Extended phenotypes of intra-tumoral and peripheral T-cells following adoptive transfer of FLY-1A TCR T cells.

A subset of triple negative breast cancer (TNBC) patients shows resistance to standard neoadjuvant chemotherapy (NAC), resulting in high relapse and mortality risk. This highlights the need for predictive biomarkers and alternative treatment strategies. Targeted molecular profiling was performed on post-NAC resection specimens from 138 TNBC patients, diagnosed across multiple centers between 2013 and 2022, all exhibiting extensive poor response, defined as >50% residual tumor and the development of distant metastasis. Integrated immunohistochemistry and genomic analyses were conducted to identify potentially targetable alterations. Most post-NAC TNBCs (60%) were HER2-ultralow or HER2-low. Among 85 patients with successful DNA sequencing, 2640 variants were detected, with TP53 mutations being most frequent (94%). Mutation count ranged from 3 to 1668 per patient (median n = 11). Several altered genes, including ERBB2, BRCA1/2, PIK3CA, and RB1, have been associated with favorable responses to targeted therapeutics in clinical trials. Moreover, 208 potential neo-peptide targets (median per patient n = 3) were detected across recurrently mutated genes such as ATM, CREBBP, IRS2, KEAP1, MSH6, NOTCH1, NOTCH2, POLD1, TP53, and TSC2. Molecular profiling of residual disease in extensively poor responding TNBC post-NAC revealed multiple potentially targetable variant, supporting the use of next-generation sequencing to guide personalized strategies for these high-risk TNBC patients.

Purpose: Anti-programmed death ligand-1 monoclonal antibody (anti-PD-L1 mAb) therapy has demonstrated notable clinical success. However, efficacy is often limited by transient interactions at the tumor-immune interface, limiting sustained immune activation. To address this, we hypothesized that liposomal delivery of anti-PD-L1 mAbs could enhance therapeutic efficacy. Nanosystems may increase binding avidity and prolong cell-surface retention, and enable multivalent antibody presentation through conjugation of multiple antibodies onto a single liposome. Methods: In this study, we engineered multivalent anti-PD-L1 liposomes (anti-PD-L1 LPs) by conjugating FDA-approved anti-PD-L1 mAbs (atezolizumab) at varying densities onto a clinically relevant liposomal formulation. The biophysical properties, cellular interactions, and therapeutic potential of anti-PD-L1 LPs were evaluated using two melanoma cell line models (BLM and MZ2Mel43), employing multiple analytical techniques. Results: Our results demonstrated that high-density anti-PD-L1 LPs exhibited superior binding avidity, prolonged membrane retention, and reduced non-specific cellular interaction with PD-L1-expressing cells compared to both low-density and non-targeted LPs. Additionally, in 3D tumor spheroid models, high-density anti-PD-L1 LPs showed deeper penetration, suggesting improved tissue accessibility compared with low-density formulations. Moreover, compared to free antibodies, anti-PD-L1 LPs displayed a higher association rate (k_on) and a significantly lower dissociation rate (k_off), resulting in an overall improved (lower) dissociation constant (K_D). Functional assays confirmed that anti-PD-L1 LPs achieved superior PD-L1 blockade compared to free antibodies. Importantly, in co-cultures of human peripheral blood mononuclear cells and tumor cells, anti-PD-L1 LPs maintained immunomodulatory activity comparable to free anti-PD-L1 antibodies. Conclusion: This study highlights the critical role of ligand density in enhancing binding strength, tumor retention, and tissue penetration of the anti-PD-L1 LP system. Our nanosystem offers a promising improvement over conventional anti-PD-L1 mAbs, supporting the broader application of this modular liposomal platform to other therapeutic antibodies in melanoma and other solid tumors.

Supplementary Materials and Methods

Retrieval of ROPN1/B-specific T-cells and TCR genes.

Supplementary Table 2: Overview of the data.

Background: Early-stage cutaneous melanoma (stage I/II) generally has a favourable prognosis, yet a substantial proportion of patients develop distant metastases. We investigated whether spatial immune features provide prognostic value independent of established clinical factors in early-stage melanoma, using the largest sample collection of stage I/II primary melanomas specifically designed to study prognosis. Methods: We analysed the discovery dataset of the Dutch Early-Stage Melanoma (D-ESMEL) study, comprising matched case-control pairs of stage I/II primary melanomas. Cases developed distant metastases; controls remained distant metastasis-free. Matching was performed on Breslow thickness, ulceration, sex, and age. Multiplex immunofluorescence (IF) was performed on 230 whole tissue sections (115 pairs) using a six-marker panel (CD8, CD3, CD79a, CD68, MelanA, DAPI) with spatial analysis. Bulk RNA sequencing with immune deconvolution and T-cell and B-cell receptor repertoire analysis was performed on 356 samples (178 pairs). Findings: Spatial analysis demonstrated significantly higher CD8+ T-cell density in controls (median 87·2 vs 59·1 cells/mm2, adjusted p = 0·029) and closer CD8+ T-cell proximity to melanoma cells (median mean nearest neighbour distance 117·5 vs 134·1 µm, adjusted p = 0·039). Controls more frequently exhibited an infiltrated spatial immune phenotype (46% vs 27%, p = 0·006), while cases predominantly displayed desert phenotypes (58% vs 42%, p = 0·033). In contrast, bulk immune deconvolution and T-cell and B-cell receptor repertoire diversity revealed no differences between matched cases and controls. Interpretation: In the largest multiplex IF study of stage I/II primary melanomas to date, spatial immune organisation captures prognostic information independent of established staging factors that bulk transcriptomic approaches cannot detect. These findings provide a foundation for developing spatial biomarkers to complement existing risk stratification in early-stage melanoma.

BACKGROUND:T cell-based immunotherapies have had limited success in glioma thus far. Here, we evaluate the literature on abundance, spatial distribution and phenotypical characteristics of T cells in the tumor micro-environment (TME) of IDH-mutant and IDH-wildtype glioma, with the aim to understand how these measures relate to immunotherapy resistance and to aid the development of immunotherapies for glioma. METHODS:Medline, Embase, Web of Science Core Collection, Google Scholar and the Cochrane Central Register of Controlled Trials were systematically searched up to May 6, 2025. Out of 4303 articles screened, 85 studies examining T cell immunity in human glioma were selected. We collected information about tumor subtype, grade, methods, T cell abundance, spatial distribution, phenotypes and prognostic significance. RESULTS:T cells are present in the glioma TME, but densities are heterogeneous and generally low, especially in IDH-mutant glioma. T cell abundance increases with higher WHO grade and upon recurrence. T cells cluster around blood vessels, especially in IDH-mutant glioma. Glioma-infiltrating T cells largely display a late-differentiated phenotype (CD45RA-CCR7-C62L-), expressing markers that signify sustained antigen activation and exhaustion (PD-1, CTLA-4, TIM-3, LAG-3, CD39, and TIGIT). This phenotype coincides with decreased anti-tumor cytotoxicity and is spatially enriched in the myeloid-rich, hypoxic tumor core. Prognostic significance remains controversial. CONCLUSIONS:T cells in glioma are scarce, generally fully differentiated and functionally inert. Understanding and reinvigorating the deficient T cell response will be essential for successful immunotherapies. Future research should incorporate functional and spatial immune profiling to optimize and personalize immunotherapeutic strategies for glioma patients.

Top-ranked ROPN1/B peptides and their scores for immunogenicity, non-cross reactivity, HLA-A2 binding and immunogenicity.

Expressions of ROPN1/B, NY-ESO1 and MAGE-A4 target antigens in healthy tissues, TNBC and other tumor types.

Supplementary Figure 7: Survival plots for TP3-TP2 delta CD4+ T cells.

Supplementary Table 1: Lung cancer representativeness table.

The extent of recognition of the ROPN1 target by FLY-1A TCR T cells correlates with level of expression.

This file contains supplementary methods and references.

ROPN1/B peptides identified with immunopeptidomics.

FLY-1A TCR T-cells do not recognize ROPN1-negative and/or HLA-A2-negative tumor cells.

Supplementary Figure 3: Correlation between the percentages obtained by manual gating and our computational pipeline.

Treatment with adoptively transferred T cells is challenged by limited longevity of therapeutic cells within tumors. To enhance the durability of anti-tumor T cell products, we have created T cell receptors (TCRs) with built-in co-stimulatory molecules. We observed that TCRs coupled to ICOS mediated exceptionally long-term responses, including delay of tumor recurrence and cures in a mouse melanoma model. TCR:ICOS T cells showed enhanced and antigen-specific production of inflammatory cytokines, enrichment for a stem-like state and resistance to exhaustion. TCR:ICOS-mediated activation of PI3K and NFκB, yet restrained activation of AKT. Genetic ablation of the ICOS-PI3K pathway neutralized the long-term anti-tumor effects. To translate TCR:ICOS to human T cells, we identified a single amino acid change in the cytosolic tail which enabled functional surface expression without proneness to TCR mispairing nor competition for CD3. Notably, the optimized receptor sustained functional performance of human T cells upon repeated stimulation across multiple tumor antigens. Collectively, we present a novel and uniformly applicable TCR:ICOS format that supports fitter T cell products for adoptive cell therapy. Newly designed TCR, with extracellular TCR-V and C domains, CD28 transmembrane domain, and ICOS and CD3ε intracellular domains (in short TCR:ICOS) shows: highly durable anti-tumor response and T cell persistence in mouse model inflammatory T cell phenotype, stem-like state and resistance to T cell exhaustion effects via activation of PI3K and NFκB, yet restraining activation of AKT translation to human T cells upon single amino acid mutation in tail no TCR mispairing nor competition for endogenous CD3 extension to multiple TCRs while preserving T cell fitness

Supplementary Figure 6: Validation of the delta-Log10NLR between time point (TP) 2 and 3 with counts obtained from Complete Blood Count (CBC) analysis.