Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
The ICOS domain promotes trogocytosis and fratricide, explaining the poor persistence and function of ICOS CAR-T cells observed in this trial and raise significant caution for using CAR constructs that rely on ICOS for CAR activation.
Abstract
CAR-T therapy targeting B-cell antigens is effective for relapsed hematologic malignancies, but antigen escape remains a challenge. We previously showed dual CD19/CD20-4-1BB CAR-T therapy was highly effective (Nat Med, 2020). We further developed trispecific CAR-T cells (CD19/CD20/CD22) incorporating ICOS signaling (Sci Transl Med, 2021). Despite promising preclinical activity, trispecific-ICOS CAR-T cells failed to expand and showed no efficacy in a Phase I trial (NCT05094206; ASH 2024). Here, we investigated mechanisms underlying this failed response.
Based on our IRB approved protocol and using patient-derived PBMCs and leftover samples from treated patients, we generated tested bispecific-4-1BB, bispecific-ICOS and trispecific-ICOS CAR-T cells.
NSG mice receiving bispecific- or trispecific-ICOS CAR-T cells had worse survival than those with 4-1BB CAR-T cells. In vivo, ICOS CAR-T cells failed to expand and showed an exhaustion phenotype. When co-culture with Raji cells, ICOS CAR-T cells significantly enhanced surface-FMC63 loss compared to 4-1BB counterparts. Surface-FMC63 expression was mostly recovered at 40 hours on 4-1BB CAR-T cells, but not on ICOS CAR-T cells. Since trogocytosis can mediate CAR internalization and antigen loss on tumor, we examined CD19 transfer. ICOS CAR-T cells displayed higher and more sustained CD19 uptake and fratricide reflected by apoptosis than 4-1BB cells. Consistently, when co-culture with patient autologous B cells, ICOS CAR-T cells again showed greater CAR loss and apoptosis. Extended co-culture mimicking chronic antigen stimulation revealed expansion of 4-1BB but not ICOS CAR-T cells, the latter showing reduced proliferation (Ki67) and elevated exhaustion markers.
In conclusion, the ICOS domain promotes trogocytosis and fratricide, explaining the poor persistence and function of ICOS CAR-T cells observed in our trial. Thus, our findings raise significant caution for using CAR constructs that rely on ICOS for CAR activation.
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Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
This first-in-human study demonstrated the feasibility of an "off-the-shelf" base-edited CAR T cell approach and informs future multiantigen strategies against AML.
C. Georgiadis, Robert Chiesa, Hebatalla Rashed et al.· Science Translational Medici...· 0 citations
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Current Immunotherapies, including chimeric antigen receptor (CAR) T cell strategies, are limited by the scarcity of antigens that are present on AML cells but absent from hematopoietic stem and progenitor cells (HSPCs). To identify a potential therapeutic target, we analyzed two publicly available single-cell RNA sequencing datasets and found that CD4 is frequently expressed on AML blasts but absent from HSPCs, supporting its potential as a novel CAR target. We developed a CD4-targeted CAR using CD3+CD4⁻CD8⁻ double-negative T cells (CAR4-DNTs). DNTs offer a unique therapeutic platform: they naturally lack CD4, preventing fratricide, and elicit endogenous anti-leukemic activity. Their feasibility and safety as an allogeneic cell therapy platform was demonstrated in a Phase I trial. This work aims to establish CAR4-DNTs as a next-generation, off-the-shelf CAR-T cell therapy for AML.
Donor-derived DNTs were engineered to express a CD4-CAR. Cytotoxicity, persistence, and cytokine secretion were evaluated against AML cell lines, CD4+ and CD4-knockout AML variants, and primary AML samples. NSG-SGM3 mice engrafted with luciferase+ MV4-11 cells received CAR4-DNTs, untransduced (UT) DNTs, or PBS. Disease progression was monitored by bioluminescence imaging and survival analysis.
CAR4-DNTs were successfully manufactured without fratricide. They exhibited significantly enhanced and more durable cytotoxicity than UT-DNTs against AML cell lines and patient samples. Importantly, CAR4-DNTs retained endogenous cytotoxicity towards CD4-negative AML cells. 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.
These findings establish CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development.
Distinguished Doctoral Recruitment Scholarship, The C17 Council, Alberta Cancer Foundation, Alberta Children’s Hospital Research Institute
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Shanshan Wang, Michele Nawata, Juan Arteaga et al.· Journal of Immunology· 0 citations
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.
Alan E. Bers, I. Gillanders, Eden Bobilev et al.· Clinical Cancer Research· 0 citations
A trispecific CAR targeting CD19, CD20, and CD22 with OX40 co-stimulatory domain with overall response rate was 50%, including complete responses in 83% of lymphoma patients, and one-year overall survival rate was 61%, with durable remissions observed in lymphoma.
S. Vasu, N. Denlinger, No-Joon Song et al.· Blood Cancer Discovery· 0 citations
CD19-CAR-T cells are a major therapy for relapsed/refractory B-cell malignancies, yet ~50% of patients relapse after infusion. Clinical data suggest that tisa-cel treatment is associated with higher rates of CD19 mutation than axi-cel, though direct comparisons have been lacking. Although both products target the FMC63 epitope, they employ different structures of hinge/transmembrane/costimulatory domains (CD8α/CD8α/4-1BB in tisa-cel, CD28/CD28/CD28 in axi-cel). Here, we show that CAR structure is a critical determinant of CD19 loss in tumor B cells. Specifically, repeated exposure to CD19-4-1BB-based CAR-T cells, but not CD19-CD28-based counterparts, drives FMC63-epitope and total CD19 protein loss. Consistent with clinical observations, resistance to CD19-4-1BB-based treatment is associated with aberrant splicing, loss of heterozygosity, and frameshift/missense mutations in CD19 in exons encoding the FMC63 epitope. Mathematical modeling indicates that the failure of CD19-4-1BB-based CAR-T cells to eliminate CD19low cells, unlike CD28-based CARs, promotes the expansion of resistant populations. These results suggest that optimizing recognition of CD19low cells could prevent the emergence of resistant clones and reduce relapse rates. We also show that commonly used diagnostic anti-CD19 monoclonal antibodies detect epitopes distinct from FMC63 and may therefore give misleading CD19-positive results, underscoring the need for FMC63-specific assessment to guide diagnostic and therapeutic decisions.
Marta Krawczyk, K. Fidyt, Narcís Fernandez-Fuentes et al.· Leukemia· 0 citations
The potential of circRNA-based CD19-targeted CAR-NK therapy as an effective approach for enhancing the safety and efficacy of cancer immunotherapy is supported.
Qisheng Dong, Ying Liu, Na Chen et al.· International Immunopharmaco...· 0 citations
