Abstract INTRODUCTION: 4-1BB (CD137) is an inducible co-stimulatory molecule that plays crucial roles in immune activation. Upon ligand binding, 4-1BB forms trimeric signaling complexes triggering receptor activation. However, therapeutic monoclonal antibodies targeting 4-1BB often face challenges due to peripheral toxicities arising from systemic receptor activation. To address this, bispecific antibodies (bsAbs) have been developed to selectively activate 4-1BB in a tumor-associated antigen (TAA)-dependent manner. This report investigates whether a combination of bsAbs, pairing 4-1BB and TAA targeting arms, can further enhance 4-1BB clustering and activation in the tumor microenvironment. METHODS: A panel of Fc-silent bsAbs targeting different domains on 4-1BB and HER2 were generated and tested in combination for optimal 4-1BB signaling. For that purpose, 4-1BB reporter cells were co-cultured with HER2-expressing tumor cells. Functional in vitro assays were conducted using primary human T cells isolated from peripheral blood mononuclear cells to assess cytokine production and their tumoricidal activity. In vivo efficacy was evaluated in PBMC-engrafted NOG mice inoculated with JIMT-1, a HER2-expressing tumor cell line. RESULTS: The simultaneous binding of two bsAbs to the same epitope on 4-1BB, while targeting distinct epitopes on HER2, markedly enhances 4-1BB signaling, compared to the single bsAbs. 4-1BB signaling triggered by the bsAbs is driven by HER2 expression, with strong IL-2 release observed only in the presence of HER2-positive cells. In JIMT-1 xenograft models, combination of the bsAb pair with a T cell engager (TCE) resulted in superior antitumor activity and enhanced CD4+ and CD8+ T-cell infiltration within the tumor microenvironment, compared to either single bsAb plus TCE or the TCE alone. CONCLUSIONS: This study introduces a novel bsAb combination strategy that amplifies TAA-driven activation of 4-1BB+ T cells and may unlock similar opportunities to other TNFR superfamily targets such as OX40 and CD40. Citation Format: Lucie Diby, Pauline Malinge, Valery Moine, Lise Nouveau, Laurence Chatel, Krzysztof Masternak, Limin Shang, Walter Ferlin, Nicolas Fischer, Jose Saro, Mikael Pittet, Vanessa Buatois, Eric Hatterer. Tumor-selective activation of 4-1BB receptor via bispecific antibody combinations [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 5553.

Dendritic cells (DCs) are central orchestrators of anti-tumor immunity, yet their therapeutic utility has been limited by dysfunction and insufficient activation within solid tumors. Here, we report chimeric antigen receptor-engineered DCs (CAR-DCs) that potently prime T cells and reprogram the tumor microenvironment. We identify the intracellular domains of CLEC9A or DEC205 as essential for functional CAR-DC activity, enabling enhanced antigen uptake, cross-presentation, and robust cytokine production that drive strong T cell expansion and effector differentiation. Mechanistically, CARs incorporating CLEC9A or DEC205 intracellular domains triggered SYK phosphorylation and downstream signaling pathways. In both murine and humanized models, CAR-DC therapy suppressed tumor growth and promoted expansion of tumor-reactive T cells. These findings establish CAR-DCs as a scalable and clinically translatable platform for next-generation solid tumor immunotherapy.

Locally advanced rectal cancer (LARC) is treated with neoadjuvant chemoradiotherapy (nCRT), but only a minority of patients achieve a pathological complete response (pCR). Predictive biomarkers of response could help guide treatment decisions, yet none have reached clinical practice. In this exploratory study, we integrated six publicly available transcriptomic datasets and applied machine learning to derive a 186-gene signature predictive of nCRT response. The signature showed good performance in cross-validation (AUC 0.80) and was associated with consensus molecular (CMS4) and immune (iCMS3) subtypes enriched in responders. Gene set enrichment analyses highlighted pathways involved in tumor growth, immune regulation, and resistance. Spatial transcriptomic profiling of pre-treatment biopsies further identified compartment-specific markers, with tumor-associated genes showing greater predictive value. These results provide biological insights into response mechanisms and generate hypotheses for future validation. Larger prospective studies are required to assess the clinical utility of this approach.

Venn plots and selected GSEA pathways.

Head and neck squamous cell carcinoma (HNSCC) shows variable response to anti-programmed cell death protein 1 (PD-1) therapy, which can be partially explained by a combined positive score (CPS) of tumor and immune cell expression of programmed death-ligand 1 (PD-L1) within the local tumor microenvironment (TME). To better define TME immune determinants associated with treatment efficacy, we conduct a study of n = 48 HNSCC tumors from patients prior to pembrolizumab therapy. Our investigation combines a rapid bioorthogonal multiplex staining method with computational analysis of whole-slide imaging to capture the single-cell spatial heterogeneity and complexity of the TME. Analyzing 6,316 fields of view (FOVs), we provide comprehensive PD-L1 phenotyping and cell proximity assays across the entirety of tissue sections. While none of the PD-L1 metrics adequately predict response, we find that the spatial organization of CCR7+ dendritic cells (DCs) in niches better predicts overall patient survival than CPS alone. This study highlights the importance of understanding the spatial context of immune networks for immunotherapy.

Relationship between pathological response and circulating biomarker levels in the plasma of treated patients.

Longitudinal analysis of circulating biomarkers in the plasma of treated patients.

Immunofluorescence staining of residual PDAC in pathological responders and non-responders in FFX+CRT and losartan+FFX+CRT.

Immunotherapy has shown limited success in recurrent ovarian cancer (OC), with prognostic insights largely derived from treatment-naive tumors. We analyzed 697 tumor samples (566 primary and 131 recurrent) from 595 OC patients across five independent cohorts, capturing tumor-infiltrating lymphocytes (TILs) heterogeneity and identifying four immune phenotypes linked to prognosis and TIL:myeloid networks driving malignant progression. We found that in preclinical mouse models, mirroring inflamed human OCs, the recurrent Brca1mut tumors maintained activated TILs:dendritic cells (DCs) niches but evaded immune control through upregulation of COX/PGE2 signaling. Conversely, recurrent Brca1wt tumors displayed loss of TILs:DCs niches and accumulated immunosuppressive tumor microenvironment (TME) networks featuring Trem2/ ApoEhigh tumor associated macrophages (TAMs) and Nduf4l2high/Galectin3high malignant states. Recurrent tumors recapitulate the immunogenic landscapes of original cancers. Our findings reveal BRCA-dependent TIL:myeloid crosstalk as key to persistent immunogenicity in recurrent OC and propose new targets to enhance chemotherapy efficacy.

Heatmap showing differentially expressed genes (DEGs) and their expression in each patient in FFX+CRT and losartan+FFX+CRT-treated groups.

Macrophages can foster pro- or anti-tumor immune environments. In this issue of Immunity, Clark et al. report that altering the composition of the mitochondrial electron transport chain reprograms macrophages toward a CXCL9hiSPP1lo immunostimulatory phenotype, thus amplifying anti-tumor immunity.

Quantitative analysis of immunofluorescence staining in PDAC lesions from FFX+CRT-treated patients.

Top 30 upregulated and 30 downregulated genes in treatment group versus untreated.

BACKGROUNDImmune checkpoint blockade (ICB) is an effective treatment in a subset of patients diagnosed with head and neck squamous cell carcinoma (HNSCC); however, the majority of patients are refractory.METHODSIn a nonrandomized, open-label Phase 1b clinical trial, participants with recurrent and/or metastatic (R/M) HNSCC were treated with low-dose 5-azacytidine (5-aza) daily for either 5 or 10 days in combination with durvalumab and tremelimumab after progression on ICB. The primary objective was to assess the biologically effective dose of 5-aza as determined by molecular changes in paired baseline and on-treatment tumor biopsies; the secondary objective was safety.RESULTSThirty-eight percent (3 of 8) of participants with evaluable paired tissue samples had a greater-than 2-fold increase from baseline in IFN-γ signature and CD274 (programmed cell death protein 1 ligand, PD-L1) expression within the tumor microenvironment (TME), which was associated with increased CD8+ T cell infiltration and decreased infiltration of CD4+ T regulatory cells. The mean neutrophil-to-lymphocyte ratio (NLR) decreased by greater than 50%, from 14.2 (SD 22.6) to 6.9 (SD 5.2). Median overall survival (OS) was 16.3 months (95% CI 1.9, NA), 2-year OS rate was 24.7% (95% CI: 4.5%, 53.2%), and 58% (7 of 12) of treated participants demonstrated prolonged OS of greater than 12 months.CONCLUSIONOur findings suggest that low-dose 5-aza can reprogram systemic host immune responses and the local TME to increase IFN-γ and PD-L1 expression. The increased expression of these established biomarkers correlated with prolonged OS upon ICB rechallenge.TRIAL REGISTRATIONClinicalTrials.gov NCT03019003.FUNDINGNIH/NCI P01 CA240239.

Quantitative analysis of immunofluorescence staining in PDAC lesions from FFX+CRT and losartan+FFX+CRT-treated groups.

Neutrophils, previously considered a homogeneous immune cell population, exhibit substantial heterogeneity. Their diverse phenotypic and functional states are shaped by tissue microenvironments and disease-specific signals. However, the lack of robust fate-mapping methods and standardized classification criteria has led to overlapping and ambiguous descriptions of neutrophil heterogeneity. The growing number of neutrophil subpopulations reported in recent years highlights the need for a standardized framework to report how they might relate to each other. Here, we propose a framework that integrates maturation, tissue localization, and functional adaptations. This standardized system aims to harmonize research efforts, foster clearer cross-disciplinary communication, and accelerate both fundamental discoveries in neutrophil biology and the development of targeted therapies.

Differentially expressed genes (DEG) in losartan+FFX+CRT versus FFX+CRT, losartan+FFX+CRT versus untreated, and FFX+CRT versus untreated.

Surgical tumor resection is often the only curative option for the nearly 20 million newly diagnosed patients with cancer every year. Fluorescence-guided surgery techniques are being developed in an effort to improve margin detection and surgical resection outcomes, with several systemically administered imaging agents having gained clinical approval. However, it has been challenging to overcome limited margin contrast with current approaches and to navigate procedural complexities of intravenous contrast delivery. We hypothesized that "spray-on probes" with specificity for fibroblast activation protein alpha in peritumoral fibroblasts could improve fluorescence-guided surgery, detect smaller tumors, improve imaging accuracy, and reduce the amount of times a patient is hospitalized. We show that this strategy increases achievable tumor margin contrast by 5- to 10-fold and detects even microscopic cancer deposits. These improvements have the potential to transform patient outcomes by enabling more accurate cancer surgeries, reducing the number of follow-up surgeries, and leading to personalized treatment plans.

Gene sets associated with overall survival in losartan+FFX+CRT and FFX+CRT-treated groups.