Cell-type-specific promoters are used in gene therapy to restrict expression of the therapeutic payload. However, these promoters often have suboptimal strength, selectivity and size. Here, leveraging recent insights into the function of enhancers, we developed synthetic super-enhancers (SSEs) by assembling functionally validated enhancer fragments into multipart arrays. Focusing on the core SOX2-driven and SOX9-driven transcriptional regulatory network in glioblastoma stem cells (GSCs) 1 , we engineered SSEs with robust activity and high selectivity. Single-cell profiling, biochemical analyses and genome-binding data indicated that SSEs integrate neurodevelopmental and signalling-state transcription factors to trigger the formation of large multimeric complexes of transcription factors. Moreover, GSC-selective expression of a combination of cytotoxic (HSV-TK and ganciclovir) and immunomodulatory (IL-12) payloads, delivered using adeno-associated virus vectors, as a single treatment led to curative outcomes in a mouse model of aggressive glioblastoma. Notably, IL-12 induced an immunological memory that prevented tumour recurrence. The activity and selectivity of the adeno-associated virus and SSE were validated using primary human glioblastoma tissue and normal cortex samples. In summary, SSEs harness the unique core transcriptional programs that define the GSC phenotype and enable precision immune activation. This approach may have broader applications in other contexts when precise control of transgene expression in specific cell states is necessary.

DNA mutations are a well-characterized source of neoepitopes in immunotherapy. Here, we examined the contribution of dysregulated RNA processing to neoantigen production. Leveraging multi-omics and checkpoint inhibitor (CPI) response data from >1,000 patients, we identified reduced activity of the nonsense-mediated mRNA decay (NMD) pathway kinase SMG1 as a predictor of improved CPI response. NMD inhibition through SMG1 targeting stabilized transcripts containing premature termination codons, most of which were of non-mutational origin. This reshaped the major histocompatibility complex class I (MHC class I)-bound immunopeptidome and increased neoantigen abundance to levels comparable to high mutation burden tumors. Functionally, NMD inhibition drove antigen-dependent T cell-mediated tumor cell killing in vitro, promoted activation of tissue-resident T cells in patient-derived models ex vivo, and improved CPI efficacy in vivo. Our findings establish NMD inhibition as a strategy to harness a previously inaccessible source of canonical and non-canonical neoantigens, with the potential to increase tumor immunogenicity across cancers.

Multiple myeloma (MM) is associated with skewed T cell activation and function which is present in asymptomatic myeloma precursor conditions, but underlying mechanisms of progression remain undefined. Here, we assemble a large single-cell RNA sequencing dataset of the bone marrow and blood from patients with MM, precursor conditions, and non-cancer controls. We demonstrate that, unlike solid cancers, MM is not characterized by T cell exhaustion, but by antigen-driven terminal memory differentiation. This is influenced by tumour-intrinsic features including tumour burden and expression of antigen-presentation genes. Expanded TCR clones accumulating in MM are not enriched with viral specificities but accumulate in effector states in highly-infiltrated marrows. Additionally, we identify a role for T cell dynamics in patients treated with autologous stem cell transplantation and demonstrate T cell features predict progression from precursor to symptomatic MM. Together, these results suggest that anti-tumour immunity drives a distinctive form of cancer-associated T cell differentiation in MM.

Cancers rarely respond completely to immunotherapy. While tumors consist of multiple genetically distinct clones, whether this affects the potential for immune escape remains unclear due to an inability to isolate and propagate individual subclones from human cancers. Here, we leverage the multi-region TRACERx lung cancer evolution study to generate a patient-derived organoid - T cell co-culture platform that allows the functional analysis of subclonal immune escape at single clone resolution. We establish organoid lines from 11 separate tumor regions from three patients, followed by isolation of 81 individual clonal sublines. Co-culture with tumor infiltrating lymphocytes (TIL) or natural killer (NK) cells reveals cancer-intrinsic and subclonal immune escape in all 3 patients. Immune evading subclones represent genetically distinct lineages with a unique evolutionary history. This indicates that immune evading and non-evading subclones can be isolated from the same tumor, suggesting that subclonal tumor evolution directly affects immune escape.

Immunotherapy has revolutionized cancer treatment, yet only a minority of individuals respond clinically, necessitating alternative strategies that can benefit these patients. Novel immuno-oncology targets may achieve this through bypassing resistance mechanisms to standard therapies. We introduce Mining Immunotherapy Drug tArgetS (MIDAS), a multimodal graph neural network system for immuno-oncology target discovery. MIDAS leverages gene interactions, multi-omic patient profiles, immune cell biology, antigen processing, disease associations and phenotypic consequences of genetic perturbations. It generalizes to time-sliced data, outcompetes state-of-the-art baselines (including OpenTargets) and ranks approved targets above those in clinical development. Moreover, MIDAS recovers immunotherapy-response-associated genes in unseen patients, thereby capturing immunotherapy response determinants. Interpretability analyses reveal a reliance on autoimmunity, regulatory networks and immuno-oncology pathways. Functionally perturbing oncostatin M-oncostatin M receptor signalling, a proposed MIDAS target, in TRACERx melanoma-patient-derived explants yielded reduced dysfunctional CD8(+) T cells, which associate with immunotherapy response, and reduced CCL4levels. Furthermore, oncostatin M and oncostatin M receptor expression is associated with altered T cell and macrophage profiles in bulk transcriptomic data from patient samples. These data are consistent with a role for oncostatin M-oncostatin M in modulating the tumour microenvironment towards immunosuppressive, tumour-promoting phenotypes. Our results present a machine learning framework for analysing multimodal data for immuno-oncology target discovery.

Current evidence indicates that Parkinson's disease (PD) involves T cell-mediated inflammation, which plays a fundamental role in promoting neuroinflammation and neurodegeneration in patients and animal models. These T cells are specific to α-synuclein-derived antigens, including nitrated α-synuclein (NαSyn). Here, we sought to develop an experimental immunotherapy for PD based on the generation of regulatory T cells (Treg) specific to NαSyn, using the chimeric antigen receptor (CAR) technology. Accordingly, we first obtained an antibody specific to human α-synuclein containing three nitrated tyrosine residues (3NY-hαSyn), which displayed specific immunoreactivity in the serum of PD patients which correlated with the clinical score. Afterward, we generated CAR-Treg specific to 3NY-hαSyn and tested them in two PD models involving human α-synuclein. The CAR-Treg therapy substantially inhibited the inflammatory T cell response specific to α-synuclein-derived antigens, neuroinflammation, neurodegeneration, and the motor decline. This preclinical study indicates that the CAR-Treg therapy represents a promising therapeutic strategy for treating PD patients.

The immunosuppressive transmembrane protein PD-L1 was shown to traffic via the multivesicular body (MVB) and to be released on exosomes. A high-content siRNA screen identified the endosomal sorting complexes required for transport (ESCRT)-associated protein ALIX as a regulator of both EGFR activity and PD-L1 surface presentation in basal-like breast cancer (BLBC) cells. ALIX depletion results in prolonged and enhanced stimulation-induced EGFR activity as well as defective PD-L1 trafficking through the MVB, reduced exosomal secretion, and its redistribution to the cell surface. Increased surface PD-L1 expression confers an EGFR-dependent immunosuppressive phenotype on ALIX-depleted cells. An inverse association between ALIX and PD-L1 expression was observed in human breast cancer tissues, while an immunocompetent mouse model of breast cancer revealed that ALIX-deficient tumors are larger and show an increased immunosuppressive environment. Our data suggest that ALIX modulates immunosuppression through regulation of PD-L1 and EGFR and may, therefore, present a diagnostic and therapeutic target for BLBC.

Neoantigen vaccines are under investigation for various cancers, including epidermal growth factor receptor (EGFR)-driven lung cancers1,2. We tracked the phylogenetic history of an EGFR mutant lung cancer treated with erlotinib, osimertinib, radiotherapy and a personalized neopeptide vaccine (NPV) targeting ten somatic mutations, including EGFR exon 19 deletion (ex19del). The ex19del mutation was clonal, but is likely to have appeared after a whole-genome doubling (WGD) event. Following osimertinib and NPV treatment, loss of the ex19del mutation was identified in a progressing small-cell-transformed liver metastasis. Circulating tumour DNA analyses tracking 467 somatic variants revealed the presence of this EGFR wild-type clone before vaccination and its expansion during osimertinib/NPV therapy. Despite systemic T cell reactivity to the vaccine-targeted ex19del neoantigen, the NPV failed to halt disease progression. The liver metastasis lost vaccine-targeted neoantigens through chromosomal instability and exhibited a hostile microenvironment, characterized by limited immune infiltration, low CXCL9 and elevated M2 macrophage levels. Neoantigens arising post-WGD were more likely to be absent in the progressing liver metastasis than those occurring pre-WGD, suggesting that prioritizing pre-WGD neoantigens may improve vaccine design. Data from the TRACERx 421 cohort3 provide evidence that pre-WGD mutations better represent clonal variants, and owing to their presence at multiple copy numbers, are less likely to be lost in metastatic transition. These data highlight the power of phylogenetic disease tracking and functional T cell profiling to understand mechanisms of immune escape during combination therapies.

Checkpoint blockade immunotherapy has transformed cancer treatment. However, many patients fail to respond to these therapies, underscoring the urgent need for new therapeutic strategies. Targeting alternative pathways and immune cell subsets within the tumor microenvironment (TME) holds promise for improving outcomes in these patients. One key mechanism of anti-tumour immunity is the phagocytosis of tumor cells by myeloid cells, which can be suppressed by the interaction between the SIRPα receptor on myeloid cells and its ligand CD47, a "do not eat me" signal, on tumor cells. Inhibition of the SIRPα/CD47 axis restores myeloid cell activity, promotes antigen presentation and enables tumor cell elimination. In this study, we evaluated a novel bispecific antibody that simultaneously blocks SIRPα and PD-L1 (SIRPαxPDL1), critical immune checkpoint molecules frequently overexpressed in the TME. SIRPαxPDL1 demonstrated enhanced macrophage-mediated phagocytosis of tumour cells in vitro. Moreover, in the MC38 murine tumor model, SIRPαxPDL1 significantly improved tumor control and survival, compared to monotherapies targeting either PD-L1 or SIRPα alone or in combination. High-dimensional flow cytometry revealed the unique ability of SIRPαxPDL1 to promote monocyte-to-macrophage differentiation whilst enhancing CD8+ T cell infiltration and activation within the TME compared to the combination of SIRPα and PDL1 antibodies. Remarkably, SIRPαxPDL1 synergized with a tumour-targeting antibody, further augmenting tumor control and survival. These findings highlight, for the first time, the potential of SIRPαxPDL1 to remodel the myeloid compartment and activate antitumor immunity, offering a promising novel avenue in cancer immunotherapy and combination approaches. Mariela Navarrete, Cristobal Costoya, Erik van Buijtenen, Simone Wouters, Joost Kreijtz, Paul Vink, Hans van Eenennaam, Sergio A. Quezada. A novel SIRPαxPD-L1 bispecific antibody remodels the innate and adaptive tumor microenvironment, and enhances tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2236.

Effector/Treg ratio is significantly lower in tumor tissues and CD8+ in metastatic tissue is significantly more regulated compared with other tissue types. A, The horizontal black bars indicate the median values for each compartment. The ratio of effectors to regulatory T cells was significantly lower in primary or metastatic tumor tissues compared with the normal tissue (CD8/Treg) and PBMCs (CD4eff/Treg). B, Single level expression and co-expression of B7 and TNFR superfamily co-inhibitory and co-stimulatory molecules on T-cell subsets were quantified by flow cytometry in matched peripheral blood mononuclear cells (PBMC), normal tissue, primary, and metastatic tumor tissues obtained from all patients. Displayed is a heatmap depicting the mean percentage of CD8+, CD4eff (CD4+ FoxP3–), and Treg (CD4+ FoxP3+) cells expressing individual immune checkpoint molecules and proliferating markers in each tissue sample. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

PURPOSE:Efficacy of immune checkpoint inhibitors in unselected patients with metastatic castration-resistant prostate cancer (mCRPC) is limited. The NEPTUNES study evaluated combination nivolumab and ipilimumab in patients with immunogenic signature-positive (ImS+) mCRPC. MATERIALS AND METHODS:This open-label, 2-cohort, phase II trial enrolled patients with ImS+ mCRPC progressing on ≥1 previous line of treatment. ImS+ was defined by (1) mismatch repair deficiency (MMRD); (2) DNA damage repair gene loss; and/or (3) high inflammatory infiltrate (HII). Patients received four doses of nivolumab 1 mg/kg + ipilimumab 3 mg/kg (C1) or nivolumab 3 mg/kg + ipilimumab 1 mg/kg (C2) followed by nivolumab 480 mg once every 4 weeks up to 10 cycles. The primary end point was composite response rate (CRR) assessed radiologically, biochemically, and by reduction of circulating tumor cells. Secondary end points included toxicity, progression-free survival, overall survival, and duration of response. RESULTS:Between May 2018 and June 2022, 35 (C1) and 36 (C2) patients commenced treatment. The CRR in C1 was 14/35 (40%, 90% CI, 26% to 55%) and in C2 was 9/36 (25%, 90% CI, 14% to 40%). The overall CRR was 23/71 (32%, 90% CI, 23% to 43%). Response rates were higher in patients with MMRD (7/10), BRCA2 loss (4/8), and HII ± other ImS+ features (13/30). Duration of response for patients with HII without other ImS+ features, DNA repair gene loss without MMRD, and MMRD was 2.6, 17.3, and 10 months, respectively. Grade 3 to 4 treatment-related adverse events occurred in 22/35 (63%) in C1 and 12/36 (33%) patients in C2. There were no treatment-related deaths. CONCLUSION:Nivolumab 1 mg/kg + ipilimumab 3 mg/kg is an active treatment in ImS+ pretreated mCRPC. Nivolumab 3 mg/kg + ipilimumab 1 mg/kg has less toxicity but may have lower efficacy. HII is a promising prospectively tested predictive biomarker in prostate cancer that could be integrated into future trials.

SiNET mutational load. This shows a comparison of the siNET mutational load against 33 TCGA cohorts from the Multicenter Mutation Calling in Multiple Cancers (MC3; ref. 63) project.

Regulatory T cells (Tregs) accumulate in the tumor microenvironment, where they suppress antitumor immunity and hinder immunotherapy efficacy. Antibody-mediated Treg depletion has emerged as a promising strategy, but its clinical translation has been hampered by incomplete mechanistic understanding, target overlap with effector T cells, and toxicity concerns. This review evaluates key determinants of Treg-depleting therapies, including the choice of target, antibody isotype and engineering, and the Fc gamma receptor landscape that governs effector function. We examine advances in next-generation antibodies targeting CTLA-4, CD25, CCR4, and CCR8, highlighting preclinical insights, early clinical outcomes, and lessons from toxicity profiles. Among next-generation approaches, Fc-optimized anti-CTLA-4 and CCR8 antibodies demonstrate selective intratumoral Treg depletion with partially improved tolerability, fueling progression into phase II/III trials. Continued refinement through novel designs, such as conditionally activated or bispecific antibodies, will be essential to balance efficacy and safety. Together, these strategies hold potential to establish Treg depletion as a viable therapeutic modality in cancer.

CD8+ in metastatic tissue is more regulated compared with other tissue types. Co-expressions of key B7 and TNFR superfamily co-inhibitory and co-stimulatory molecules on T-cell subsets were quantified by flow cytometry with matched PBMCs, normal tissue, and tumors from primary and metastatic sites. A, SPICE analysis of all CD8+ and CD4eff T cells displaying the mean co-expression of checkpoint molecules across tissue types. B, Unsupervised FlowSOM of CD8 and CD4eff clustering demonstrates that PBMCs (red box) are distinctly different to other tissues types. C, UMAP distribution of each FlowSOM CD8 and CD4eff population (blue) on each tissue subtype (orange).

Acquisition of cytotoxic activity in CD4+ T cells (TCTX) can promote potent anti-tumor activity thus holding promise as a therapeutic approach. However, how this activity is regulated remains poorly understood. Here, we demonstrate that tumor-infiltrating CD4+ TCTX activity is restrained by a post-transcriptional regulatory checkpoint. In untreated tumors, CD4+ TCTX exist in a poised state, characterized by abundant Gzmb mRNA but limited Granzyme B (GzmB) protein. Differentiation into poised TCTX is regulated by the Blimp-1-Bcl6 axis and requires type-I interferon signaling. Treatment with anti-CTLA-4 or anti-LAG-3 plus anti-PD-1 removed the block to GzmB protein production by repressing expression of the post-transcriptional regulator Zfp36l1 . Constitutive Zfp36l1 expression abrogated the effects of anti-CTLA-4 while deletion of Zfp36l1 and its paralog Zfp36 triggered GzmB protein production and promoted tumor control. These data identify ZFP36/ZFP36L1 as a key post-transcriptional regulatory checkpoint of CD4+ TCTX activity and a potential immunotherapy target in cancer. ### Competing Interest Statement M.T. has a funded collaboration with AZ on a topic unrelated to this study. The remaining authors declare no competing interests Cancer Research UK, C416/A18088, CANCTA-2022/100001, C36463/A22246, C36463/A20764, C33499/A20265 Medical Research Council, MR/W002337/1, MR/T028270/1 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BBS/E/B/000C0407

The geographical immune landscape in siNET shows predominantly peri-tumoral T cells. A and B, IHC staining of siNET. T-cell subsets stained are CD8+ T cells (red), CD4+ T cells (brown), and FoxP3+ (blue), and tumor cells are stained for Cytokeratin (green). These images of two siNET show examples of a tumor with intra-tumoral T cells (A) or with mainly peri-tumoral T cells (B). Examples of intra-tumoral or peri-tumoral T cells are circled in black in A and B, respectively. C, Plots of T-cell counts per mm2 showing that T cells are predominantly peri-tumoral rather than intra-tumoral in these tumors. D, Respective CD8/Treg ratio (Log10) in primary versus metastatic, peri-tumoral, and intra-tumoral. Horizontal bars represent the mean; error bars show ± standard error of the mean (SEM). *, P < 0.05; ****, P < 0.0001.

Clinicopathological features of siNET cohort.

Effector T cells in metastatic tissue are significantly more regulated compared with other tissue types. Co-expressions of key B7 and TNFR superfamily coinhibitory and costimulatory molecules on T-cell subsets were quantified using PD-1 as backbone by flow cytometry with matched normal tissue and tumors from primary and metastatic sites. A, Graphs depict frequency of co-expression with PD-1 and ICOS, CTLA-4, and Granzyme B (GzmB) on CD8+. Horizontal bars represent the mean; error bars show ± standard error of the mean (SEM). B, Graphs depict frequency of high levels of PD-1 (PD-1hi) on CD8+. Horizontal bars represent the mean; error bars show ± SEM. C, Graphs depict frequency of co-expression with PD-1 and ICOS on CD4eff. Horizontal bars represent the mean; error bars show ± SEM. D, Graphs depict frequency of high levels of PD-1 (PD-1hi) on CD4eff. Horizontal bars represent the mean; error bars show ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

Glioblastoma is invariably lethal and responds poorly to immune checkpoint blockade. Here, we examined the impact of regulatory T (Treg) cell depletion on glioblastoma progression and immunotherapy responsiveness. In human glioblastoma, elevated Treg cell signatures correlated with poorer survival outcomes, with these cells expressing high levels of CD25. In Nf1-'-Pten-'-EGFRvIII+ glioblastoma-bearing mice, a single dose of non-interleukin-2 (IL-2) blocking (NIB) anti-CD25 (anti-CD25NIB) antibody depleted Treg cells and promoted CD8+ T cell clonal expansion and partial tumor control, further enhanced by programmed cell death-1 (PD1)-blockade. Treg cell depletion induced interferon-y (IFN-y)-dependent tumor microenvironment remodeling, increasing Fcy receptor (FcyR) expression on intratumoral myeloid cells and enhancing phagocytosis. Combination of anti-CD25NIB with anti-EGFRvIII tumor-targeting antibodies resulted in complete tumor control. Anti-human CD25NIB treatment of glioblastoma patient-derived tumor fragments effectively depleted Treg cells and activated CD8+ T cells. These findings underscore the therapeutic relevance of Treg targeting in glioblastoma and unveil potent combination strategies for anti-CD25NIB based on innate cell activation.

While antagonistic antibodies targeting CTLA-4 and PD-1 have demonstrated significant success in cancer immunotherapy, agonistic antibodies such as anti-OX40 have shown limited efficacy. This disparity is potentially due to a lack of understanding of how antibody Fc regions interact with FcγRs on innate immune cells. Anti-OX40 antibodies currently in clinical trials are predominantly human IgG1, a strong FcγR engager. This design assumes differential surface OX40 expression will result in regulatory T cell (Treg) depletion and CD4+ effector T cell (Teff) activation, yet the therapeutic outcomes remain suboptimal. Using transplantable murine cancer models, we compared the efficacy of anti-OX40 isotypes with distinct FcγR-binding affinities. Weak FcγR-binding mouse IgG1 triggered robust pan-T cell activation and expansion within the tumor microenvironment while reducing Treg suppressive capacity and destabilizing their transcriptional program. Conversely, strong FcγR-binding mouse IgG2a resulted in the depletion of both Tregs and Teffs from tumors, compromising therapeutic efficacy. Notably, combining anti-OX40 mouse IgG1 with anti-PD-1 resulted in synergistic responses, achieving nearly 100% complete tumor rejection, while anti-OX40 mouse IgG2a failed to show such synergy. These findings indicate that anti-OX40 antibodies with strong FcγR-binding may hinder optimal anti-tumor activity. A more effective approach could involve using weak FcγR-binding isotypes, which preserve effector T cells while activating agonistic signaling and impairs Treg function, thereby enhancing therapeutic efficacy without off-target effects. Future studies will validate these results in patient-derived tumor models to inform the development of anti-OX40-based therapies. Cristobal Costoya, Mariela Navarrete, Aniruddh Tyagi, Dafne Franz Demane, Maria Vila de Mucha, Sergio A. Quezada. Non-depleting anti-OX40 antibodies boosts tumor-infiltrating T cells and impairs Tregs without effector T cell depletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2251.