Abstract In this study, feeder cell-free PM21 particle activation of Natural Killer (NK) cells was combined with cytokine activation and TGF-β conditioning to produce highly proliferative and cytotoxic NK cells that can infiltrate solid tumors. Development of NK cell therapeutics is a promising anti-cancer therapy. For clinical applications, NK cells are typically either ex vivo activated with cytokines or expanded with feeder cells or by feeder cell-free methods. One such method for feeder cell-free expansion of highly cytotoxic NK cells uses plasma membrane particles containing surface IL-21 and 41BBL (PM21). Additionally, NK cells expanded with IL-12, IL-15, and IL-18 in combination with PM21 stimulation (CAP-NK cells) exhibit robust proliferation, potent cytotoxicity, and memory-like features. To further improve their activity against solid tumors—a setting often resistant to NK cells—we introduced TGF-β conditioning. Previous studies have shown that ex vivo TGF-β conditioning induces IFNγ hypersecretion. To assess whether this could enhance anti-tumor function, PM21- and CAP-NK cells were expanded in the presence of TGF-β. The CAP-based expansion method still resulted in enhanced NK-cell expansion, even with TGF-β conditioning. CAP-based expansion remained highly effective even with TGF-β conditioning, achieving an average 4000±700-fold expansion by day 14 compared to 1800±60-fold for PM21-NK cells. TGF-β conditioned NK cells retained high viability after cryopreservation (>80% immediately and at 16 h post-thaw) and 99±1% expressed CD25 by day 7. These cells produced significantly more IFNγ upon stimulation than unconditioned CAP-NK cells. Moreover, TGF-β conditioned CAP-NK cells acquired a tissue-resident-like phenotype (CD103+, CD49a+, CD300a−) not observed in PM21- or CAP-NK cells, which would be expected to enhance their ability to infiltrate tumors. To test this, labeled TGF-β conditioned CAP-NK and PM21-NK cells were co-cultured with large lung tumor spheroids and monitored via live-cell imaging. TGF-β conditioned NK cells penetrated tumors faster and deeper than PM21-NK cells, which remained mostly at the periphery, resulting in greater spheroid killing—even in tumors overexpressing TGF-β.TGF-β conditioning also enhanced cytotoxicity against multiple solid tumor cell line spheroids, including lung, pancreatic, and neuroblastoma. Overall, these findings support TGF-β conditioned CAP-NK cells as a potent cellular therapy candidate for solid tumors. Citation Format: Jeremiah L. Oyer, Tayler J. Croom-Perez, Javier A. Rivera-Huertas, Brian P. Tullius, Alicja J. Copik. Combination of cytokines, PM21-particle stimulation, and TGF-β conditioning results in Natural Killer cells with enhanced cytotoxicity and infiltration of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 151.

Natural killer (NK) cell adoptive immunotherapy is a promising therapeutic approach in which NK cells perform targeted lysis of tumor cells. Oncolytic viruses are also effective cancer therapeutic agents due to their ability to selectively target and kill tumor cells. Combination therapies that integrate NK cells and oncolytic viruses have been shown to enhance tumor killing compared to individual treatment strategies alone. Using in vitro expanded human NK cells (PM21-NK cells), we tested the relative ability of tumor cells infected with WT parainfluenza virus 5 (PIV5), which is a poor inducer of type 1 interferon (IFN-I), versus PIV5 P/V gene mutant, which is a strong inducer of IFN-I synthesis, to modulate NK cell activities. Both WT and P/V mutant viruses were capable of infecting PM21-NK cells and caused extensive cytopathic effects. Co-culturing of PM21-NK cells with virus-infected tumor cells resulted in spread of WT PIV5 to naïve NK cells, but NK cells were protected from spread of the P/V mutant virus by IFN-I induction. Direct treatment of PM21-NK cells with IFN-I or media from P/V-virus-infected tumor cells enhanced NK cell cytotoxicity, at least in part due to upregulation of the death ligand, TRAIL. IFN-I-treated PM21-NK cells also showed a decrease in IFN-γ secretion, a cytokine we have previously shown to reduce PM21-NK cell tumor killing. Our results highlight multiple mechanisms by which an IFN-I-inducing oncolytic virus can enhance NK-cell-mediated killing of target virus-infected and uninfected tumor cells.

NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both in vitro and in vivo. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications.

Background: Pembrolizumab has recently emerged as a PD-1 blockade immunotherapy treatment for lung cancer. It is critical that such treatment strategies for lung cancer should be chosen not only on the basis of histopathological features and the expression of targetable cell surface proteins (such as PD-1), but should rather be selected based on other determinants of treatment success or risk factors for poor prognosis. One method to forecast cancer trajectory is the identification of biomolecular signatures such as microRNAs (miRNAs), non-protein-coding RNA molecules that play a regulatory role in gene expression by modulating the translation or stability of messenger RNA. Methods: To find out which miRNAs have an important influence on anti-PD-1 treatment outcomes, we evaluated miRNA levels in sera from 38 lung cancer patients undergoing 3 months of pembrolizumab treatment. We selected a panel of miRNAs previously shown to be involved in lung cancer or PD-1 signaling and performed qPCR analysis. Results: Overall, we observed a significant decrease in the levels of miR126-5p (4-fold), let-7a (5-fold), miR133a-3p (4-fold), miR3615 (2-fold), miR4516 (3-fold), miR16 (3-fold), miR34c-5p (2-fold), miR20b-5p (5-fold), miR106b-5p (5-fold), miR146a-5p (3-fold) and miR181b-5p (3-fold) in response to treatment indicating effectiveness of immunotherapy. Within our selected panel of miRNAs, we identified two markers relevant to cancer prognosis: miR-217, which is negatively associated with patient survival, and let-7a, which is positively associated with patient survival. Conclusions: Our findings suggest that circulating miRNAs can be used for future treatment evaluation and lung cancer prognosis, with potential as therapeutic targets.

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells.

In this study we propose to combine feeder cell-free PM21 particle activation of Natural Killer (NK) cells with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce highly proliferative, persistent memory-like NK cells with great therapeutic potential. Development of NK cell therapeutics has risen to prominence in recent years for use as potential anti-cancer therapy. For clinical applications, NK cells are typically either ex vivo activated with cytokines or expanded with feeder cells or by feeder cell-free methods. One such method for expansion uses plasma membrane particles containing surface IL-21 and 41BBL (PM21) that allows for feeder cell-free expansion of highly cytotoxic NK cells. Activation of NK cells with IL-12, IL-15, and IL-18 has been shown to induce memory-like properties in NK cells with enhanced proliferative potential and persistence. In this study, use of cytokines with PM21 particles for NK cell expansion (CAP-NK cells) led to an increase from an average 2,400-fold expansion with PM21-particles alone (PM21-NK cells) to 13,600-fold in 14 days (p<0.01) when grown in flasks. Using G-Rex® Well Plates, a scalable clinically relevant platform, CAP-NK cells could be expanded an average 33-fold in just 7 days compared to 6-fold for PM21-NK cells (p<0.005). CAP NK cells had enhanced proliferation, decreased doubling time, and an increase in the percent of CD25+41BB+ NK cells (14% of PM21-NK cells vs 82% of CAP-NK cells; p<0.0001). CAP-NK cells also demonstrated higher levels of basal and compensatory glycolysis and increased maximal respiration compared to PM21-NK cells. CAP-NK cells demonstrated enhanced effector functions; they were more cytotoxic against ovarian (SKOV-3) and lung (A549) cancer cells compared to PM21-NK cells (SKOV-3: 58% vs 42%, p=0.0002; A549: 77% vs 65% at 24 h at 1:1 E:T, p=0.001) and had increased IFNγ production after stimulation with cytokines or K562 cells. Importantly, CAP-NK cells also demonstrated memory-like properties. After expansion for 14 days, NK cells were cold-washed and rested for 7 days with 1 ng/mL IL-15. Rested CAP-NK cells had increased IFNγ production when re-stimulated with IL-12, IL-15 and IL-18 (MFI 5353 vs 3679; p<0.01) and higher potency against K562 (EC50 0.58 vs 1.17; p<0.05) as compared to rested PM21-NK cells. Finally, cryopreserved CAP-NK cells showed improved persistence 21 days after i.p. injection with no exogenous cytokine support in NSG mice. Additionally, CAP-NK cells could home to and control tumor growth in an orthotopic K562 model. Taken together, the CAP-NK method results in highly proliferative, highly cytotoxic memory-like NK cells amenable to clinical use. Citation Format: Jeremiah L. Oyer, Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Md Faqrul Hasan, Sarah B. Gitto, Joanna M. Mucha, Deborah A. Altomare, Robert Y. Igarashi, Alicja J. Copik. Combination of cytokines and PM21-particle stimulation results in robust expansion of memory-like Natural Killer cells with enhanced survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2896.

Cellular senescence, a hallmark of aging, has been implicated in the pathogenesis of many major age-related disorders, including neurodegeneration, atherosclerosis, and metabolic disease. Therefore, investigating novel methods to reduce or delay the accumulation of senescent cells during aging may attenuate age-related pathologies. microRNA-449a-5p (miR-449a) is a small, noncoding RNA down- regulated with age in normal mice but maintained in long-living growth hormone (GH)-deficient Ames Dwarf (df/df) mice. We found increased fibroadipogenic precursor cells, adipose-derived stem cells, and miR-449a levels in visceral adipose tissue of long-living df/df mice. Gene target analysis and our functional study with miR-449a-5p have revealed its potential as a serotherapeutic. Here, we test the hypothesis that miR-449a reduces cellular senescence by targeting senescence-associated genes induced in response to strong mitogenic signals and other damaging stimuli. We demonstrated that GH downregulates miR-449a expression and accelerates senescence while miR-449a upregulation using mimetics reduces senescence, primarily through targeted reduction of p16Ink4a, p21Cip1, and the PI3K-mTOR signaling pathway. Our results demonstrate that miR-449a is important in modulating key signaling pathways that control cellular senescence and the progression of age-related pathologies.

In this study we propose knockout of the inhibitory receptor TIGIT on Natural Killer (NK) cells enhances their anti-tumor response and prevents NK cell fratricide when combined with ADCC-competent antibodies targeting TIGIT. T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a major inhibitory receptor of both T cells and NK cells and is an emerging target for immune checkpoint blockade for the treatment of cancer. Although preclinical studies using combinations of anti-TIGIT and anti-PD-(L)1 showed promising results, the phase III SKYSCRAPER-01 trial using the combination of tiragolumab and atezolizumab for NSCLC did not meet its co-primary endpoint of progression free survival (PFS) while the overall survival (OS) at the time of this publication was immature with the study still ongoing. There is critical need to further understand the mechanism of function of TIGIT on immune cells to support its clinical use. Most of the current understanding of TIGIT regulation of immune cell function has been based on T cells, despite comparable or higher TIGIT expression on NK cells. Recent murine studies have shown the therapeutic efficacy of PD-1 and TIGIT blockade depended on the presence of NK cells, indicating they are an important immune population to consider in the efficacy of TIGIT blockade treatments. Furthermore, the majority of TIGIT antibodies have a humanized IgG, which binds to Fc receptors (CD16) and induces antibody-dependent cellular cytotoxicity (ADCC), a major contributor to their efficacy. Since both CD16 and TIGIT are highly expressed on activated NK cells, there is a potential for ADCC-driven fratricide of TIGIT+ NK cells with ADCC-competent anti-TIGIT. In this study, TIGIT knockout was performed in ex vivo-expanded primary human NK cells and anti-tumor response measured. Compared to WT NK cells, TIGIT knockout NK cells had improved in vitro killing of 3D lung cancer spheroids and increased ADCC when combined with cetuximab. TIGIT KO NK cells had upregulated mTORC1 signaling, increased basal glycolytic rate, and increased degranulation. Moreover, fratricide of WT NK cells was observed in the presence of ADCC-competent anti-TIGIT and could be ameliorated by TIGIT knockout. Altogether, this study demonstrated that the highly cytotoxic fratricide resistant TIGIT knockout NK cells have translation potential alone or in combination with ADCC-competent anti-TIGIT antibodies to enhance the efficacy of anti-TIGIT therapeutics. Citation Format: Md Faqrul Hasan, Tayler J. Croom-Perez, Jeremiah L. Oyer, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Alicja J. Copik. TIGIT knockout enhances anti-tumor response of Natural Killer cells and prevents fratricide when combined with therapeutic ADCC-competent TIGIT antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2908.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the most powerful mechanisms for Natural Killer (NK) cells to kill cancer cells or virus-infected cells. A novel chimeric protein (NA-Fc) was created, which when expressed in cells, positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. This NA-Fc chimera was tested with PM21-NK cells, produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Real time viability assays revealed higher PM21-NK killing of both ovarian and lung cancer cells expressing NA-Fc, which correlated with increased release of TNF-α and IFN-γ cytokines from NK cells and was dependent on CD16-Fc interactions. Lentivirus delivery of NA-Fc to target cells increased the rate of PM21-NK cell killing of A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells. This NA-Fc-directed killing was extended to virus infected cells, where delivery of NA-Fc to lung cells that were persistently infected with Parainfluenza virus resulted in increased killing by PM21-NK cells. In contrast to its effect on PM21-NK cells, the NA-Fc molecule did not enhance complement mediated lysis of lung cancer cells. Our study lays the foundation for application of the novel NA-Fc chimera that could be delivered specifically to tumors during oncolytic virotherapy to mark target cells for ADCC by co-treatment with adoptive NK cells. This strategy would potentially eliminate the need to search for unique cancer specific antigens for development of new antibody therapeutics.

In this study we test if knockout of DNAM-1 competing inhibitory receptors on Natural Killer (NK) cells improves cytotoxic response against neuroblastoma cell lines. Neuroblastoma and other peripheral nervous cell tumors account for 6% of all pediatric cancer. While the 5-year survival rate has greatly improved, the prognosis of children with high-risk neuroblastoma at diagnosis is less than 50%. Immune checkpoint therapy is a promising new application for pediatric oncology; however, neuroblastoma is often immunologically “cold” and current immune checkpoint targets are not abundant in pediatric solid tumors. To improve response to checkpoint blockade and directly target tumor cells for elimination, this study evaluated the use of NK cells. NK cells prime the immune system for successful response to cancer immunotherapy. NK cells activated with particle technology (PM21-NK cells) are highly cytotoxic, produce IFNγ and induce PD-L1 on cancer cells. Although highly cytotoxic, PM21-NK cells express inhibitory receptors that compete for ligands that trigger NK cell lysis, such as PVR, a ligand highly expressed on neuroblastoma cells. We knocked out expression of multiple PVR inhibitory receptors in PM21-NK cells to further enhance cytotoxicity and prevent exhaustion. Three inhibitory receptors on NK cells that bind PVR - TIGIT, CD96, and PVRIG were knocked out alone or in combination and effector functions of these edited NK cells tested. Cytotoxicity against 3D spheroids of the neuroblastoma cell line SK-NA-S cells was assessed and compared to wild type PM21-NK cells. Knockout (KO) of TIGIT enhanced NK cell killing compared to wildtype NK cells (72%±10 vs 55%±3% killing) as well as PVIRG KO (84±3% vs 46±6% killing) and CD96 KO (62%±9% vs 46±6% killing) at a 1:3 E:T ratio at 72 h. Double or triple KO of these receptors resulted in further increased killing of cancer cells. The knockouts of each of the receptors could affect cytokine production and cytokine responsiveness and this will be examined. Overall, PM21-NK cells show promise for therapeutic use against neuroblastoma and preventing the PVR-mediated inhibitory axis signaling enhances their function. Citation Format: Tayler J. Croom-Perez, Jeremiah L. Oyer, Md Faqrul Hasan, Liza D. Robles-Carrillo, Brian P. Tullius, Alicja J. Copik. Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2909.

Background Neuroblastoma is the most common extra-cranial solid tumor occurring in children and accounts for 15% of all pediatric cancer-related deaths. While the 5-year survival rate has greatly improved, the prognosis of children with high-risk neuroblastoma at diagnosis is less than 50%. Immune checkpoint therapy is a promising new application for pediatric oncology; however, neuroblastoma is often immunologically 'cold' and current immune checkpoint targets are not abundant in pediatric solid tumors. To improve response to checkpoint blockade and directly target tumor cells for elimination, this study evaluated the use of Natural Killer (NK) cells. NK cells prime the immune system for a successful response to cancer immunotherapy. NK cells activated with PM21-particle technology (PM21-NK cells) are highly cytotoxic, produce IFNγ and induce PD-L1 on cancer cells. Although highly cytotoxic, PM21-NK cells express inhibitory receptors that compete for ligands that trigger NK cell lysis, such as PVR, a ligand highly expressed on neuroblastoma cells. In this study we hypothesize knockout of inhibitory receptors that compete with the activating receptor DNAM-1 will enhance their anti-tumor effects against neuroblastoma. Methods CRISPR-based Knockout (KO) of TIGIT, PVRIG and/or CD96 in PM21-NK cells was performed and effector functions of these gene-edited NK cells were tested. Cytotoxicity against 3D neurosphere models using neuroblastoma cell lines derived from patients with a range of clinical phenotypes was assessed and compared to wild-type PM21-NK cells using a kinetic live-cell imaging assay. Other effector functions such as cytokine production and resistance to exhaustion will be examined. Results TIGIT, PVRIG, and CD96 receptors could be knocked out alone or in double or triple combinations with on average 89% efficiency. Each receptor as a single knockout improved NK cell cytotoxicity, with TIGIT KO followed by PVRIG KO having the most profound effect. Double knockout of TIGIT and PVRIG outperformed single knockouts of those receptors, with triple knockout of TIGIT, PVRIG, and CD96 incrementally improved cell killing. The knockouts of each of the receptors could affect cytokine production and resistance to exhaustion and these will be examined. Conclusions Overall, PM21-NK cells show promise for therapeutic use against neuroblastoma, and engineering to prevent the PVR-mediated inhibitory axis signaling further enhances their function. Acknowledgements We would like to thank the FL DOH (Grant #22L04) and the UCF Preeminent Postdoctoral Program for funding. Ethics Approval This study was conducted in accordance with the University of Central Florida Institutional Biosafety Committee and all biological materials used were approved under BARA 19–27 and Safety Protocol SPROTO202200000044. The University of Central Florida Institutional Review Board determined this study is not research involving human subjects (STUDY00005502, STUDY00004553 and STUDY00004071).

Abstract In this study we propose inhibition of NKG2A signaling enhances the anti-tumor response of primary human NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. In this study ex vivo-expanded primary human NK cells were shown to have significantly elevated expression of NKG2A. These expanded NK cells also secreted high levels of IFNγ and could induce expression of HLA-E on tumor cells. Suppression of NKG2A by either antibody blockade or gene deletion increased the cytotoxicity of NK cells against the lung cancer cell line A549 stably expressing HLA-E. NKG2A knockout cells had an almost 10-fold decrease in EC50 compared to wild-type NK cells (EC50 0.15 vs 0.94 at 48 h), meaning nearly log-fold fewer NKG2A knockout cells are needed to kill the same number of tumor cells. The effect of NKG2A knockout on cytotoxicity of NK cells against other cancer cell lines stably expressing HLA-E will be presented along with effects of NKG2A suppression on NK cell phenotype, cytokine secretion and degranulation. These initial data suggest that blockade of the CD94/NKG2A inhibitory complex can improve the function of ex vivo expanded human NK cells and could provide an effector population with the potential for enhanced therapeutic efficacy. Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Md Faqrul Hasan, Alicja J. Copik. NKG2A suppression enhances the function of primary human Natural Killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2910.

Treatments targeting TIGIT have gained a lot of attention due to strong preclinical and early clinical results, particularly with anti-PD-(L)1 therapeutics. However, this combination has failed to meet progression-free survival endpoints in phase III trials. Most of our understanding of TIGIT comes from studies of T cell function. Yet, this inhibitory receptor is often upregulated to the same, or higher, extent on NK cells in cancers. Studies in murine models have demonstrated that TIGIT inhibits NK cells and promotes exhaustion, with its effects on tumor control also being dependent on NK cells. However, there are limited studies assessing the role of TIGIT on the function of human NK cells (hNK), particularly in lung cancer. Most studies used NK cell lines or tested TIGIT blockade to reactivate exhausted cells obtained from cancer patients. For therapeutic advancement, a better understanding of TIGIT in the context of activated hNK cells is crucial, which is different than exhausted NK cells, and critical in the context of adoptive NK cell therapeutics that may be combined with TIGIT blockade. In this study, the effect of TIGIT blockade on the anti-tumor activities of human ex vivo-expanded NK cells was evaluated in vitro in the context of lung cancer. TIGIT expression was higher on activated and/or expanded NK cells compared to resting NK cells. More TIGIT+ NK cells expressed major activating receptors and exerted anti-tumor response as compared to TIGIT- cells, indicating that NK cells with greater anti-tumor function express more TIGIT. However, long-term TIGIT engagement upon exposure to PVR+ tumors downregulated the cytotoxic function of expanded NK cells while the inclusion of TIGIT blockade increased cytotoxicity, restored the effector functions against PVR-positive targets, and upregulated immune inflammation-related gene sets. These combined results indicate that TIGIT blockade can preserve the activation state of NK cells during exposure to PVR+ tumors. These results support the notion that a functional NK cell compartment is critical for anti-tumor response and anti-TIGIT/adoptive NK cell combinations have the potential to improve outcomes.

BackgroundInhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in the development of cancer immunotherapeutics. Although early results of anti-TIGIT and its combinations with antiprogrammed death-ligand 1 were highly exciting, results from an interim analysis of phase III trials are disappointing. With mixed results, there is a need to understand the effects of therapeutic anti-TIGIT on the TIGIT+immune cells to support its clinical use. Most of the TIGIT antibodies in development have an Fc-active domain, which binds to Fc receptors on effector cells. In mouse models, Fc-active anti-TIGIT induced superior immunity, while Fc receptor engagement was required for its efficacy. NK-cell depletion compromised the antitumor immunity of anti-TIGIT indicating the essential role of NK cells in the efficacy of anti-TIGIT. Since NK cells express TIGIT and Fc-receptor CD16, Fc-active anti-TIGIT may deplete NK cells via fratricide, which has not been studied.MethodsCRISPR-Cas9-based TIGIT knockout (KO) was performed in expanded NK cells. Phenotypic and transcriptomic properties of TIGIT KO and wild-type (WT) NK cells were compared with flow cytometry, CyTOF, and RNA sequencing. The effect of TIGIT KO on NK-cell cytotoxicity was determined by calcein-AM release and live cell imaging-based cytotoxicity assays. The metabolic properties of TIGIT KO and WT NK cells were compared with a Seahorse analyzer. The effect of the Fc-component of anti-TIGIT on NK-cell fratricide was determined by co-culturing WT and TIGIT KO NK cells with Fc-active and Fc-inactive anti-TIGIT.ResultsTIGIT KO increased the cytotoxicity of NK cells against multiple cancer cell lines including spheroids. TIGIT KO NK cells upregulated mTOR complex 1 (mTORC1) signaling and had better metabolic fitness with an increased basal glycolytic rate when co-cultured with cancer cells compared with WT NK cells. Importantly, TIGIT KO prevented NK-cell fratricide when combined with Fc-active anti-TIGIT.ConclusionsTIGIT KO in ex vivo expanded NK cells increased their cytotoxicity and metabolic fitness and prevented NK-cell fratricide when combined with Fc-active anti-TIGIT antibodies. These fratricide-resistant TIGIT KO NK cells have therapeutic potential alone or in combination with Fc-active anti-TIGIT antibodies to enhance their efficacy.

All supplementary data - four figures and supplementary methods

Natural Killer cells (NK cells) are a key component of the innate immune system and are key effectors of immunosurveillance. NK cells not only have the inherent ability to directly kill malignant, compromised, or virally infected cells, but also recruit and coordinate responses by other immune cells to prime the adaptive immune response. Given this potent anti-tumor response and good safety profile, adoptive NK cell therapy is an emerging cancer treatment modality. Direct killing of tumor cells is major mode of action for NK cell anti-tumor activity and measuring changes in NK cell cytotoxic response in vitro is a critical step in pre-clinical evaluation of novel NK cellular products. Here, we provide a detailed protocol for a live-cell imaging assay for testing NK cell cytotoxicity against a broad range of adherent and 3D in vitro tumor models. Compared to other methods for measuring in vitro cytotoxicity, this method offers real-time dynamic tracking of and provides a multiparameter readout for more robust understanding of NK cell tumor killing.

In this study we propose to circumvent HLA-E/NKG2A mediated Natural Killer (NK) cell inhibition by suppression of NKG2A, to enhance cytotoxicity of ex vivo expanded NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. The CD94/NKG2A inhibitory complex is in balance with CD94/NKG2C stimulatory complex. HLA-E binds both of these complexes and the cytolytic activity of NK cells is influenced by the relative ratios of CD94 complexed to NKG2A or NKG2C. Thus, altering the ratio to increase NKG2C will increase NK cell cytotoxicity. In this study we show that NK cells ex vivo expanded with PM21-particle technology are highly cytotoxic and have elevated expression of NKG2A. These expanded NK cells also secrete high levels of IFNγ, inducing expression of HLA-E in tumor cells. To increase the cytotoxic potential of these ex vivo expanded NK cells, the potential to form CD94/NKG2A inhibitory complex was suppressed by either antibody blockade of NKG2A or deletion of the NKG2A gene by CRISPR/Cas9 editing. Cytotoxicity of NK cells against a lung cancer cell line stably expressing HLA-E were determined by kinetic live-cell imaging. Multiple NK cell:target (NK:T) ratios were used to show an increase in cytotoxicity over control NK cell conditions (alone or with isotype control) in the presence of NKG2A blocking antibodies with 39% increase observed at 1:10 NK:T ratio at 96h with anti-NKG2A and 56% increase with NKG2A knockout NK cells. These data suggest that blockade of CD94/NKG2A complex can improve cytotoxic activity of ex vivo expanded NK cells and could provide a promising effector population with the potential for enhanced therapeutic efficacy. Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Alicja J. Copik. Suppression of NKG2A mediated inhibition in ex vivo expanded natural killer cells increases their cytotoxicity [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 2819.

Background Antibody blockade of the Natural Killer cells (NK cells) and T cell inhibitory receptor TIGIT has been shown to enhance tumor control and survival in preclinical mouse models and early clinical trials; however, there is a lack of understanding of the effect of TIGIT engagement on anti-tumor functions of activated primary human NK cells. Additionally, the majority of TIGIT antibodies in clinical development have a humanized IgG, which induces antibody-dependent cellular cytotoxicity (ADCC) and likely contributes to their efficacy. However, the potential consequences of these Fc-competent antibodies when binding NK cells, such as fratricide, have not been well characterized. Fratricide could deplete NK cells upon treatment, a detrimental effect to the overall efficacy since NK cells play a critical role in the success of checkpoint blockade immunotherapies. Recent efforts have focused on developing adoptive NK cell therapy and combinatorial immune-oncology therapies in order to enhance response. Adoptive transfer of TIGIT KO NK cells could provide a treatment strategy to mitigate potential negative effects of TIGIT blockade on NK cells. In this study, TIGIT knockout in ex vivo PM21-particle expanded human NK cells was performed and the effect on anti-tumor activity alone or in combination with Fc-competent TIGIT antibody blockade was evaluated and compared to WT NK cells. Methods CRISPR was used to make a targeted TIGIT knockout (KO) in ex vivo PM21-particle expanded NK cells (PM21-NK cells). TIGIT KO NK cells were compared to wild type (WT) NK cells to determine changes cytotoxicity, ADCC, and IFNγ, TNFα, and the degranulation marker CD107a expression. Glycolytic rate and mitochondrial stress were measured. TIGIT KO or WT PM21-NK cells were combined with Fc-competent or non-Fc-competent anti-TIGIT and fratricide and cytotoxicity were measured. Results TIGIT KO PM21-NK cells showed improved killing compared to WT against 3D spheroids from multiple cancer cell lines. ADCC increased proportionally in TIGIT KO NK cells. TIGIT KO PM21-NK cells had increased CD107a surface expression after cancer spheroid exposure and increased basal glycolytic rate after stimulation. TIGIT Knockout prevented Fc-competent anti-TIGIT driven NK cell fratricide and prevented decrease in NK cell cytotoxicity when combined with Fc-competent anti-TIGIT. Conclusions Knockout of TIGIT in ex vivo expanded PM21-NK cells resulted in NK cells with improved anti-tumor activity and metabolic fitness. TIGIT KO prevented ADCC driven NK cell fratricide and inhibition of cytotoxicity when combined with Fc-competent anti-TIGIT. TIGIT KO PM21-NK cells are a potential cellular product for therapeutic use in combination with TIGIT blockade. Acknowledgements We would like to thank the FL DOH (Grant #9JK04); Kiadis Pharma, a Sanofi company; and the UCF Preeminent Postdoctoral Program for funding and MaxCyte for providing instrument for initial testing.

There is a great interest in developing natural killer (NK) cells as adoptive cancer immunotherapy. For off-the-shelf approaches and to conduct multicenter clinical trials, cryopreserved NK cells are the preferred product. However, recent studies reported that cryopreservation of NK cells results in loss of cell motility and, as a consequence, cytotoxicity which limits the clinical utility of such products. This study assessed the impact of cryopreservation on the recovery and function of PM21-particle expanded NK cells (PM21-NK cells) as well as their antitumor activity in vitro using 2D and 3D cancer models and in vivo in ovarian cancer models, including patient-derived xenografts (PDX). Viable PM21-NK cells were consistently recovered from cryopreservation and overnight rest with a mean recovery of 73 ± 22% (N = 19). Thawed and rested NK cells maintained the expression of activating receptors when compared to expansion-matched fresh NK cells. Cryopreserved NK cells that were thawed and rested showed no decrease in cytotoxicity when co-incubated with tumor cells at varying effector-to-target (NK:T) ratios compared to expansion-matched fresh NK cells. Moreover, no differences in cytotoxicity were observed between expansion-matched cryopreserved and fresh NK cells in 3D models of tumor killing. These were analyzed by kinetic, live-cell imaging assays co-incubating NK cells with tumor spheroids. When exposed to tumor cells, or upon cytokine stimulation, cryopreserved NK cells that were thawed and rested showed no significant differences in surface expression of degranulation marker CD107a or intracellular expression of TNFα and IFNγ. In vivo antitumor activity was also assessed by measuring the extension of survival of SKOV-3-bearing NSG mice treated with fresh vs. cryopreserved NK cells. Cryopreserved NK cells caused a statistically significant survival extension of SKOV-3-bearing NSG mice that was comparable to that observed with fresh NK cells. Additionally, treatment of NSG mice bearing PDX tumor with cryopreserved PM21-NK cells resulted in nearly doubling of survival compared to untreated mice. These data suggest that PM21-NK cells can be cryopreserved and recovered efficiently without appreciable loss of viability or activity while retaining effector function both in vitro and in vivo . These findings support the use of cryopreserved PM21-NK cells as a cancer immunotherapy treatment.

Background Immunotherapeutic strategies, such as checkpoint blockade of PD-1/PD-L1, have become a focal point of immunotherapy in oncology. Recent studies highlight the importance of Natural Killer (NK) cells in the success of these immunotherapies and adoptive NK cellular therapy is being explored to enhance response to these treatments. Antibodies targeting PD-L1 are mostly Fc silent but some, such as Avelumab, can engage FcγR (CD16) receptor on NK cells resulting in killing cancer cells via antibody-dependent cellular cytotoxicity (ADCC). PM21-particle expanded NK (PM21-NK) cells are an optimal NK cell product to consider for a combination strategy as these NK cells lack PD-L1 but can induce PD-L1 on tumors cells. Previous reports, however, have shown that PD-L1 can be induced on NK cells. This could potentially lead to fratricide of NK cells in the presence of Fc-competent, PD-L1 targeting antibodies and mitigate their cytotoxic response. This study determined if PD-L1 can be induced on PM21-NK cells and what effect PD-L1 engagement had on their activity and potential for fratricide in both WT and PD-L1 knockout PM21-NK cells. Methods CRISPR-based Knockout (KO) of PD-L1 in PM21-NK cells was performed and efficiency was determined after overnight incubation of WT or PD-L1 KO PM21-NK cells with cytokines to induce PD-L1 expression. Cancer cells were incubated with NK cells in the presence of non-competent or Fc-competent α-PD-L1 and cytotoxicity was measured using a kinetic live-cell imaging assay. NK cell fratricide was measured in cultures of WT or PD-L1 KO PM21-NK cells induced for PD-L1 expression with non-competent or Fc-competent α-PD-L1. Results PM21-NK cells were found to express low levels of PD-L1 (< 20%) after exposure to various cancer cell line monolayers or K562 co-culture. Exposure to SKOV-3 spheroids or to a cytokine combination of IL12, IL15, and IL18 led to a significant induction in PD-L1 in WT PM21-NK cells (> 80%). PD-L1 knockout prevented the induction of PD-L1 on NK cells after cytokine exposure and enhanced cytotoxicity. Fratricide was decreased and cytotoxicity increased in PD-L1 KO PM21-NK cells in combination with Fc-competent α-PD-L1. Conclusions Knockout of PD-L1 in ex vivo expanded PM21-NK cells prevented the induction of PD-L1 on NK cells without negative effects on cytotoxicity. PD-L1 KO PM21-NK cell cytotoxicity was further enhanced in combination with Fc-competent α-PD-L1 compared to WT NK cells. PD-L1 knockout PM21-NK cells are a potential cell product for therapeutic use in combination with PD-L1 targeting antibodies. Acknowledgements We would like to thank the FL DOH (Grant #9JK04); Kiadis Pharma, a Sanofi company; and the UCF Preeminent Postdoctoral Program for funding and MaxCyte for providing instrument for initial testing.