Engineered PD1-NKG2D Dual-CAR NK92 cells broaden antitumor target recognition in preclinical tumor models

Multifunctional-engineered NK cells overcome tumor immunosuppression by combining PD-L1 and HLA-E targeting and endogenous IL15 production

The immunosuppressive tumor microenvironment (TME) poses a significant challenge to effective cancer immunotherapy, as it enables tumor escape through redundant checkpoint pathways, metabolic constraints, and direct inhibition of effector cells. To overcome these barriers, we developed a next-generation NK cell platform using a tri-cistronic retroviral vector that enhances NK cell activation, recruitment, survival, and metabolic fitness. Pan-cancer transcriptomic analyses reveal consistent co-expression of PD-L1 and HLA-E across tumors, which correlates with immune infiltration accompanied by strong immunosuppression, highlighting these molecules as key targets for immune evasion. To improve NK cell recruitment, activation, cytolytic function, survival, and metabolic fitness in the TME, we developed a next-generation NK cell platform using a tri-cistronic retroviral vector that encodes an extracellular PD-1 domain (exPD1) fused to the intracellular portion of NKG2D with the costimulatory molecule 4-1BB, and expressing soluble IL15 and NKG2A single-chain variable fragments (scFv). Thus, the exPD1 allows the recognition of cells expressing PD-L1, while the intracellular costimulatory signaling transforms the inhibitory interaction PD-1/PD-L1 into an activating one. This strategy effectively targets PD-L1-positive tumor cells and induces de novo PD-L1 expression in otherwise negative tumors. To further enhance anti-tumor activity, we incorporated a module encoding soluble NKG2A-scFv to mask the NKG2A receptor on NK cells and hinder NKG2A/HLA-E inhibitory interaction. Additionally, we improved NK cell survival and expansion by delivering controlled low doses of IL15, which prevents NK cell exhaustion and extends their presence in vivo. This integrated strategy may provide a novel, ready-to-use allogenic mature NK cell therapy that could overcome checkpoint-mediated inhibition, metabolic suppression, and immune escape, offering a promising model for treating high-risk and treatment-resistant tumors.

Combinatorial therapy with resveratrol sensitizes glioblastoma to NKG2D CAR-T cells

Chimeric antigen receptor T-cell (CAR-T) therapies have shown potential in clinical trials for glioblastoma, yet treatment responses vary due to heterogeneous antigen expression and post-treatment immune escape. NKG2D−based CAR-T cells have exhibited a favorable safety profile in patients with hematologic malignancies and demonstrated potent antitumor activity in xenograft models, including those of glioblastoma. Nevertheless, glioma cells could evade immune recognition by downregulating or proteolytically shedding NKG2D ligands. To enhance the efficacy of NKG2D CAR-T therapy, we investigated its combination with preclinical agents capable of penetrating the blood-brain barrier that could upregulate NKG2D ligands on glioma cells. Our study revealed that resveratrol (RSV), a bioactive polyphenol, significantly increased the surface expression of NKG2D ligands on glioblastoma cells. RSV pretreatment sensitized these cells to NKG2D CAR-T-mediated killing in vitro. Additionally, the combination of RSV with NKG2D CAR-T cells demonstrated potent antitumor activity in vivo. Mechanistically, RSV potentially induced NKG2D ligand expression via activation of the p53 signaling pathway. These preclinical findings identify RSV as a promising pharmacological adjuvant that enhances NKG2D CAR-T efficacy in glioblastoma, supporting further translational and clinical evaluation of this combinatory approach.

Nicotinamide mononucleotide potentiates the anti-tumor efficacy of CAR-NK cell therapy targeting MSLN in ovarian cancer

Coexpression of GITRL confers resistance to Treg cell-mediated immunosuppression to anti-CD19 CAR-NK cells

Introduction Regulatory T cells play a pivotal role in shaping immune interactions, limiting antitumor immunity, and impairing the activity of engineered effector cells. Overcoming regulatory T (Treg) cell–mediated immunosuppression is critical to unlock the full therapeutic potential of chimeric antigen receptor (CAR)-cell therapies. Here, we develop “armored” anti-CD19 CAR-NK cells coexpressing GITR ligand (GITRL) (CAR19-GITRL-NK) to both target malignant B cells and locally counteract Treg-mediated suppression. GITR is highly expressed on Tregs, and engagement by its ligand GITRL can modulate their function, providing a rational strategy to reprogram the tumor microenvironment. Methods We generated anti-CD19 NK-92 and primary peripheral blood NK (PB-NK) cells with or without GITRL coexpression using lentiviral vectors, and evaluated their functionality and therapeutic potential. CAR19-GITRL-NK-92 cells were analyzed regarding their expansion, metabolism, cytotoxicity, cytokine secretion, degranulation, and in vivo antitumor potential. The cytotoxic activity of CAR19-GITRL PB-NK cells was examined, and their growth was measured in the presence of T CD4⁺ cells transduced with FoxP3 expression vector (iTreg-FoxP3) used as a model for Treg cells. Results CAR19-GITRL-NK-92 cells displayed superior expansion and enhanced metabolic fitness. Functionally, both CAR constructs increased the cytotoxicity of NK-92 and cytokine secretion against CD19⁺ Nalm-6 and Namalwa cells in vitro. RNA-Seq revealed 54 upregulated and 34 downregulated genes in CAR19-GITRL-NK-92 versus CAR19-NK-92 cells, with enrichment of pathways linked to immune activation, proliferation, chemotaxis, and cytotoxicity, including increased expression of NCR2, CD2, CMKLR1, IRF5, KIT, and PTPN3. In vivo, CAR19-GITRL-NK-92 cells achieved superior tumor control and the longest overall survival. In primary NK cells, CAR19-GITRL enhanced killing of CD19⁺ targets, and GITRL expression attenuated iTreg-FoxP3 mediated inhibition in CAR-NK cells, indicating a resistance mechanism to Treg suppression. Discussion Collectively, these data position CAR19-GITRL-NK cells as a potent and innovative strategy that couples direct tumor targeting with active undermining of Treg-driven immunosuppression for the treatment of B-cell leukemias and lymphomas.

Decoding the Hallmark Features of Modular CAR Domains and Their Impact on CAR T Cell Tumoricidal Activity 2305603

Engineering T cells to express a Chimeric Antigen Receptor (CAR) directs potent tumor cytotoxicity, and this cellular immunotherapy has yielded robust success in hematological malignancies. However, in solid tumor models, including neuroblastoma, regression after CAR administration is only transient. The linked domains of CARs determine their activation threshold and can be optimized to overcome efficacy barriers, including exhaustion, persistence, and activation threshold. Yet, as to how each modular domain and its variants influence T cell phenotype and function is poorly understood. We generated a library of 12 constructs that combinatorially link 3 distinct hinge regions (CD8, CD28, or IgG), 2 transmembrane domains (CD8 or CD28), and 2 co-stimulatory regions (41BB or CD28), all frequently used in FDA-approved CARs. We used a scFv targeting the oncofetal antigen GPC2, an antigen with moderate to low expression in neuroblastoma. Expressing these unique CARs in human T cells, we will evaluate differences in T cell cytokine production, killing capacity, phenotype, and persistence using preclinical models of neuroblastoma. These assays will generate a high-dimensional dataset, and we will employ machine learning to identify an in vitro biosignature of CAR T cells that predicts in vivo CAR T cell killing, as well as a favorable phenotype for persistence and effector qualities. Preliminary results identified a broad range of CAR T cell cytotoxicity against neuroblastoma lines in vitro, with the transmembrane domain driving the greatest variation in killing, as well as significant interaction effects between domains. Overall, we aim to create a model to establish the role of each domain and how they interact to propagate an activation signal, recruit a unique signalosome, and dictate the phenotype and function of CAR T cells. NCI Intramural Research Program Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract B056: DLL3 CAR-T and CAR-NK cell combination therapy promotes synergistic antitumor activity in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive form of lung cancer, with most patients showing poor response rates to immunotherapies, resulting in limited treatment options. Profiling of the SCLC TIME shows immunologic heterogeneity, with the majority of SCLC being neuroendocrine (NE) subtype and exhibiting low MHC-I expression, and a minority being non-neuroendocrine (non-NE) subtype, with higher MHC-I expression. Recent incorporation of T cell engagers targeting DLL3 and CD3 have shown promising results, however, MHC-I downregulation can potentially limit endogenous T cell activity and therapeutic responses. Chimeric antigen receptor (CAR) based therapies enable the genetic engineering of T or NK cells to target tumor-specific antigens independently of MHC-I, representing a promising therapeutic strategy for SCLC. However, CAR-NK cells’ proliferation and lifespan is limited compared to CAR-T cells. CAR-T might benefit from a combination with CAR-NK providing a rapid antitumor response to promote infiltration and expansion. In this study we aimed to use 3D micro-physiological systems to test whether a combination of CAR-T and CAR-NK could overcome CAR therapy limitations in solid tumors. DLL3 surface protein expression was characterized in SCLC cell lines by flow cytometry. Then, DLL3-targeting CAR-T and CAR-NK cells were generated through viral transduction and validated in both 2D and 3D tumor models. The combination of the CAR-T and CAR-NK was tested using a co-culture spheroids model in a 3D microfluidic device. Finally, infiltration of the CARs across a blood vessel barrier into the TME was investigated by creating a microphysiological system of SCLC TME. We found that DLL3 expression was heterogenous across SCLC cell lines, with NE SCLC exhibiting higher DLL3 expression than non-NE SCLC. Both DLL3 CAR-T and CAR-NK cells demonstrated potent antitumor activity in 2D and 3D culture, with CAR-NK cells showing more rapid cytotoxic activity compared to CAR-T cells, killing via antigen recognition but also recognizing the absence of MHC-I expression. We then sought to combine both therapies to investigate cooperative activity between the two effector-cell platforms. At a 6-day timepoint, without exogenous IL-2 stimulation, we found that the DLL3 CAR-T and CAR-NK cell combination showed the greatest anti-tumor activity in the DLL3+ NE cell lines, compared to either therapy alone when tested in the 3D microfluidic model, suggesting synergistic and bystander effector function. These results correlated with higher IL-2 and IFN-g production detected in conditioned media only in the CAR-T and CAR-NK cell combination treatment. These results suggest that DLL3-targeting CAR therapies represent a promising approach for treating SCLC, particularly given their ability to function independently of MHC-I. In addition, a combination of CAR-T and CAR-NKs could be an efficacious approach to overcoming current limitations of CAR therapies in solid tumors, including immune cell exclusion, persistence, and exhaustion. Alan E. Bers, Ian Gillanders, Eden Bobilev, Carla Stornante, Mubin Tarannum, Navin R. Mahadevan, Rizwan Romee, Marco Campisi, David A. Barbie. DLL3 CAR-T and CAR-NK cell combination therapy promotes synergistic antitumor activity in small cell lung cancer [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B056.