Journal for Immunotherapy of Cancer最新文献

Background: Although immune checkpoint inhibitors (ICIs) have significantly improved outcomes for patients with certain cancers, their efficacy is largely confined to "hot" tumors characterized by robust infiltration of tumor-specific CD8⁺ T cells. Conversely, tumors expressing B7-H4 often exhibit an immunologically "cold" tumor microenvironment with poor T cell infiltration, contributing to primary resistance to programmed cell death protein 1 (PD-1) blockade.

Methods: We evaluated the association between B7-H4 expression and clinical outcomes in ICI-treated patients using public immunotherapy datasets. The role of B7-H4 in mediating resistance to PD-1 therapy was examined in mouse tumor models. A fully human anti-B7-H4 monoclonal antibody (clone A8) was generated via phage display screening from a non-immunized human single-chain variable fragment library. In vitro assays assessed antibody-induced tumor cell death and immune activation, while in vivo efficacy was tested in MC38-mH4 and SKOV3-hH4 tumor models, as well as human colorectal cancer organoids. Statistical analyses included Student's t-test, one-way analysis of variance, and Kaplan-Meier survival analysis, with p<0.05 considered significant.

Results: High B7-H4 expression was associated with inferior prognosis in patients receiving ICI therapy. In MC38-mH4 tumors, B7-H4 expression conferred resistance to anti-PD-1 treatment. We identified A8, a novel antibody targeting the IgV-like domain of B7-H4, with cross-reactivity to both human and mouse B7-H4. A8-hIgG1 and its Fab fragment induced dynamin-dependent endocytosis of B7-H4, resulting in lysosomal accumulation, altered lysosomal membrane permeabilization and intracellular acidification, ultimately triggering ferroptosis, a form of immunogenic cell death. A8 binding was enhanced under acidic conditions (pH 5.5), promoting lysosome-dependent degradation of B7-H4. A8-induced ferroptosis enhanced dendritic cell maturation, macrophage phagocytosis, and T cell activation. In vivo, A8 promoted CD8⁺ T cell and HER2 chimeric antigen receptor-T cell infiltration, inhibited tumor growth, and synergized with PD-1 blockade to overcome primary resistance in multiple preclinical models. This immunogenic and lysosome-dependent cell death mechanism was unique to A8 among the anti-B7-H4 antibodies tested.

Conclusions: Our study identifies a novel mechanism by which a fully human anti-B7-H4 antibody induces lysosome-dependent immunogenic tumor cell death. These findings support the therapeutic potential of A8 as a single agent or in combination with PD-1 blockade to overcome immune resistance in B7-H4-expressing "cold" tumors.

Background: Granzyme K (GZMK) is a serine protease known for its perforin-dependent cytotoxicity. However, the non-cytotoxic role of GZMK in lung adenocarcinoma (LUAD) remains largely elusive.

Methods: Multiomics datasets were integrated to investigate the clinical relevance of GZMK and its association with programmed death-ligand 1 (PD-L1) in LUAD. Recombinant human GZMK (rhGzmK) was applied in tumor-CD8⁺ T cell co-culture systems, with its effects on PD-L1 expression and CD8⁺ T-cell function evaluated via flow cytometry. Key signaling proteins were analyzed by Western blotting. To evaluate the therapeutic potential of GZMK inhibition, a selective GZMK inhibitor was combined with anti-programmed cell death protein 1 (anti-PD-1) therapy in both C57BL/6 and human peripheral blood mononuclear cells (huPBMC)-reconstituted NVSG humanized mouse models. Finally, multiplex immunofluorescence analysis was conducted on paired pretreatment and post-treatment specimens from a clinical cohort of patients with LUAD receiving immunotherapy to assess the spatial dynamics of GZMK expression in response to treatment.

Results: GZMK upregulated PD-L1 expression on tumor cells and enhanced PD-L1/PD-1 binding. Furthermore, GZMK promoted CD8⁺ T-cell dysfunction through the induction of apoptosis, the promotion of CD8⁺ T-cell exhaustion and suppression of proliferation. Mechanistically, cleavage of F2R-like trypsin receptor 1 (F2RL1) by GZMK activated the AKT Serine/Threonine Kinase (AKT) /glycogen synthase kinase-3β/β-catenin and Janus kinase 2/signal transducer and activator of transcription 1 (JAK2/STAT1) pathways, triggering nuclear accumulation of β-catenin and phosphorylated STAT1, which ultimately drove PD-L1 transcription. Additionally, F2RL1 signaling upregulated COPS8, stabilizing PD-L1 through inhibition of its ubiquitin-mediated degradation. In vivo, pharmacological inhibition of GZMK synergized with anti-PD-1 therapy to suppress tumor growth and enhance CD8⁺ T-cell infiltration and function. Clinically, high baseline GZMK expression correlated with an improved response to immunotherapy, and anti-PD-1 treatment modulated the spatial distribution of GZMK within the tumor microenvironment.

Conclusion: In the absence of perforin, GZMK acquires an immunosuppressive function through F2RL1 activation on tumor cells, which in turn promotes the formation of an immune-suppressive niche. Accordingly, combined targeting of the GZMK/F2RL1 axis and the PD-1/PD-L1 pathway represents a promising synergistic strategy to overcome immune evasion in LUAD.

Background: Engineering chimeric antigen receptor (CAR) T cells with logic-gated synthetic Notch (synNotch) receptor circuits can enhance specificity and mitigate on-target/off-tumor toxicity. However, the conventional synNotch system uses two lentiviral vectors encoding the synNotch receptor and inducible CAR, requiring dual transduction and cell sorting, which limits clinical translation. Integrating the synNotch-CAR circuit into a single lentiviral vector could overcome this limitation, yet manufacturing CAR T cells with large transgenes remains challenging, as increasing transgene size drastically reduces lentiviral titers and T cell transduction efficiency. Current production workflows compensate for low transduction efficiency by sorting transduced cells, further impeding clinical translation. Consequently, these constraints have limited the broader development of synNotch-CAR T cell therapies.

Methods: We engineered a single-vector synNotch (svsNotch) system that integrates all components of the conventional dual-vector circuit into one lentiviral vector to facilitate clinical translation. To overcome the low lentiviral titers and T cell transduction efficiency caused by the large svsNotch transgene, we established an optimized CAR T cell production workflow for effector T cells with large lentiviral transgenes.

Results: Our optimized workflow increased T cell transduction rates by up to 14.8-fold and enabled the production of effector T cells with lentiviral transgenes exceeding the effective packaging capacity limit of 9.2 kb. As a proof of concept, we engineered human epidermal growth factor receptor 2 (HER2)-mesothelin (MSLN) svsNotch (9.2 kb), in which a synNotch receptor targeting HER2 regulates the expression of a second-generation 4-1BBζ CAR against MSLN to enable selective targeting of double-positive HER2⁺MSLN⁺ ovarian tumors. In vitro, HER2-MSLN svsNotch T cells demonstrated superior specificity to conventional dual-vector synNotch-CAR T cells, with selective cytotoxicity against HER2⁺MSLN⁺ but not HER2^koMSLN⁺ tumor cells. To enable in vivo monitoring, we engineered HER2-MSLN-click beetle green (CBG) svsNotch (10.1 kb) incorporating CBG luciferase. In mouse models using constitutive CAR T cells as controls, HER2-MSLN-CBG svsNotch T cells exhibited minimal cytotoxicity in the absence of HER2 and superior efficacy against HER2^lowMSLN^high and HER2^highMSLN^high tumors.

Conclusion: These data establish a framework for engineering logic-gated single-vector immunotherapies and provide an optimized workflow for generating CAR T cells with transgenes that exceed current size limitations.

Adoptive cell therapy (ACT) has demonstrated curative potential in select cancers, but its translation to solid tumors such as ovarian cancer (OC) has been hindered by multiple factors, including tumor heterogeneity, immune exclusion, and a profoundly immunosuppressive tumor microenvironment. This review provides a comprehensive analysis of current ACT modalities, including tumor-infiltrating lymphocytes, T cell receptor-engineered, and chimeric antigen receptor-T cell therapies, as well as emerging approaches such as bispecific T cell engager (BiTE)-secreting T cells, dual-targeting platforms, and synthetic antigen receptors. We examine their application in OC and contextualize relevant findings using insights from other solid tumors. Key barriers, including limited T cell persistence, antigen escape, and T cell exhaustion, are explored alongside strategies to enhance efficacy through cytokine armoring, checkpoint modulation, metabolic reprogramming, and gene editing. We further highlight innovations in safety engineering, including logic-gated and self-regulating synthetic circuits, to mitigate toxicity and improve precision. Additional attention is given to the evolving role of allogeneic products and in vivo engineering as scalable solutions. Finally, we emphasize the critical value of integrating high-dimensional tools such as spatial transcriptomics, single-cell profiling, and machine learning to refine ACT design, identify biomarkers of response, and support patient selection and stratification. Collectively, these advances offer a roadmap for overcoming the unique immunologic barriers to ACT in OC and accelerating the development of more potent, durable, and personalized T cell-based strategies.

Peptide presentation on human leukocyte antigens (HLAs) is essential for initiating T-cell responses and all consequences of this presentation including anticancer immunity or immune escape. Many studies have relied on in silico prediction tools rather than biological measurement of HLA presentation to study these effects. To better assess the frequency and consequences of neoantigen presentation, we overexpressed 125 combinations of full-length neoantigens and one HLA class I allele to experimentally validate presentation of mutated and non-mutated HLA ligands through HLA ligand isolation followed by tandem mass spectrometry. A successful presentation was observed only in 22% of predicted cases with strong implications on previously described downstream effects. For example, the association of HLA loss of heterozygosity with predicted neoepitopes was challenged for 58% (73/125) of combinations. Furthermore, when testing 51 sequences used for personalized messenger RNA neoepitope vaccines, we observed that clinical responses were independent of the presentation status of the neoepitopes. Even a presumably neoepitope-specific and strongly expanded T cell receptor clone from a neoantigen vaccination study could not be linked to a successfully presented neoepitope. Overall, these data highlight the importance of validating the presentation of neoepitopes to fully understand our interpretation of clinical mutation-specific responses and their related effects, including immune evasion.

Background: Oncolytic herpes simplex virus (oHSV) therapy is a live virus-based immunotherapy that lyses tumor cells which release antigens and activate antitumor immunity. oHSV therapy has been shown to increase ATP production and release of extracellular ATP (eATP). In the extracellular tumor microenvironment, eATP functions as an immune-activating damage-associated molecular pattern but is hydrolyzed to extracellular adenosine (eADO), which can be immune-suppressive. eADO is generated by the sequential action of ectoenzymes CD39 and CD73 (NT5E). Here, we examined the role of immunosuppressive eADO signaling in regulating antitumor immune efficacy of oHSV.

Methods: We evaluated changes in eADO signaling in vitro and in patient specimens after virotherapy. A genetic CD73 knock-out mouse model and blocking antibodies were used to assess the impact of CD73 on virotherapy in two different solid tumor models. Single-cell RNA sequencing was employed to assess changes in immune cell infiltration and communication. Flow cytometric immunophenotyping and immunofluorescent imaging were utilized to confirm single-cell sequencing predicted changes in tumor microenvironment.

Results: Transcriptomic analysis of patient tumors pre-virotherapy and post-virotherapy with CAN-3110 revealed increased expression of the adenosine receptor gene ADORA2B after treatment. High NT5E gene expression, as well as gene signatures suggestive of adenosine signaling, correlated with a significantly worse prognosis for patients with solid tumors. Single-cell sequencing of immune cells recruited to tumor-bearing brain hemispheres in CD73 knockout mice revealed an increase in macrophage-mediated antigen presentation and CD4⁺ T cell cross-communication. Intracranial tumor-bearing CD73 knock-out mice treated with oHSV showed significant therapeutic improvement as the result of oHSV compared with wild-type mice. Combination of virotherapy with CD73 antibody blockade also resulted in enhanced antitumor efficacy.

Conclusions: Here, we identify that immunosuppressive eADO signaling in the TME is a major barrier to oHSV therapy and CD73 blockade prevents tumor immune escape. The combination of oHSV with CD73 blockade supports the development of an antitumor immune memory response in solid tumors. This study supports clinical development of this combination strategy.

Shared tumor-associated antigen (TAA) vaccines are the legacy of several generations of cancer immunologists who have labored to bring them to patients with cancer to improve their disease outcome and eventually use them to prevent cancer. TAA vaccines failed as monotherapy but the development of checkpoint inhibitors and other reagents that can modify immunosuppressive tumor microenvironment, warrants their reassessment in combination immunotherapy trials. They now sit on shelves and in freezers of academic labs and pharmaceutical companies, but if shown effective in the new setting, could be quickly turned into broadly applicable, inexpensive, off-the-shelf vaccines. Shared TAA vaccines also provide a unique opportunity to address the global cancer pandemic by repurposing them for cancer prevention. The opportunity that shared TAA vaccines provide to help patients now and to protect millions globally from the agony of cancer diagnosis in the future, is either not fully recognized, recognized but ignored, or at best, being put on hold.

Background: Mismatch repair deficiency (dMMR) colorectal cancer (CRC) is characterized by abundant tumor-infiltrating lymphocytes and tertiary lymphoid structures (TLSs). However, while B cells are pivotal for TLS formation, their function and the signaling pathways driving their activation in dMMR CRCs remain undefined.

Methods: Data from The Cancer Genome Atlas (TCGA) database analyzed by XCELL method and multiplex immunofluorescence (MIF) staining tissue slides were used to compare the abundance and distribution of TLSs and B cell populations between dMMR and proficient MMR cohorts. Then MLH1 knockdown models both in vitro and in vivo were used to mimic dMMR/high microsatellite instability (MSI-H) tumors and explore the influence of tumor cells on B cell behavior.

Results: TCGA analysis and MIF staining revealed a significant association between memory B cell abundance, TLS formation, and improved prognosis in dMMR CRCs. In vivo MLH1 knockdown models showed that B cell depletion enhanced tumor growth and reduced the efficacy of anti-PD-1 treatment in dMMR CRCs. Furthermore, in vitro experiments demonstrated a dsDNA/STING/type I interferon (IFN)/STAT1/ccl19 signaling pathway mediating the dMMR-induced increase in memory B cells.

Conclusions: In conclusion, these findings show that CCL19 generated by STING/type I IFN/STAT1 pathway in dMMR/MSI-H CRC cells can promote the expansion of memory B cells, which suppresses tumor growth and enhances the efficacy of PD-1 blockade.

Tumor-infiltrating microbes are emerging as a novel dimension of cancer biology, with growing evidence suggesting their potential as prognostic and predictive biomarkers. In this issue, Chen et al demonstrate associations between microbial signatures and treatment response in triple-negative breast cancer (TNBC). They join a growing list of examples whereby tumor-infiltrating microbes influence therapeutic efficacy, with mechanisms ranging from drug metabolism to immune modulation. Here, we explore the known mechanisms, as well as the methodological and conceptual challenges facing microbial biomarker research, including contamination risk, detection sensitivity, and the functional validation of microbial activity. As the field advances, integrating microbial profiling with genomic and immunological data, alongside foundational microbiological techniques, will be essential to clarify the role of microbes in cancer progression and treatment response. Ultimately, a deeper understanding of these microbial ecosystems may open new avenues for precision oncology in TNBC and beyond.