NK cell tumor infiltration is associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). NK cells recognize and kill targets by a process called natural cytotoxicity. We hypothesized that promoting an antigen-specific synapse with coactivation may enhance NK cell function in mCRPC. We describe a tri-specific killer engager (TriKE) construct that engages with the activating receptor CD16 on NK cells and prostate-specific membrane antigen (PSMA) on mCRPC cells and has an IL15 moiety that is essential for NK cell survival, proliferation, and priming. We show that the PSMA TriKE specifically binds to PSMA-expressing cells and significantly enhances expansion, degranulation, and cytokine production of NK cells derived from healthy donors or patients with prostate cancer. Bystander killing of PSMA-negative tumor cells was also achieved with PSMA TriKE treatment when cocultured with PSMA-positive cells, suggesting potential PSMA TriKE benefit in controlling tumor antigen escape. When tested under physiologic conditions recapitulating the mCRPC tumor microenvironment, NK cells treated with PSMA TriKE and prolonged exposure to hypoxia or myeloid-derived suppressor cells maintained their potent function whereas IL15-treated NK cells showed greatly impaired cytotoxicity. Finally, in vivo testing of PSMA TriKE showed improved tumor control and survival of mice as compared with IL15-treated and untreated control groups. In conclusion, PSMA TriKE demonstrates potential as a new therapy for advanced prostate cancer by providing additional signals to NK cells to maximize their antitumor potential in prostate cancer, especially in the setting of a hostile tumor microenvironment.

Supplemental Table 1 and Supplemental Figures 1-5

155 Background: Recombinant human (rh) IL-15, the homeostatic factor for natural killer (NK) cells, is being clinically developed, but it has little antitumor activity alone. B7H3 (CD276) is an immune checkpoint inhibitor that is associated with poorer prognosis and is highly-expressed on prostate cancer. NK cells can be given as allogeneic products and, unlike T cells, do not induce cytokine-release syndrome or neurotoxicity. Here we developed a B7H3 targeting Tri-Specific Killer Engager (TriKE) as a novel dual camelid (cam) TriKE containing WT IL-15 and two cam engagers targeting CD16 on NK cells and B7H3 on tumor targets, making NK cells antigen specific. We have previously demonstrated that NK cells infiltrate prostate cancer tumors. As proof of concept, a clinical trial of a CD33-targeted TriKE for AML (NCT03214666) induced endogenous NK cell expansion and activation in refractory AML patients. Methods: Prostate cancer cell lines or patient-derived xenografts (PDX) were incubated with healthy donor or prostate cancer patient NK cells with or without B7H3 TriKE. PDX were propagated in NSG mice and then homogenized for in vitro assays. NK cell function was measured by flow cytometry and IncuCyte live tumor imaging assays. Castration-sensitive and -resistant prostate cancer (CSPC and CRPC, respectively) patient or normal donor peripheral blood mononuclear cells (PBMC) were immunophenotyped using 42-marker NK specific or broad immune cytometry time-of-flight (CyToF) panels. Results: B7H3 TriKE resulted in a dose-dependent proliferation of NK cells, but not T cells. This was in marked contrast to rhIL-15, which stimulated both cell types. camB7H3 was broadly expressed on prostate, head and neck, ovarian and glioblastoma cancers as well as multiple myeloma. We observed a B7H3 TriKE dose-dependent increase in CD107a degranulation and inflammatory cytokines to all B7H3 positive targets that was highly specific, with no response seen with B7H3 negative hematologic targets and CRISPR KO controls. Compared to rhIL-15, B7H3 TriKE given at molar equivalent dosing induced B7H3+ target killing in in a dose-dependent manner above that seen with rhIL-15 induced natural cytotoxicity. Using CSPC and CRPC patient PBMC (n=11-15), we demonstrated that there is no significant loss in NK cell degranulation/interferon gamma production or target cytotoxicity compared to healthy age- and sex-matched donors when treated with B7H3 TriKE. CyToF analysis of CSPC and CRPC patient PBMC is ongoing. In vivo activity in xenogeneic models of human tumor is underway. Conclusions: B7H3 TriKE delivers an NK cell specific IL-15 signal to expand NK cells and is highly specific against B7H3+ prostate cancer cell lines and PDX. Clinical-grade B7H3 TriKE is undergoing validation and a Phase 1/2 clinical trial is planned to open in the 3rd Quarter of 2024 for CRPC patients progressing on one or more therapies the CRPC setting.

Abstract With an annual incidence rate exceeding 660,000 cases, and a death toll surpassing 325,000 per year, head and neck cancer (HNC) ranks as the seventh most common cancer in the world. Surgery, radiation and chemotherapy are used to treat HNC patients with modest and variable clinical success. However, these treatments prove less effective for human papillomavirus negative (HPV-) HNC patients, a subset of HNC patients with markedly worse prognosis. While significant advancements have been made in cancer immunotherapy over the past decade, its success also remains elusive for HNC due to factors such as the hypoxic solid tumor microenvironment (TME). To address the critical need for an improved therapeutic intervention, we leveraged the ability of natural killer (NK) cells in killing cancer cells without prior sensitization by developing a novel tri-specific killer engager (TriKE) that can direct NK cell killing of tumor within the hypoxic solid TME. The TriKE is composed of three domains: a humanized nanobody arm binding the activating receptor CD16 on NK cells, an interleukin (IL)-15 moiety that can drive expansion of NK cells, and a nanobody arm binding B7H3, a protein which high expression can be negatively correlated with overall survival of HPV- HNC patients. B7H3 is a prime target candidate because it is highly expressed on HPV- HNC cells, but minimally expressed on healthy tissues. In vitro testing using HPV- HNC patient blood samples revealed that B7H3 TriKE enhances activation (measured by NK cell degranulation and interferon-gamma production) and expansion of NK cells from these patients at levels equivalent to those observed in healthy controls. Furthermore, B7H3 TriKE is efficacious in its ability to drive high NK cell cytotoxicity in both acute (<48-hours) and prolonged (7-days) hypoxic (1% oxygen) models of HNC. Under acute hypoxia, B7H3 TriKE induces significantly more killing of targets by NK cells compared to IL-15 treatment, where NK cell cytotoxicity is impaired. In addition, B7H3 TriKE can boost the killing efficacy of NK cells exposed to prolonged hypoxia, surpassing limitations seen with IL-15 treatment. These findings strongly suggest that the B7H3 TriKE can bypass hypoxic suppression of NK effector functions in the solid TME. In vivo studies using immunocompromised mice engrafted with HPV- HNC cells revealed that B7H3 TriKE treatment significantly extends the survival of mice, compared to IL-15 treatment. Moreover, NK cells persisted in the blood of B7H3 TriKE-treated mice 28 days post-NK cell injection, highlighting the promising clinical translation of this immunotherapy. More in-depth characterization of NK cells from HPV- HNC patients are underway but altogether, these robust pre-clinical data present a novel avenue for the management of HNC for these patients. We plan to translate results from these studies to clinical trials in fall 2024. Citation Format: Melissa Khaw, Nicholas A. Zorko, Carly Selleck, Laura Bendzick, Zachary Davis, Peter Hinderlie, Madison Shackelford, Ann Lu, James Lim, Naomi Fujioka, Margaret MacMillan, John Wagner, Martin Felices, Jeffrey S. Miller. Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1241.

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rβ when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/−) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/− iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/− HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.

Many tumors exist in vivo as three-dimensional masses. In order to better model the dynamics of three-dimensional tumor growth and immune cell invasion, cancer cell lines are grown in low-adhesion plates that force them to interact with each other, forming a spheroid. After spheroids form in culture, immune effectors and drugs are added to the wells and their effect on the tumor mass is measured by transmitted light or fluorescence microscopy in a live-cell image system (IncuCyte, Sartorius Inc).

New treatments are required to enhance current therapies for lung cancer. Mesothelin is a surface protein overexpressed in non-small cell lung cancer (NSCLC) that shows promise as an immunotherapeutic target in phase I clinical trials. However, the immunosuppressive environment in NSCLC may limit efficacy of these therapies. We applied time-of-flight mass cytometry to examine the state of circulating mononuclear cells in fourteen patients undergoing treatment for unresectable lung cancer. Six patients had earlier stage NSCLC (I-IVA) and eight had highly advanced NSCLC (IVB). The advanced NSCLC patients relapsed with greater frequency than the earlier stage patients. Before treatment, patients with very advanced NSCLC had a greater proportion of CD14- myeloid cells than patients with earlier NSCLC. These patients also had fewer circulating natural killer (NK) cells bearing an Fc receptor, CD16, which is crucial to antibody-dependent cellular cytotoxicity. We designed a high affinity tri-specific killer engager (TriKE®) to enhance NK cytotoxicity against mesothelin+ targets in this environment. The TriKE consisted of CD16 and mesothelin binding elements linked together by IL-15. TriKE enhanced proliferation of lung cancer patient NK cells in vitro. Lung cancer lines are refractory to NK cell killing, but the TriKE enhanced cytotoxicity and cytokine production by patient NK cells when challenged with tumor. Importantly, TriKE triggered NK cell responses from patients at all stages of disease and treatment, suggesting TriKE can enhance current therapies. These pre-clinical studies suggest mesothelin-targeted TriKE has the potential to overcome the immunosuppressive environment of NSCLC to treat disease.

BackgroundNatural killer (NK) cells are being leveraged in the clinic due to their safety profile and their ability to mediate tumor killing without prior priming. However, lack of antigen-specific targeting, decreased numbers, and suppressive signals derived from the tumor microenvironment (TME) of Prostate Cancer (PCa), can impact NK cell efficacy. To bypass this issue, we designed a novel tri-specific killer engager (TriKE®) molecule that consists of three parts: an arm that engages with CD16, an activating receptor of NK cells, an arm that binds to tumor antigens express in PCa (PSMA or B7H3), and an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, priming and motility (figure 1A).MethodsTriKE molecules were generated in mammalian (Expi293) manufacturing systems. Peripheral Blood Mononuclear Cells (PBMCs) from normal donors or prostate cancer patients were either used directly or magnetically enriched for NK cells in assays co-culturing cells with PCa cell lines in the presence or absence of PSMA TriKE or B7H3 TriKE. Flow cytometry-based readouts evaluated NK cell activation and tumor killing while impedance and imaging assays were also used to dynamically measure tumor killing. Hypoxic (1% oxygen) culture condition and cytokine-induced myeloid-derived suppressor cells (MDSC) were also incorporated in some assays. Pre-clinical xenogeneic mouse models were also used to evaluate efficacy of TriKEs in vivo.ResultNormal donor and PCa patient NK cells display better, specific, degranulation against PCa cell lines in the presence of PSMA (figure 1B) or B7H3 TriKEs. NK cell cytotoxicity is also improved, even in the presence of enzalutamide resistant lines (figure 1C), hypoxia (figure 1D), or Myeloid Derived Suppressor Cells. Finally, the TriKE molecules display improved tumor control, compared to IL-15 control or no treatment, in xenogeneic models of prostate cancer.ConclusionsOur findings indicate that TriKE molecules improve PCa control in several systems and in the presence of varied TME-specific stresses. These pre-clinical studies highlight the potential for using TriKE molecules in the setting of metastatic Prostate Cancer and pave the way for future, targeted, NK cell immunotherapeutic interventions in this setting.AcknowledgementsThis work was carried out using funds from DoD grant W81XWH-20–1-0659. We would like to acknowledge the Translational Therapy Laboratory (TTL), the Data and Biospecimen Utilization Committee (DBUC), and the Clinical Informatics Shared Services (CISS) at the University of Minnesota for their excellent service in helping access de-identified Prostate Cancer patient samples from the Solid Tumor Cancer Specimen Bank (2016NTLS035).Ethics ApprovalPeripheral blood mononuclear cells (PBMCs) from the blood of deidentified healthy donors were obtained, after participants gave informed consent, from Memorial Blood Centers (Minneapolis, Minnesota, USA) and used in compliance with the Committee on the Use of Human Subjects in Research (IRB# 9709 M00134) and in accordance with the Declaration of Helsinki. The in vivo mouse studies were conducted in accordance with the Institutional Animal Care and Use Committee at the University of Minnesota (IACUC# 1908–37330A).

Abstract Immune checkpoint blockade (ICB) has changed the standard of care for many patients with cancer, yet no ICB is approved for ovarian cancer. We hypothesized that maintenance therapy with an IL15 “superagonist” (N-803) and ICB in combination could induce potent immune activation in ovarian cancer. Using flow cytometry, cytometry by time of flight analysis, and cytotoxicity assays, we analyzed patient samples from women with advanced epithelial ovarian cancer treated with N-803 for indications of PD-1/PD-L1 upregulation with this treatment. In addition, ICB and N-803 were evaluated in preclinical studies to determine the functional impact of combination therapy on natural killer (NK) cells in vitro and in vivo. We observed that N-803 stimulated initial NK-cell expansion in patient samples; however, proliferation was not sustained beyond 2 weeks despite continued treatment. This result was reverse translated back to the laboratory to determine the functional relevance of this finding. The addition of ICB with an antibody-dependent cellular cytotoxicity IgG1 antibody against PD-L1 (avelumab) or an IgG4 antibody against PD-1 (pembrolizumab) enhanced N-803 induced NK-cell function in vitro. Using models of human ovarian cancer and NK-cell adoptive transfer in mice, we showed enhanced antitumor control with N-803 and ICB, as well as a combination effect that enhanced NK-cell persistence and expansion in vivo. This work suggests that PD-1/PD-L1 blockade combined with IL15 signaling may overcome resistance to cytokine therapy in ovarian cancer.

Background Acute myeloid leukemia (AML) incidence increases with age. Five year survival for those over 65 is less than 11%, highlighting the need for safe interventions to improve outcomes. Adoptive natural killer (NK) cell products have achieved success as a 'bridge to transplant' in refractory leukemia and lymphoma, inducing remission to a point where patients are eligible for stem cell transplantation. Multiplexed-engineered induced pluripotent stem cells (iPSCs) are a reproducible source of highly functional NK cells (iNK) for on-demand treatment and broad patient access. Clinical trials are currently testing iNK cells with therapeutic antibodies for the treatment of leukemia and lymphoma (NCT04023071, NCT04614636, NCT04714372). We have developed a protocol for the production of highly functional iNK cells, engineered for greater anti-tumor effect. However, we hypothesized that performing NK cell lineage commitment under physiological oxygen conditions found in bone marrow (5%) would create a niche that could support the generation of a more functional cell product. Methods iPSCs are matured into CD34+ precursors, then differentiated into iNK cells that are subsequently expanded to clinically-relevant quantities (figure 1A). We have previously published on iNK cells that consist of three unique edits: high-affinity non-cleavable CD16, membrane bound IL-15 and knockout of CD38. Using these cells, we performed stage specific differentiation from CD34+ precursors in 5% oxygen ('physoxic iNK') or conventional 20% oxygen, with subsequent expansion in 20% oxygen. iNK cells were compared for their phenotype (CyTOF), proliferation (flow cytometry), cytotoxicity (live cell imaging), metabolic stability (reactive oxygen species staining by flow cytometry) and ability to control tumor (xenograft mouse model). Results CyTOF analysis revealed a more naive phenotype in physiological oxygen conditions that persisted after expansion. However, these cells were equally capable of natural cytotoxicity and antibody-dependent cellular cytotoxicity. In a xenograft model of AML (NSG mice with HL60-GFP/luciferase; figure 1B) there was greater persistence of physoxic iNK cells in blood and bone marrow (figure 1C), correlating with greater tumor control within the bone marrow (figure 1D) and across the whole animal (figure 1E). When exposed to oxidative stress, physoxic iNK cells were more resilient, with lower reactive oxygen species detected in their mitochondria, suggesting greater tumor control arose from greater persistence within the animal, rather than better cytotoxicity. Conclusions These data suggest that manufacturing therapeutic NK cells in a physiological environment at a unique stage of lineage commitment can generate resilient cells with a greater durability for anti-tumor activity. Acknowledgements This project was supported by grants from the Department of Defense (CA200922) and National Institutes of Health (R35 CA197292, P01 CA111412). Parts of the figure were drawn using images from Servier Medical Art and from Biorender. Ethics Approval The University of Minnesota human research protection program determined this was not human research (institutional review board ID: STUDY00013106)

Immunotherapies for solid tumors need to overcome barriers within the solid tumor microenvironment (TME), including stromal cells, cancer-associated fibroblasts, endothelial cells, pericytes, and immune cells. These can comprise a significant portion of the tumor mass in many common carcinomas and contribute to immune cell dysfunction within the tumor. Stromal cells support cancer cells through several mechanisms and can promote metastasis. Tumor vasculature supplies cancer cells with nutrients and oxygen necessary for tumor growth and dissemination. Thus, targeting these components of cancer can enhance tumor rejection directly and increase sensitization of surviving tumors to immune infiltration and chemotherapeutic treatments through weakening of the tumor architecture. While there are current antiangiogenic therapies being tested clinically, they have associated toxicities in normal angiogenic processes, limiting clinical efficacy. Tumor Endothelial Marker 8 (TEM8, encoded by the ANTXR1 gene) is a highly conserved integrin-like adhesion molecule that was discovered on the endothelium of colorectal cancer, and subsequent studies identified its expression on multiple stromal cells—endothelial cells, fibroblasts, and pericytes—in the tumor microenvironment of diverse human cancer types. Little to no expression has been demonstrated in normal human stroma, indicating that this would be a good target for immunotherapy. Using a mammalian expression system, we designed and expressed a TEM8 targeting TriKE (cam1615TEM8) consisting of a humanized anti-CD16 single domain antibody, a wild-type IL-15 moiety, and an anti-TEM8 scFv. cam1615TEM8 induces NK cell degranulation and cytokine production against TEM8+ tumor and stromal cell lines (endothelial cells and fibroblasts). Using TEM8- cancer lines and a TEM8-CRISPR knockout version of A549 lung cancer lines, we show that cam1615TEM8 only activates NK cells in the presence of tumor antigen. cam1615TEM8 also preferentially stimulates TEM8+ tumor spheroid killing. Moreover, the camelid anti-CD16 nanobody selectively enhances signaling by the IL-15 moiety to NK cells, specifically promoting NK cell survival and proliferation in vitro and in vivo. In an in vivo xenogeneic mouse model, containing human NK cells and tumor, cam1615TEM8-treated mice had enhanced NK cell tumor infiltration, significantly decreased tumor growth, and enhanced survival when compared to IL-15 treated mice. Since TEM8 is highly conserved, and the scFv within the TriKE is reactive to mouse TEM8, we were able to show reduced tumor endothelial density in the tumors of mice treated with cam1615TEM8. Given the findings of this study cam1615TEM8 could be leveraged in several clinical settings by inducing NK cell specific targeting of the tumor itself and/or the tumor endothelium and stroma. Citation Format: Martin Felices, Michael Kaminski, Peter Hinderlie, Rachel Hopps, Laura Bendzick, Melissa Geller, Jeffrey S. Miller. Mediating NK cell function against solid tumors, via targeting of tumor stroma, using a TEM8-targeting Tri-specific Killer Engager (TriKE®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2878.

NK cell exhaustion is caused by chronic exposure to activating stimuli during viral infection, tumorigenesis, and prolonged cytokine treatment. Evidence suggests that exhaustion may play a role in disease progression. However, relative to T cell exhaustion, the mechanisms underlying NK cell exhaustion and methods of reversing it are poorly understood. Here, we describe a potentially novel in vitro model of exhaustion that uses plate-bound agonists of the NK cell activating receptors NKp46 and NKG2D to induce canonical exhaustion phenotypes. In this model, prolonged activation resulted in downregulation of activating receptors, upregulation of checkpoint markers, decreased cytokine production and cytotoxicity in vitro, weakened glycolytic capacity, and decreased persistence, function, and tumor control in vivo. Furthermore, we discovered a beneficial effect of NK cell inhibitory receptor signaling during exhaustion. By simultaneously engaging the inhibitory receptor NKG2A during activation in our model, cytokine production and cytotoxicity defects were mitigated, suggesting that balancing positive and negative signals integrated by effector NK cells can be beneficial for antitumor immunity. Together, these data uncover some of the mechanisms underlying NK cell exhaustion in humans and establish our in vitro model as a valuable tool for studying the processes regulating exhaustion.

Background The tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to cure because of tumor-promoting stromal cells, but current therapies targeting tumor stromal cells are constrained by modest efficacy and toxicities. TEM8 is a surface antigen selectively upregulated on tumor and tumor stromal cells, endothelial cells and fibroblasts that may be targeted with specific natural killer (NK) cell engagement. Methods A Tri-specific Killer Engager (TriKE) against TEM8-'cam1615TEM8'-was generated using a mammalian expression system. Its function on NK cells was assessed by evaluation of degranulation, inflammatory cytokine production, and killing against tumor and stroma cell lines in standard co-culture and spheroid assays. cam1615TEM8-mediated proliferation and STAT5 phosphorylation in NK cells was tested and compared with T cells by flow cytometry. NK cell proliferation, tumor infiltration, and tumor and tumor-endothelium killing by cam1615TEM8 and interleukin-15 (IL-15) were assessed in NOD scid gamma (NSG) mice. Results cam1615TEM8 selectively stimulates NK cell degranulation and inflammatory cytokine production against TEM8-expressing tumor and stromal cell lines. The increased activation translated to superior NK cell killing of TEM8-expressing tumor spheroids. cam1615TEM8 selectively stimulated NK cell but not T cell proliferation in vitro and enhanced NK cell proliferation, survival, and tumor infiltration in vivo. Finally, cam1615TEM8 stimulated NK cell killing of tumor and tumor endothelial cells in vivo. Conclusions Our findings indicate that the cam1615TEM8 TriKE is a novel anti-tumor, anti-stroma, and anti-angiogenic cancer therapy for patients with solid tumors. This multifunctional molecule works by selectively targeting and activating NK cells by costimulation with IL-15, and then targeting that activity to TEM8+ tumor cells and TEM8+ tumor stroma.

While acute myeloid leukemia (AML) treatment has improved, relapse is still common, even after reduced intensity conditioning allogeneic transplantation. Natural killer (NK) cells are a promising allogeneic cell immunotherapy without the risk of graft-versus-host disease or cytokine release syndrome. We and others have reported that haploidentical NK cells can safely result in 30-50% complete remissions in advanced AML patients when given with high-dose lymphodepleting chemotherapy and exogenous cytokine support. However, while NK cell natural cytotoxicity has a clinical signal in AML, increased specificity and multi-dosing are likely to enhance the efficacy and durability of this treatment strategy. A number of stress ligands, including the MHC-I polypeptide-related sequence A and B (MICA/B), bind to the NK cell activating receptor NKG2D and initiate NK cell killing of transformed and infected cells, and several groups have shown promising results in clinical trials by developing NK cells that carry a NKG2D chimeric antigen receptor (CAR). However, the distal α1 and α2 domains of MICA/B that are recognized by NKG2D can be proteolytically cleaved from the surface of the tumor by metalloproteases, allowing for escape from NK cell-mediated detection and elimination. Wucherpfennig and colleagues have described that the membrane proximal α3 domain remains on the cell surface and may be a potential target for immunotherapy across many cancers, including AML. However, since AML is heterogenous, we hypothesized that clinical success would require dual-targeting and selected CD33 as the ideal complementary targeting approach since CD33 is a validated marker expressed on greater than 80% of AML blasts. To this end, our lab has developed a Tri-specific Killer Engager (TriKE) capable of agonistically ligating the CD16 Fc receptor to CD33 found on AML along with IL-15 co-stimulation to further activate the NK cell response. Here, we demonstrate the efficacy of a multiplexed-gene edited induced pluripotent stem-cell (iPSC) derived NK cell (iNK) product that expresses a high-affinity, non-cleavable version of CD16 (hnCD16), a membrane-bound IL-15 fusion receptor, CD38 knockout to enhance metabolic fitness, and a CAR against the α3 domain of MICA/B (α3 MICA/B) to drive a potent response against AML alone and when combined with anti-CD33 TriKE. In the initial study, the iNK cell backbone (iNK cells without the α3 MICA/B CAR) induced potent activity against the AML cell line HL60, and displayed further enhancement of activity with the addition of anti-CD33 TriKE (GTB-3650), representing combined effects of natural cytotoxicity and antibody-dependent cellular cytotoxicity. To show specificity for α3 MICA/B targeting, the AML cell line THP-1 was stained for the presence of MICA/B by flow cytometry using the 6D4 clone that recognizes the α1/2 domains of MICA/B or the 7C6 clone that uniquely recognizes the α3 domain +/- proteolytic cleavage with trypsin (Fig. 1A-B). Significant expression of the α3 domain was observed on THP-1 cells using the 7C6 antibody, which was highly expressed even after protease treatment. On the other hand, the α1/2 domains of MICA/B were detectable at lower levels using the 6D4 antibody and were undetectable after protease treatment (Fig. 1A-B). To assess a dual targeting approach, THP-1 cells were used as targets in live imaging functional assays under standard conditions (Effector:Target [E:T] 2:1). The iNK cell backbone had modest natural cytotoxicity at 3 hours that was significantly enhanced by the addition of α3 MICA/B CAR. The effect was further improved with more rapid killing kinetics when combined with the anti-CD33 TriKE (Fig. 1C). To evaluate the efficacy of dual-targeting in a more stringent physiologic manner, we mimicked "stress” conditions by minimizing the E:T ratio to 0.25:1. While all effector conditions induced immediate killing in 4 hours at the low E:T ratio, sustained tumor control was only observed with dual-antigen targeting (Fig. 1D). Studies with primary AML targets, +/- preincubation of decitabine and all-trans retinoic acid, known to upregulate NKG2D ligands in AML, are in progress and will be discussed. In summary, dual-targeting strategies using off-the-shelf CAR NK cells targeting α3 MICA/B in combination with antigen-specific TriKE targeting CD33 represent an ideal clinical strategy to enhance efficacy and durability of treatment in advanced AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression of programmed cell death protein 1 (PD-1) on natural killer (NK) cells has been difficult to analyze on human NK cells. By testing commercial clones and novel anti-PD-1 reagents, we found expression of functional PD-1 on resting human NK cells in healthy individuals and reconstituting NK cells early after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Peripheral blood samples from healthy individuals and transplant recipients were stained for PD-1 expression using the commercial anti-PD-1 clone PD1.3.1.3, fluorescein isothiocyanate (FITC)-labeled pembrolizumab, or an FITC-labeled single-chain variable fragment (scFv) reagent made from pembrolizumab. These reagents identified low yet consistent basal PD-1 expression on resting NK cells, a finding verified by finding lower PD-1 transcripts in sorted NK cells compared with those in resting or activated T cells. An increase in PD-1 expression was identified on paired resting NK cells after allo-HSCT. Blockade of PD-1 on resting NK cells from healthy donors with pembrolizumab did not enhance NK function against programmed death-ligand 1 (PD-L1)-expressing tumor lines, but blocking with its scFv derivative resulted in a twofold increase in NK cell degranulation and up to a fourfold increase in cytokine production. In support of this mechanism, PD-L1 overexpression of K562 targets suppressed NK cell function. Interleukin-15 (IL-15) activity was potent and could not be further enhanced by PD-1 blockade. A similar increase in function was observed with scFv PD-1 blockade on resting blood NK cells after allo-HSCT. We identify the functional importance of the PD-1/PD-L1 axis on human NK cells in which blockade or activation to overcome inhibition will enhance NK cell-mediated antitumor control.

Clinical studies validated antibodies directed against HER2, trastuzumab, and pertuzumab, as useful methodology to target breast cancer cases where HER2 is expressed. The hope was that HER2 targeting using these antibodies in ovarian cancer patients would prove useful as well, but clinical studies have shown lackluster results in this setting, indicating a need for a more comprehensive approach. Immunotherapy approaches stimulating the innate immune system show great promise, although enhancing natural killer (NK) function is not an established mainstream immunotherapy. This study focused on a new nanobody platform technology in which the bispecific antibody was altered to incorporate a cytokine. Herein we describe bioengineered CAM1615HER2 consisting of a camelid VHH antibody fragment recognizing CD16 and a single chain variable fragment (scFv) recognizing HER2 cross-linked by the human interleukin-15 (IL-15) cytokine. This tri-specific killer engager (TriKETM) showed in vitro prowess in its ability to kill ovarian cancer human cell lines. In addition, we demonstrated its efficacy in inducing potent anti-cancer effects in an in vivo xenograft model of human ovarian cancer engrafting both cancer cells and human NK cells. While previous approaches with trastuzumab and pertuzumab faltered in ovarian cancer, the hope is incorporating targeting and cytokine priming within the same molecule will enhance efficacy in this setting.

For Natural Killer (NK) cell-based immunotherapy to succeed against solid tumors, NK cells need to enter a highly immunosuppressive tumor microenvironment (TME) and retain functionality. The objective of this study is to evaluate how hypoxia exerts an immunosuppressive effect on NK cells. We used the novel AVATAR™ system to model oxygen levels of three key tissues that NK cells inhabit in vivo: the peripheral blood (12% O2), the bone marrow (5% O2) and the TME (1% O2). NK cells incubated at 1% O2 have decreased proliferation and cytotoxicity compared to NK cells incubated at higher oxygen conditions. To assess what contributes to these changes, we conducted a mass cytometry (CyTOF) analysis, real time imaging assays, metabolic assays and gene expression (RNA-seq and ATAC-seq) analysis. We observed oxygen dependent changes in expression of activating receptors, Ki-67, perforin and granzyme. Hypoxia impacts aggregation of perforin and granzyme granules at the immune synapse. Under hypoxic conditions, NK cell metabolism resembles cancer cell metabolism with increased glycolysis, amino acid synthesis and central carbon metabolism. These changes are accompanied by mitochondrial defects. Gene expression analysis revealed that changes in NK cell metabolism and function are mediated at the epigenetic level by histone demethylases. Treatment with histone demethylase inhibitors rescued NK cell cytotoxicity as measured using an IncuCyte machine. These results indicate that NK cells who enter the TME are fundamentally different than those in the bone marrow or blood stream. The insights gained from this study will be leveraged to overcome hypoxia induced immune suppression in the TME to enhance NK cell-based immunotherapy for solid tumors.