Jul 2026· Signal Transduction and Targeted Therapy· Vol 11· 0 citations· 66 references
Medicine
TL;DR
A next-generation NK cell platform using a tri-cistronic retroviral vector that encodes an extracellular PD-1 domain fused to the intracellular portion of NKG2D with the costimulatory molecule 4-1BB and induces de novo PD-L1 expression in otherwise negative tumors is developed.
Abstract
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.
BACKGROUND
Oncolytic virus M1 encoding a mutant IL-18 decoy (OVM18) represents a novel virotherapy that integrates selective oncolysis with localized activation of the IL-18 pathway. However, the heterogeneity of therapeutic responses suggests that host immune determinants influence its efficacy.
METHODS
To elucidate these determinants, we performed integrated immune profiling using bulk and single-cell transcriptomics, flow cytometry, and T cell receptor (TCR) repertoire analyses. The functional contribution of specific immune subsets was validated using Batf3⁻/⁻ mice and FTY720-mediated lymphocyte trafficking blockade. Potential synergistic immunotherapies were identified through transcriptomic database screening and confirmed in tumor-bearing mice receiving combined OVM18 and immune checkpoint blockade.
RESULTS
We identified intratumoral IL-18R1+CD8+ T cells as an important effector population, whose IL-18R1 expression and intratumoral abundance positively correlate with OVM18 efficacy. Integrated single-cell RNA-sequencing and cytometry analyses revealed that IL-18R1+CD8+ T cells are clonally expanded, tumor antigen-enriched, polyfunctional cytotoxic T cells. Their generation requires conventional type 1 dendritic cells (cDC1)-dependent priming within tumor-draining lymph nodes, followed by trafficking into tumors to mediate antitumor responses. Notably, CTLA-4 blockade enhances cDC1-derived IL-12 and promotes IL-18R1+CD8+ T-cell expansion, thereby overcoming resistance to OVM18 therapy. Combination treatment with OVM18 and anti-CTLA-4 enhances intratumoral IL-18R1+CD8+ T cell infiltration, delays tumor progression, and prolongs survival in poor-responsive models.
CONCLUSIONS
Our findings establish IL-18R1+CD8+ T cells as an important effector population in OVM18 therapy, thereby providing a strong rationale for combining OVM18 with CTLA-4 blockade to overcome therapeutic resistance and achieve durable antitumor responses.
Unknown authors· Journal for ImmunoTherapy of...· 0 citations
Abstract Background Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with dismal outcomes, especially in relapsed/refractory settings. Chimeric antigen receptor natural killer (CAR-NK) cell therapy holds promise but is constrained by the immunosuppressive tumor microenvironment (TME), where adenosine-mediated suppression is a key barrier. Objective To develop a novel CAR-NK construct cotargeting AML cells and the adenosine-rich TME to enhance antileukemia efficacy. Methods Ex vivo expanded primary NK cells were used to compare the effects of CD39 versus CD73 blockade on NK cell function via messenger RNA-electroporated antibodies. A CD33-CD73 dual-function CAR-NK construct (integrating CD33-specific lysis and anti-CD73scFv secretion for TME disruption) was designed and transduced into NK cells via retrovirus. Engineered NK cells were characterized for transduction efficiency, expansion, purity, viability, and CAR stability. In vitro cytotoxicity against AML cell lines and primary blasts was assessed, and in vivo efficacy was evaluated in a MOLM-13 xenograft mouse model. Results CD73 blockade more potently enhanced NK cell activity than CD39 blockade. Retroviral transduction achieved >50% efficiency, and expansion with K562-4-1BBL-mbIL-21/−15 feeder cells yielded NK cells with ≥6,000 fold expansion, >93% purity, >98% viability, and stable CAR expression. At an effector-to-target ratio of 0.5:1, CD33-CD73 CAR-NK cells mediated ~80% specific lysis, with superior cytotoxicity vs conventional CD33 CAR-NK cells. In xenografts, CD33-CD73 CAR-NK cells achieved robust tumor clearance, extended median survival by 24.5 days (59.5 vs 35 days) versus standard CD33 CAR-NK cells, and five out of six mice achieved long-term survival (>50 days). Conclusion The CD33-CD73 dual-targeting CAR-NK platform synergistically targets AML cells and the adenosine-rich TME, exhibiting superior anti-leukemia efficacy. This strategy advances AML immunotherapy and provides a translational blueprint for TME-targeted therapies in other cancers.
Lu Wang, S. Gong, Jun Wang et al.· Journal for ImmunoTherapy of...· 0 citations
While cancer immunotherapies have transformed treatment outcomes, most patients fail to respond primarily due to immune resistance mechanisms. Targeting tumor antigens along with suppressive factors may overcome such barriers. Glycoprotein A repetitions predominant (GARP) is a cell surface receptor that anchors latent transforming growth factor beta and contributes to immunosuppression, with its expression mostly to regulatory T cells (Tregs), cancer cells, and platelets. This study examines the therapeutic impact of engaging effector T cells to target GARP-expressing tumor cells along with Tregs, utilizing our unique anti-GARP antibody (PIIO-1) that targets tumors and Tregs but spares platelets.
We developed an anti-human GARP/CD3 bispecific T-cell Engager (BiTCE) with a tandem single-chain variable fragment construct. Human T cells armed with BiTCE were co-cultured with GARP+ tumor cells to assess cytotoxicity. In vivo efficacy and safety were evaluated using both immunocompromised and immunocompetent preclinical models that represent highly refractory diffuse large B-cell lymphoma, acute lymphoblastic leukemia, and glioblastoma. Importantly, we utilized systemic adeno-associated virus-mediated BiTCE delivery in vivo to overcome the pharmacokinetic challenges of short half-life of the typical BiTCE proteins without Fc.
We demonstrate that elevated GARP expression in human tumors correlates with poor survival and higher relapse rates after first-line therapies. Our anti-GARP/CD3 BiTCE shows strong in vitro and in vivo efficacy against both GARP-expressing hematologic and solid tumor models without significant toxicity.
Our study provides a novel BiTCE therapeutic platform that enables us to achieve long-term tumor control and overcome tumor resistance by engaging CD3 on effector T cells to target GARP-expressing cancer and Tregs. The Anti-GARP/CD3 platform therefore holds the promise as a novel therapy of cancer immunotherapy worthy for clinical translation.
NIH R01
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Ziyu Wang, Yaa S Amankwah, J. Mandula et al.· Journal of Immunology· 0 citations
Natural Killer (NK) cells are potent effectors of the innate immune system that kill tumor cells via natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). In acute myeloid leukemia (AML), tumor heterogeneity and antigen escape challenge current therapies, resulting in a 5-year survival rate of < 40%. To overcome these barriers and build on our ongoing clinical trial of a CD33-targeted Tri-Specific Killer Engager (TriKE), we engineered TriKEs with Poly Antigen Cytokine Complexes (TriKE-PACCs), which combine dual AML-specific targeting with NK activation and persistence.
TriKE and TriKE-PACC constructs were expressed in Expi293 cells and purified by affinity chromatography. TriKE-PACCs consist of an anti-CD16 sdAb, an IL-15 moiety, and an anti-AML scFv or sdAb with a PACC arm containing IL-15Rα linked to an additional anti-AML scFv or sdAb. AML antigens were evaluated on cell lines and 20 primary samples by flow cytometry. Antigen escape was modeled using CRISPR knockout AML lines or primary patient samples. NK activation (CD107a, IFNγ, TNFα) and cytotoxicity were measured using flow cytometry and Incucyte assays.
Our examination of AML patient samples revealed diverse antigen profiles, highlighting the need for modular TriKE-PACC designs. The TriKE-PACCs enhanced CD107a, IFNγ, and TNFα production and exhibited improved cytotoxicity compared to single-antigen TriKEs, demonstrating resistance to antigen escape.
TriKE-PACCs offer a customizable, modular approach to treating AML heterogeneity and antigen escape with NK cell-based immunotherapies. By combining IL-15 and CD16 signaling with dual tumor-antigen targeting, we enhance NK cell proliferation and antitumor activity compared to TriKEs or IL-15 therapies alone. These data support further preclinical development and testing of modular and personalized AML immunotherapies to improve upon our current CD33 TriKE clinical trial.
NIH/NCATS: 1T32TR004376 and 1UM1TR004405
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Anders W. Matson, Caden Caligiuri, Katherine M. Koehler et al.· Journal of Immunology· 0 citations
Abstract Background Renal cell carcinoma (RCC) frequently exhibits upregulation of CD70, a tumor-associated antigen implicated in immune evasion and disease progression, making it an attractive therapeutic target. Cusatuzumab, an Fc-enhanced anti-CD70 monoclonal antibody, has demonstrated the ability to mediate antibody-dependent cellular cytotoxicity (ADCC); however, its clinical efficacy is limited by insufficient effector cell engagement, antigen heterogeneity, and adaptive resistance mechanisms within the tumor microenvironment. Methods To overcome these limitations, we developed a universal, off-the-shelf natural killer T (NKT) cell product derived from hematopoietic stem cells (HSCs) through genetic engineering of an invariant T cell receptor (TCR) and a high-affinity, non-cleavable CD16 receptor. These CD16-enhanced HSC-derived NKT (CD16HSC-NKT) cells were generated using a feeder-free differentiation platform, yielding high purity and scalability while preserving canonical NKT cell phenotypes and effector functions. Results Functionally, CD16HSC-NKT cells exhibited potent intrinsic cytotoxicity and multimodal tumor-targeting capabilities. In combination with cusatuzumab, these cells demonstrated robust ADCC against CD70-positive RCC cells both in vitro and in vivo, significantly enhancing tumor cell killing compared to antibody monotherapy. Moreover, CD16HSC-NKT cells displayed improved persistence and functional stability, supporting sustained antitumor activity. Importantly, CD16HSC-NKT cells contributed to remodeling of the tumor microenvironment by selectively depleting immunosuppressive populations, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Safety assessments indicated a favorable profile, with no evidence of uncontrolled activation or off-target toxicity. Conclusions Collectively, our findings establish CD16HSC-NKT cells as a versatile and scalable cellular platform that synergizes with cusatuzumab to enhance therapeutic efficacy against RCC. This combinatorial strategy represents a promising approach to overcoming current limitations of monoclonal antibody therapy and advancing next-generation immunotherapies for solid tumors.
Yan-Ruide Li, Yi-Chen Zhu, Yu-Ning Chen et al.· The Oncologist· 0 citations
Natural killer (NK) cells are central mediators of antitumor immunity and possess the unique ability to recognize and eliminate malignant cells independently of major histocompatibility complex (MHC) restriction. Compared with T-cell-based approaches, NK-cell immunotherapies generally exhibit a more favorable safety profile, highlighting their growing therapeutic potential in oncology. Among the ligands that regulate NK-cell activity, B7-H6 has emerged as a particularly attractive target because of its highly restricted expression in normal tissues and frequent upregulation across diverse malignancies. Initially identified as a ligand for the activating NK-cell receptor NKp30, B7-H6 is now recognized as a multifunctional molecule with roles extending beyond immune recognition. Accumulating preclinical evidence suggests that B7-H6 may contribute to tumor progression by regulating signaling pathways involved in proliferation, survival, migration, invasion, and immune evasion. Furthermore, the existence of soluble B7-H6 adds an additional layer of biological complexity and may influence both NK-cell function and therapeutic responsiveness. In this review, we summarize current knowledge regarding the regulation, expression patterns, and biological functions of B7-H6, with particular emphasis on its dual roles in tumor immunology and cancer cell biology. We discuss the emerging significance of soluble B7-H6, evaluate the current landscape of B7-H6-targeted therapeutic strategies, including bispecific engagers and cellular immunotherapies, and highlight key translational challenges that may influence clinical development. Collectively, current findings position B7-H6 as a promising immuno-oncologic target at the intersection of immune surveillance, immune escape, and malignant progression, warranting continued investigation as a next-generation therapeutic axis in cancer immunotherapy.
Ziyi Yang, Qi Zhao· Cancer Biome and Targeted Th...· 0 citations
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