours at 37°C and 5% CO₂. Effector cells were combined with DU145 or hPrSCS-44 target cells at an effector–target (E:T) ratio of 1:1 (1.5×10⁵ effector and 1.5×10⁵ target cells, respectively) and incubated with or without anti-TROP2 or B12 (5 μg/mL) in SFM for 2 hours at 37°C and 5% CO₂. After incubation, the cells were centrifuged and cell supernatants were collected for IFNγ determination using the human IFNγ flex set (BD Biosciences, catalog no. #560111) and a BD LSR II flow cytometer. Analysis was performed using FlowJo software.

Flow cytometric analysis was performed on FACSCelesta and BD LSR II instruments using BD FACSDiva Software (BD Biosciences) or the Beckman Coulter Cytoflex flow cytometer. CD64 expression was determined using the human anti-CD64 clone 10.1 (BioLegend, catalog no. 305014). To analyze CD3ζ expression, cells were fixed and permeabilized using the Tonbo Foxp3/Transcription Factor Staining Buffer Kit (Tonbo, TNB-0607-KIT) and stained with anti-CD247(BioLegend, catalog no. 644106). For controls, fluorescence minus one was used as well as appropriate isotype-matched antibodies. An FSC-A/SSC-A plot was used to set an electronic gate on cell populations, and an FSC-A/FSC-H plot was used to set an electronic gate on single cells. To distinguish live versus dead cells, 7AAD was used as per the manufacturer's instructions (BioLegend, catalog no. 420403). Analysis was performed using FlowJo software.

ADCC was determined using the DELFIA EuTDA cytotoxicity assay (PerkinElmer, catalog no. #AD0116) according to the manufacturer's instructions and performed as described previously (20). Cytotoxicity for each sample is represented as % specific release and was calculated using the following formula:

Conventional ADCC: CD3ζ KO and NK-92MI^CD64 cells were preincubated in SFM for 2 hours at 37°C and 5% CO₂. For the monotherapy, effector (CD3ζ KO or NK-92MI^CD64) and target cells (DU145 or hPrCSC-44) were combined at the indicated E:T ratios in SFM with or without anti-TROP2 or B12 (5 μg/mL). For the combination therapy, effector cells (NK-92MI^CD64) were combined with a mixture of target cells (1:1; 4×10³ hPrCSC-44: 4×10³ DU145 cells/well) at the indicated E:T ratios in SFM with or without anti-TROP2 and B12 (2.5 μg/mL each).

Bound mAb ADCC: NK-92MI^CD64 cells were preincubated with or without anti-TROP2 or B12 (5 μg/mL) in SFM for 2 hours at 37°C and 5% CO₂ then washed once with SFM. For the monotherapy, effector and target cells (DU145 or hPrCSC-44) were combined at the indicated E:T ratios. The experiment was repeated with NK-92MI^CD64 cells preincubated with or without a human IgG isotype control (5 μg/mL) in SFM for 2 hours at 37°C and 5% CO₂. For the combination therapy, effector cells (1:1; anti-TROP2: B12) were combined with a mixture of target cells (1:1; 4×10³ hPrCSC-44: 4×10³ DU145 cells/well) at the indicated E:T ratios.

Animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). Three- to four-week-old NOD/scid/IL-2 common γ chain -/- (NSG) mice were purchased from Jackson Labs (catalog no. #005557). Tumors were implanted by subcutaneous injection of a mixture of 2×10⁶ hPrCSC-44 and 1×10⁶ DU145 cells suspended in 100 μL of 1:1 PBS and Matrigel (Corning, catalog no. 356234) into the rear flanks of mice. NK-92MI^CD64 cells were irradiated (10 gray) before use as a treatment. Randomized mice (n = 4/arm) with tumor volumes 100 to 200 mm³ were assigned to the following treatment arms: saline; NK-92MI^CD64 cells alone; intraperitoneal injection of anti-TROP2 and B12 (6 mg/kg of each mAb) 24 hours before adoptive NK-92MI^CD64 cell therapy or adoptive NK-92MI^CD64 cell therapy with bound anti-TROP2 and B12 (1:1; anti-TROP2: B12). Treatment (1×10⁷ cells/mouse) was administered peritumorally once a week for 4 weeks. Tumor volumes were measured two to three times a week until tumors reached 1,500 mm³. For IHC, mice (n = 3/arm) were treated with the aforementioned arms. For the ADCC combination, mice were injected intraperitoneally with anti-TROP2 and B12 (6 mg/kg of each mAb) every 2 days. After 7 days, the tumors were excised and fixed for IHC analysis.

IHC was performed on formalin-fixed paraffin-embedded tissue sections using (1:400 dilution) anti-human cleaved caspase 3 (cCasp3; catalog no. 9661L, Cell Signaling Technology). Unstained sections (4 μm thick) were deparaffinized and rehydrated using standard methods. Antigen retrieval and staining were performed as described previously (27). Slides were incubated in secondary biotinylated goat anti-rabbit IgG (10 μL/mL; Vector, BA-1000) diluted in 10% blocking solution (Peroxidase, Biocare Medical, catalog no. PX968)/90% TBST. Detection of the antibody complexes was by the avidin−biotin immunoperoxidase method. Images of cCasp3-stained tumor tissue were collected using an Axiolab 5 microscope (ZEISS). Images (n = 3/mouse) were collected for each treatment group (n = 3 mice/group). Image J was used to remove background color and the total image was measured for mean grey value.

All statistical analyses were performed using Prism GraphPad software. Comparison between two groups was done using Student t test. Comparison between three or more groups was done using one-way ANOVA followed by Tukey honest significance post hoc test. Statistical significance of tumor growth was determined using a two-way ANOVA and corrected for multiple comparisons using Sidak hypothesis testing. Survival curves were constructed using the Kaplan–Meier method and statistical analysis of survival was performed using a log-rank (Mantel–Cox) test. IHC staining was quantified as mean intensity. Mean intensity = 255 – mean grey value. Results are depicted as mean ± SEM. The symbols used to represent the P values were as follows; ns, P > 0.05; *, P |\le $| 0.05; **, P |\le $| 0.01; ***, P |\le $| 0.001; ****, P |\le $| 0.0001.

NK-92MI cells are derived from NK-92 cells, which do not express endogenous FcγRs but can mediate ADCC through recombinant CD16A (28). We transduced NK-92MI cells to express recombinant human CD64 at high levels (Fig. 1A). CD64 is normally expressed by myeloid cells in which its signaling is mediated by its association with the signaling adapter FcRγ (29). We investigated whether recombinant CD64 can associate with CD3ζ in NK-92MI cells. As shown in Fig. 1B, CD3ζ was observed to coimmunoprecipitate with CD64, indicating their noncovalent association.

It is well established that CD16A expression on the cell surface requires the coexpression of either FcRγ or CD3ζ (21, 30, 31). In contrast, CD64 surface expression is not dependent on FcRγ, but its level of expression at the cell surface can be increased with co-expression of FcRγ (23). We examined the effects of FcRγ and CD3ζ coexpression on the cell surface levels of CD64. This was performed in HEK-293T cells, which lack endogenous CD64, FcRγ, and CD3ζ. HEK-293T cells transfected with CD64 cDNA alone expressed low levels of CD64, whereas its expression was markedly increased with the cotransfection of either FcRγ or CD3ζ (Fig. 1C). Taken together, we demonstrate that CD64 can be expressed in NK cells and that it can associate with the signaling adaptor CD3ζ when available. Moreover, alignment of the amino acid sequences of the transmembrane regions of CD16A and CD64 revealed a high degree of homology and the conservation of several residues that are critical for CD16A association with CD3ζ (Fig. 1D).

To assess whether CD3ζ affects CD64 function, we used CRISPR/Cas9 genome editing to knockout CD3ζ from NK-92MI cells. NK-92MI cells were transfected with a single CD3ζ CRISPR guide RNA plus Cas9 RNP or Cas9 alone as a control. The genotype of CRISPR edited NK-92MI cells was determined via Sanger sequencing. Short electropherograms of the sequence area near the sgRNA binding site were used to determine the number of indels at the site of excision and subsequently scored for knockout using ICE analysis (Fig. 2A). This revealed that 97% of the sequences in the CRISPR/Cas9 sgRNA transfected sequences contained a frameshift mutation. CD3ζ knockout in NK-92MI cells was confirmed by Western blotting and flow cytometry, which both demonstrated a dramatic reduction in CD3ζ expression (Fig. 2B and C).

Next, we evaluated the cytotoxic capacity of NK-92MI^CD64 cells lacking CD3ζ using a conventional ADCC assay. For these studies, CD3ζ knockout and Cas9-transfected control NK-92MI cells were transduced with equal amounts of viral supernatant to express CD64 and sorted to match cell surface expression levels of CD64 (Fig. 2D). A bicistronic retroviral vector was used for transduction that expresses GFP in a proportional manner, and its levels were equivalent for the NK-92MI^{CD64/CD3ζ KO} and NK-92MI^CD64 transductants (Fig. 2D). Each transductant was then incubated with the TROP2-positive prostate cancer cell line DU145 in the presence or absence of an anti-TROP2 mAb. As shown in Fig. 2E, high levels of DU145 cell cytotoxicity occurred by NK-92MI^CD64 cells in the presence but not absence of anti-TROP2. However, NK-92MI^{CD64/CD3ζ KO} cells demonstrated highly diminished levels of cytotoxicity in the presence of anti-TROP2 (Fig. 2E), indicating that CD3ζ had a key role in CD64 induction of ADCC. These findings show that CD64 in NK-92MI cells can utilize CD3ζ as a signaling adaptor. It was interesting to us that ADCC by NK-92MI^{CD64/CD3ζ KO} cells was so highly ablated, as we presumed that FcRγ in these cells would mediate signaling as well. However, we were unable to detect FcRγ expression by Western blotting, suggesting that its levels are very low in NK-92MI cells (Fig. 2F).

Having elucidated the functional activity of CD64 in NK-92MI cells and its activation mechanism, we evaluated the potential of the engineered cell therapy and ADCC-inducing mAbs as a strategy for treating mCRPC. The effector function of NK-92MI^CD64 cells was first tested against DU145, a human prostate cancer cell line that expresses TROP2, and hPrCSC-44, a human prostate cancer–associated fibroblast cell line that expresses FAP. Previously developed therapeutic mAbs against TROP2 (26) and FAP (27, 32), were used in this study. The FAP-specific mAb is known as B12. The anti-TROP2 and B12 were shown to induce ADCC by the NK-92MI^CD64 cells. Killing levels were measured using the Delfia EuTDA cell cytotoxicity assay with data represented as percent specific release (or % killing). A concentration-dependent effect of anti-TROP2 and B12 on ADCC was observed, with highest concentration the most potent (Fig. 3A). A cytotoxicity assay with different E:T ratios at the highest concentration of mAbs was performed (Fig. 3B). NK-92MI^CD64 cells had significantly higher DU145-cell killing efficiencies in the presence of anti-TROP2 than in its absence at different E:T ratios (83.28% vs. 8.67% at 20:1; 66.6% vs. 3.88% at 10:1; and 44.83% vs. 7.37% at 5:1 E:T). Similarly, NK-92MI^CD64 cells demonstrated significant hPrCSC-44-cell killing efficiencies in the presence of B12 compared with its absence at different E:T ratios (77.6% vs. 8.44% at 20:1; and 52.4% vs. 9.35% at 10:1). Next, as a negative control, cytotoxicity by NK-92MI^CD64 cells was measured using the highest concentration of anti-TROP2 in the presence of hPrCSC-44 cells, which do not express TROP2, or B12 in the presence of DU145 cells, which do not express FAP (Fig. 3C). No ADCC was observed under these conditions. Finally, IFNγ production was measured by flow cytometry to evaluate the function of NK-92MI^CD64 cells after incubation with or without anti-TROP2 or B12 (Fig. 3D). IFNγ levels increased by 20-fold in the presence of target cells and anti-TROP2 and by 4-fold in the presence of B12, respectively. These data suggest that NK-92MI^CD64-cell effector functions against cancer and associated stromal cells can be enhanced in the presence of anti-TROP2 or B12 in vitro.

Because CD64 binds IgG with high affinity and can stably bind soluble antibody (33), we evaluated the killing capacity of NK-92MI^CD64 cells. The capture of anti-TROP2 and B12 by CD64 on NK-92MI^CD64 cells in SFM was detected by flow cytometry using an APC-conjugated secondary mAb specific for human IgG1 (Fig. 4A). More than 95% of the cells incubated with either anti-TROP2 or B12 were positively stained compared with NK-92MI^CD64 cells stained with secondary mAb alone, indicating that CD64 on NK-92MI^CD64 can stably bind both mAbs. We next determined the mAb-directed killing effect using the Delfia EuTDA cell cytotoxicity assay (Fig. 4B). NK-92MI^CD64 cells with bound anti-TROP2 (anti-TROP2-NK-92MI^CD64) or B12 (B12-NK-92MI^CD64) were incubated with their respective target cells at the indicated E:T ratios. Compared with the NK-92MI^CD64 cells alone, the anti-TROP2-NK-92MI^CD64 cells had significantly higher cytotoxicity at different E:T ratios (86.94% vs. 8.67 at 20:1; and 64.63% vs. 3.88% at 10:1). B12-NK-92MI^CD64 cell cytotoxicity was also significantly higher than the NK-92MI^CD64 cells alone (67.53% vs. 8.44% at 20:1; and 36.97% vs. 9.35% at 10:1). NK-92MI^CD64 bound with human IgG1 isotype control had basal levels of cytotoxicity that were not significantly different from the untreated NK-92MI^CD64 cells for both cell lines (Fig. 4C). In conclusion, anti-TROP2 or B12 stably bound to CD64 expressed by effector cells can effectively direct the NK-92MI^CD64 cells to prostate cancer and stromal target cells, resulting in antigen-selective cytotoxicity.

With the demonstrated success of the conventional ADCC and bound mAb NK-92MI^CD64 cell monotherapies, we evaluated the efficacy of anti-TROP2 and B12 as a mixable therapeutic approach. Previous studies have shown a therapeutic effect from combination therapies that target both the tumor stroma and malignant cells compared with either monotherapy alone (12, 34, 35). To fabricate the prostate TME in an in vitro model, we combined DU145 and hPrCSC-44 target cells at a 1:1 ratio. First, the anti-TROP2 and B12 combination was tested for conventional ADCC induction with the NK-92MI^CD64 cells (Fig. 5A). We measured potency of target cell killing following coculture with the NK-92MI^CD64 cells in the presence of both mAbs compared with NK-92MI^CD64 cells alone at each E:T ratio. The combination therapy demonstrated enhanced cytotoxicity and % killing in the presence of both mAbs was significantly higher compared with coculture with NK-92MI^CD64 cells alone at different E:T ratios (74.83% vs. 10.23% at 20:1; 59.3% vs. 6.53% at 10:1; and 38.5% vs. 4.03% at 5:1). Next, we tested the bound mAb combination therapy (Fig. 5B). The anti-TROP2-NK-92MI^CD64 and B12-NK-92MI^CD64 cell combination therapy exhibited significantly higher cell killing compared with NK-92MI^CD64 cells alone (75.36% vs. 10.23% at 20:1; and 40.00% vs. 6.53% at 10:1). These data suggest that treatment with NK-92MI^CD64 cells in combination with anti-TROP2 and B12 by the conventional or bound ADCC mechanism could be effective at targeting prostate tumors.

Therapeutic efficacy of the NK-92MI^CD64 cells in combination with anti-TROP2 and B12 was evaluated in a prostate cancer xenograft mouse model (Fig. 6A). NSG mice bearing subcutaneous DU145 and hPrCSC-44 xenografts were randomly assigned to the treatment arms: saline; adoptive NK-92MI^CD64 cell transfer; intraperitoneal injection of anti-TROP2 and B12 24 hours before adoptive NK-92MI^CD64 cell transfer; and adoptive anti-TROP2-NK-92MI^CD64 and B12-NK-92MI^CD64 cell transfer. Treatment was administered peritumorally once per week for four weeks and tumor volumes were measured up to 1,500 mm³. Robust antitumor activity in the animals treated with either combination therapy was observed (Fig. 6B). At day 26 posttreatment, mice receiving the ADCC or bound mAb combination therapies had significantly reduced tumor growth by 72.1% (P = 0.0013) and 75.5% (P = 0.0016) compared with the saline control. Furthermore, treatment with either combination therapy significantly improved OS (Fig. 6C). Median survival was 26 days for the saline control versus 40 days for the conventional ADCC combination (P = 0.0069) and 48 days for the bound mAb combination (P = 0.0069). Tumor-bearing mice treated once in each of the previously mentioned arms had tumors excised after 7 days. IHC staining with anti-human cCasp3 was used to measure apoptosis in the tumor tissue (Fig. 6D). Tumors treated with either of the combination therapies had significantly higher mean intensity indicating increased cCasp3 compared with the saline control (Fig. 6E). In conclusion, adoptive NK-92MI^CD64 cell transfer with antibodies targeting the tumor stroma and malignant cells is effective in a prostate tumor model.

NK cells utilize the low affinity Fc receptor CD16A to kill antibody-opsonized tumor cells via ADCC. In humans, CD16A has two known allelic variants, CD16A-176V and CD16A-176F, with CD16A-176V having a higher affinity for IgG (∼2–3 fold). In clinical trials with tumor-targeting mAbs, patients homozygous for CD16A-176V have improved outcomes compared with homozygous CD16A-176F or heterozygous patients (36), suggesting that increasing the binding affinity of CD16A for IgG mAbs may improve the efficacy of antibody therapy. One common approach to achieve higher affinity interactions is to modify the Fc region of the therapeutic mAb; however, this requires modification and optimization of each individual antibody. Another approach that has been explored is to increase the Ab binding affinity of CD16A. We have previously generated a recombinant FcγR consisting of the extracellular region of CD64 and the transmembrane and cytoplasmic regions of CD16A to retain its signaling process for NK-cell activation (20). Here, we show that intact CD64 can be expressed in an NK cell line and induce potent ADCC. We further demonstrate that like CD16A, CD64 can utilize the CD3ζ signaling adaptor for ADCC. It is thus conceivable that CD64 could be expressed in T cells as well to induce ADCC, which has been done for CD16A (37). Of note, the alpha chains of CD16A and CD64 differ and mediate distinct signaling mechanisms (38, 39), and therefore it will be interesting to determine in future studies how these regions may differentially signal in NK cells.

Our combination approach introduces what we believe to be a new strategy for treating mCRPC, which, so far, has been largely resistant to immunotherapy. The engineered NK-92MI^CD64 cells showed enhanced effector function against prostate cancer and stromal cell lines in the presence of TROP2- and FAP-targeted antibodies, suggesting the therapeutic potential of NK cell therapies for prostate cancer. Dual targeting of stromal and cancer cells by the combination approach demonstrated potent cytotoxicity in vitro compared with NK cells alone when tested in our prostate tumor stroma model. Using the bound mAb method, CD64 captured the soluble therapeutic mAbs and effectively directed cell killing. Finally, treatment with the engineered NK-92MI^CD64 cells in combination with the therapeutic mAbs led to tumor control and prolonged survival of mice in a subcutaneous xenograft model, further indicating the clinical potential of the combination approach.

A patient's response to immunotherapy depends on a number of factors including tumor heterogeneity, previous treatment history, T-cell infiltration, mutation burden, TME-driven immunosuppression, tissue hypoxia, and density of the stromal network (1). Although prostate cancer has a low tumor mutation burden and is considered to be immunologically “cold” (40, 41), several preclinical studies show that pharmacologic targeting of the TME improves immunotherapy efficacy, suggesting that prostate cancer could be sensitized to immunotherapy (42–44). Targeting both the tumor stroma and prostate cancer cells could be a key strategy to overcome the limitations of current therapeutics given the central role of the TME in survival and progression of the disease. Recent studies demonstrating the high genomic heterogeneity found in patients with prostate cancer indicate the importance of identifying patient-specific molecular signatures to guide immunotherapy strategies (45, 46). The localization and upregulated expression of stromal biomarkers and tumor antigens, including FAP and TROP2, facilitate the development of targeted therapies. FAP is highly expressed by cancer-associated fibroblasts in mCRPC across genomic subtypes and metastatic lesion sites (27) and TROP2 is overexpressed in several epithelial tumors including mCRPC (47).

Several preclinical studies have reported a therapeutic effect from targeting FAP and various tumor antigens using combinations of vaccines, antibody–drug conjugates, and chemotherapies (11, 36, 48). Few studies, however, have used a combination of antigen-targeting cell therapies. Adoptive transfer of a combination of FAP-specific and EphA2-specific CAR T cells enhances antitumor activity and prolongs survival in a lung cancer mouse model (12). In addition, a phase I clinical trial for Nectin4- and FAP-targeted CAR T cells in solid tumors is currently ongoing (ClinicalTrials.gov Identifier: NCT03932565). Our approach is the first to our knowledge that uses engineered NK cells and a combination of antibodies to simultaneously target solid tumor cells and stromal cells. With this strategy, patients can be pretreated with therapeutic antibodies before administration of the engineered cells. In addition, CD64 can also be used as a docking platform where therapeutic antibodies are bound before treatment to direct NK cells to varied tumor antigens. Antibody attachment to CD64 thus has the advantage of dynamic tumor antigen targeting, whereas the FDA-approved CARs are restricted to a specific tumor antigen.

The NK-92 cell platform is a well-characterized human NK cell line that has received US FDA approval for testing in cancer patients. Genetically modified NK-92 cells expressing the CD16A-176V variant (referred to as haNK) are currently being investigated in clinical trials (ClinicalTrials.gov Identifier: NCT03387085, NCT03387111, NCT03586869). Advantages of NK-92 cells include unlimited clonal expansion for achieving clinical doses. However, it will be interesting to engineer other NK-cell platforms with CD64, including peripheral blood NK cells, cord blood NK cells, or induced pluripotent stem cell (iPSC)–derived NK cells that do not require irradiation prior to infusion.

H.M. Hintz reports grants from NIH/NCI during the conduct of the study, as well as a patent for 16/778,977 (compounds binding to fibroblast activation protein alpha and methods of making and using) pending. K.M. Snyder reports grants from Howard Hughes Medical Institute and Burroughs Wellcome Fund during the conduct of the study. J. Wu reports grants from NIH during the conduct of the study. B. Walcheck reports grants from NIH and Fate Therapeutics, and other support from Howard Hughes Medical Institute and Burroughs Wellcome Fund during the conduct of the study, as well as a patent for WO2019084388A1 pending and licensed to Fate Therapeutics. A.M. LeBeau reports a patent for 16/778,977 pending. No disclosures were reported by the other authors.

H.M. Hintz: Data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. K.M. Snyder: Data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. J. Wu: Conceptualization, resources, funding acquisition, methodology, writing–original draft, writing–review and editing. R. Hullsiek: Data curation, writing–review and editing. J.D. Dahlvang: Resources, data curation, formal analysis, writing–review and editing. G.T. Hart: Conceptualization, resources, supervision, funding acquisition, methodology, writing–review and editing. B. Walcheck: Conceptualization, resources, formal analysis, supervision, funding acquisition, methodology, writing-original draft, writing–review and editing. A.M. LeBeau: Conceptualization, resources, formal analysis, supervision, investigation, methodology, writing–original draft, project administration, writing–review and editing.

This work was supported by NIH/NCI T32CA009138 (to H.M. Hintz); a DOD Idea Award W81XWH-19-1-0243 (to A.M. LeBeau); a 2018 Prostate Cancer Foundation Challenge Award (to A.M. LeBeau); a 2013 Prostate Cancer Foundation Young Investigator Award (to A.M. LeBeau); NIH R01s CA237272, CA233562, and CA245922 (to A.M. LeBeau); NIH R01CA203348 (to B. Walcheck); NIH R21AI149395 (to J. Wu); a Howard Hughes Medical Institute and Burroughs Wellcome Fund Medical Research Fellowship (to K.M. Snyder); and a University of Minnesota Medical School intramural grant for studying CRISPR in NK cells (to G.T. Hart). The authors also thank Colleen Forster and the Biorepository and Laboratory Services Division at the University of Minnesota for their histology and pathology support.

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