Aug 2026· International Immunopharmacology· Vol 188, pp.
117325
· 0 citations· 46 references
Medicine
TL;DR
A humanized CD70-targeted CAR-T cell therapy is developed, A174-hu1-CAR-T, with reduced fratricide and potent antitumor activity, providing a preclinical foundation to support further translational development.
Abstract
Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in B-cell malignancies, but its application in acute myeloid leukemia (AML) and renal cell carcinoma (RCC) remains challenging due to the lack of ideal target antigens. CD70 is highly expressed on AML and RCC tumor cells, while its expression in normal tissues is largely restricted to activated lymphocytes, making it a promising immunotherapeutic target. However, CD70 expression on activated T cells can trigger fratricide in CD70-targeted CAR-T cells, posing a major challenge for their preparation and efficacy. Using an analog of the clinical-stage anti-CD70 antibody ARGX-110 as a reference, we screened a phage display library from immunized mice and identified high-affinity anti-CD70 antibodies, which were then used to construct a panel of second-generation CARs. Functional characterization identified a lead candidate, A174-CAR-T, with high CAR expression, strong in vitro antitumor activity, and low fratricide. We further humanized its single-chain variable fragment to generate A174-hu1-CAR. Compared with the parental construct, A174-hu1-CAR-T cells exhibited enhanced expansion and markedly reduced surface expression of CD70, consistent with improved cis-masking (i.e., surface shielding on the same cell). Molecular docking and surface electrostatic potential analyses suggested that humanization optimized the scFv-CD70 binding interface, which together with increased CAR surface expression contributed to the enhanced masking and reduced fratricide. Functionally, A174-hu1-CAR-T cells maintained potent cytotoxicity and showed enrichment of proliferative and central memory T-cell subsets. In xenograft models, A174-hu1-CAR-T infusion demonstrated potent antitumor efficacy against both AML and RCC, with a favorable safety profile. Overall, we developed a humanized CD70-targeted CAR-T cell therapy, A174-hu1-CAR-T, with reduced fratricide and potent antitumor activity, providing a preclinical foundation to support further translational development.
Chimeric antigen receptor (CAR) therapies have shown great success in hematological malignancies but remain largely ineffective against solid tumors such as pancreatic ductal adenocarcinoma (PDAC). A key obstacle among various aspects, is the dense stromal barrier formed by cancer-associated fibroblasts (CAFs), providing a rationale for simultaneously targeting stroma and tumor cells. Using immunohistochemistry of primary PDAC tumors and liver metastases, we confirmed high mesothelin (MSLN) expression on tumor cells, and CD70 expression on tumor cells and predominantly CAFs. Based on these results and the favorable safety profile of CAR natural killer (NK) cells over CAR T cells, we generated MSLN- and CD70-targeting IL-15-armored CAR NK cells. Both constructs mediated cytotoxicity against different pancreatic cancer and CAF cell lines with varying antigen expression in vitro, demonstrating that both, the CAR-molecule and IL-15 were required to increase functionality against more treatment-resistant cell lines. Interestingly, pooled MSLN- and CD70-CAR NK cells did not significantly improve cytolysis compared to monotherapies in an advanced 3D in vitro model or in vivo. Together these findings highlight the limitations of dual-targeting approaches and underscore the need for advanced engineering strategies to improve CAR NK cells beyond antigen targeting and cytokine support in the PDAC microenvironment.
Laura Gehrcken, J. Ott, Astrid Van Den Eynde et al.· Molecular therapy. Oncology· 0 citations
In vivo, CAR4-DNT achieved durable leukemia clearance and prolonged survival, whereas PBS-treated mice rapidly progressed and UT-DNT recipients showed transient disease control before relapse, establishing CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development.
Shanshan Wang, Michele Nawata, Juan Arteaga 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
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)
Zoe C. Schmiechen, A. Sweatt, Jangsuk Oh et al.· Journal of Immunology· 0 citations
Chimeric antigen receptor (CAR) T cell therapies have revolutionized treatment of hematologic malignancies such as lymphoma and multiple myeloma. However, their success is limited by high manufacturing costs, reliance on autologous T cells, variable product quality, and life-threatening toxicities like cytokine release syndrome. In contrast, natural killer (NK) cells offer a safer, more flexible alternative, but their clinical translation remains constrained by complex expansion protocols and high production costs. Here, we present a transformative approach using the human NK cell line YTS, which is amenable to large-scale culture, genetic manipulation, and cryopreservation. By introducing a CD19-specific CAR into YTS cells, we generate potent effector cells capable of selectively eliminating CD19-expressing targets. We demonstrate that CAR signaling in YTS cells requires intracellular activation and, in certain tumor settings, is enhanced by co-stimulation via the 2B4-CD48 pathway. Importantly, irradiation of YTS-CAR cells prevents proliferation without compromising their cytotoxic function even after freezing and thawing. In preclinical models, injections of irradiated YTS-CAR cells significantly reduced CD19+ tumor burden, underscoring their therapeutic promise. This work positions engineered YTS cells as a novel, scalable, and cost-effective "off-the-shelf" immunotherapy platform suitable for treating refractory leukemias and lymphomas. Future studies will be required to assess safety and to explore applicability to autoimmune diseases and solid tumors.
Hanan Jaber, Anas Abu Khalaf, L. Weiss et al.· Haematologica· 0 citations
Osteosarcoma is a highly malignant tumor with poor prognosis. Current CAR-T cell therapies for osteosarcoma are predominantly designed with single targets, but their efficacy remains unsatisfactory. In this study, a novel bispecific CAR-T cell was developed to provide an experimental basis for improving the therapeutic outcome of osteosarcoma. Single-cell RNA sequencing (scRNA-seq) identified two antigens highly expressed in osteosarcoma cells, ANXA2 and CD147, whose expression was further validated at the tissue level by qRT-PCR, flow cytometry, and immunohistochemistry. Based on a second-generation CAR backbone, a bispecific ANXA2/CD147 CAR-T construct was generated using magnetic bead sorting, primary T-cell culture, and lentiviral transduction, achieving a transduction efficiency of 47.1%. LDH release assays demonstrated that bispecific CAR-T cells exhibited significantly greater cytotoxicity against tumor cells than single-target and control groups. ELISA confirmed that bispecific CAR-T cells released higher levels of effector molecules, including GZMB and TNFα. In a subcutaneous CDX model, bispecific CAR-T cells displayed superior antitumor activity and greater T-cell infiltration. In a paw pad xenograft model, mice treated with bispecific CAR-T cells exhibited the smallest tumor volumes, lowest tumor weights, and reduced rates of lymph node metastasis. Furthermore, PDX models confirmed that bispecific CAR-T cells effectively suppressed osteosarcoma growth. ScRNA-seq of tumors derived from CDX models and immunohistochemistry revealed markedly increased infiltration of M1 macrophages in the bispecific group. Collectively, this study successfully generated a bispecific ANXA2/CD147 CAR-T cell with robust antitumor activity, providing a promising new strategy for the immunotherapy of osteosarcoma.
