Journal for Immunotherapy of Cancer最新文献

Background: Neoadjuvant immunotherapy has reshaped the treatment paradigm for esophageal squamous cell carcinoma (ESCC), yet mechanisms of resistance in non-responders remain poorly understood. Specifically, the spatial orchestration of the tumor microenvironment that limits effective antitumor immunity is not fully elucidated.

Methods: Integrating spatial transcriptomic analysis with large-scale tissue pathology, we mapped the spatial architecture of the immune landscape in immunotherapy-resistant ESCC to identify cellular and molecular determinants of treatment failure.

Results: We revealed a distinct immune exclusion phenotype in non-responders, characterized by peritumoral CD8⁺ T cell enrichment coupled with intratumoral depletion. At the invasive front, we identified COL11A1⁺ cancer-associated fibroblasts (CAFs) and SPP1⁺ tumor-associated macrophages as spatially correlated markers of immune exclusion, demarcating regions characterized by limited T cell infiltration. Mechanistically, a distinct subpopulation of LAMC2⁺ tumor cells localized at the tumor boundary acts as the master orchestrator of this barrier. These LAMC2⁺ cells exhibit aberrant lactate metabolism and elevated stemness, driving CAF activation via Semaphorin 3C secretion. Strikingly, this 'LAMC2⁺ boundary tumor cell-immune-privileged niche' axis exhibits pan-cancer correlations with immunotherapy resistance, positioning LAMC2 as a robust predictive biomarker.

Conclusion: In this study, we identify the tumor invasive margin as a critical determinant of immunotherapy resistance in ESCC. By defining the specific spatial markers and molecular architecture of an immune-privileged niche associated with the immune-exclusion barrier, our findings demonstrate the value of spatially resolved microenvironmental analysis. Moreover, we propose that therapeutic targeting of this boundary niche represents a promising strategy to overcome resistance in ESCC.

Background: Macrophages can eliminate cancer cells through phagocytosis via the CD47/signal regulatory protein α axis, which provides promising targets for cancer immunotherapy as innate immune checkpoints. Although CD47 is overexpressed in multiple cancer types, it remains largely unknown whether and how CD47 can be targeted by manipulating its protein stability.

Experimental design: Multiple human cancer cell lines were used to identify the function of the ubiquitin-specific protease 2 (USP2) /speckle-type POZ protein (SPOP) axis and the USP2 inhibitor on CD47 protein stability by immunoblot and immunoprecipitation, real-time quantitative PCR, in vitro deubiquitination assay, cell fractionation assay, flow cytometry, and phagocytosis assay. We investigated the antitumor immune response and immunotherapy effects of the USP2 inhibitor using multiple syngeneic and orthotopic mouse tumor models, bioluminescence imaging, immune cell depletion, tumor-infiltrating lymphocyte (TIL) isolation, and flow cytometry.

Results: Here, we report that ML364, an inhibitor of the USP2 deubiquitinase, reduces the protein abundance of CD47. Mechanistically, USP2 deubiquitinates and protects CD47 from proteasome-mediated degradation. Furthermore, we reveal that USP2 itself can be ubiquitinated by the SPOP ubiquitin E3 ligase, which leads to USP2 degradation and decreased CD47 protein abundance. Functionally, ML364 promotes macrophage phagocytosis of cancer cells by reducing the expression of CD47 and enhances the efficacy of anti-programmed cell death protein-1 (PD-1) immunotherapy, thereby inhibiting tumor growth and improving the overall survival rate in multiple syngeneic and orthotopic mouse tumor models. Bioinformatic analyses indicate that low USP2 expression or high SPOP expression predicts a better response to anti-PD-1 treatment.

Conclusion: Hence, our findings reveal a pivotal role of the SPOP/USP2 axis in regulating CD47 protein stability and advocate for combining USP2 inhibitors with anti-PD-1 immunotherapy to combat cancer.

Background: Antigen loss and tumor heterogeneity present significant challenges for successful immunotherapies. T-cell receptor (TCR)-based therapies rely on the recognition of epitopes derived from intracellular tumor proteins presented by major histocompatibility complex class I molecules on cell surface. Solid tumor cells frequently lack immunoproteasomes, which are crucial for processing and presenting certain immunogenic epitopes. An effective strategy to mitigate the risk of antigen absence and tumor heterogeneity is to simultaneously target multiple tumor antigens, thereby providing critical rescue from disease relapse. Previously, we engineered a TCR mimic monoclonal antibody (TCRm) "ESK2", specific for Wilm's tumor 1 (WT1)-derived epitope RMFPNAPYL (RMF) in the context of HLA-A2, into a new chimeric antigen receptor T-cell format, antibody-TCR receptor (AbTCR)-chimeric signaling receptor (CSR). However, the RMF epitope is largely dependent on processing by the immunoproteasomes, which can be lost from leukemia cells and sometimes absent in solid tumor cells.

Methods: To mitigate antigen loss, tumor heterogeneity and broaden the reach of AbTCR T cells, we combined ESK2 with a new TCRm for an immunoproteosome-independent epitope derived from WT1, VLDFAPPGA (VLD), in the context of HLA-A2 molecules, named ESK3. ESK2 and ESK3 were tandemly engineered into one AbTCR-CSR construct, simultaneously recognizing both the WT1 RMF and VLD epitopes. To add additional specificity and potency, a CSR in these cells was engineered with a single chain variable fragment (scFv) for either CD33 to treat leukemia or mesothelin to treat solid tumors. The specificity and efficacy of the AbTCR-CSRs were evaluated in both in vitro and in vivo.

Results: In vitro studies demonstrated that the Tri-AbTCR-CSR (CD33 CSR) T cells showed the best killing activity against most acute myeloid leukemia cells. Similar levels of cytotoxicity were exhibited by ESK3 AbTCR-CSR (mesothelin CSR) against most solid tumor cell lines when compared with the Tri-AbTCR or a combination of ESK2 and ESK3 AbTCR-CSR. In animal therapy models, trispecific AbTCR-CSR T cells showed efficacy equivalent to single ESK2-AbTCR or ESK3-AbTCR-CSR T cells, against hematopoietic or solid tumor cells, further supporting the advantage of triple targeting strategy, overcoming epitope loss variants.

Conclusions: Trispecific T cells targeting immunoproteasome-dependent and independent epitopes of WT1 peptide/HLA-A2 complexes, plus a CSR recognizing a third tumor-associated antigen, present an effective and cost-efficient approach for overcoming tumor immune evasion.

Merkel cell carcinoma (MCC) exemplifies the paradigm of immunogenic tumors that nonetheless develop sophisticated immune evasion mechanisms. This is in line with the observation that MCC exhibits remarkable susceptibility to immune checkpoint inhibitors (ICIs), with responses in approximately half of patients with advanced disease in the first-line setting. However, 40-50% of patients show primary ICI resistance, while 20-30% of patients develop acquired ICI resistance on initial disease control. Still, the advent of ICI therapy represents a revolutionary rather than evolutionary advance in MCC management, fundamentally transforming outcomes from the dismal prognosis associated with conventional chemotherapy to durable responses extending substantially beyond 2 years. Still, important questions remain: (1) Is programmed cell death protein-1 or programmed death-ligand 1 inhibition more effective? (2) How long should ICI treatment continue? (3) What is the best choice of salvage therapy for patients with primary or acquired ICI resistance? (4) Which combination regimens can increase the share of patients benefiting from ICI? (5) Which patient/tumor characteristics predict response and its duration? Finally, (6) which timing of ICI therapy, that is, the neoadjuvant, adjuvant or therapeutic setting, offers the optimal overall outcomes for patients with MCC? A number of recent studies provide initial, but unfortunately not yet definitive answers.

Background: The adoptive transfer of T cells engineered to express chimeric antigen receptors (CAR-T) has shown high efficacy and safety in treating various hematologic malignancies. However, many hematologic disorders, such as BCR::ABL1-negative myeloproliferative neoplasms (MPNs), lack effective treatment options. Some of these neoplasms are marked by a recurrent mutation that results in the expression of mutant calreticulin (mCALR), a neoantigen absent in healthy tissues, making it a highly specific and appealing target for CAR-T cell therapy.

Methods: Five distinct CARs were designed based on available monoclonal antibody sequences that target mCALR and were subsequently used to generate CAR-T cells. The most effective construct was selected through functional in vitro assays against mCALR-positive cell lines. Its efficacy was then evaluated in cell lines, patient-derived cells, and orthotopic xenograft models, assessing tumor burden, CAR-T cell infiltration, and animal survival. Bulk and single-cell RNA sequencing were performed on patient-derived cells and residual tumor cells from CART-treated mice, respectively, to investigate potential resistance mechanisms. The impact of the most relevant pathway alteration on CAR-T efficacy was also analyzed. Pharmacological rescue assays using targeted agents were then conducted.

Results: Among the five constructs, one demonstrated superior and specific cytotoxicity against mCALR-expressing cells, with no activity against mCALR-negative controls. This CAR-T cell also eliminated patient-derived MPN cells and controlled disease progression in xenograft models, which correlated with the persistence of CAR-T cells and tumor infiltration. Transcriptomic profiling of patient samples and residual tumor cells in spleens of treated mice revealed upregulation of anti-apoptotic proteins. Functional assays confirmed reduced CAR-T efficacy in Bcl-2 high cells, which was restored by co-treatment with venetoclax, indicating a viable combination approach to overcome resistance.

Conclusions: This study demonstrates, for the first time, the successful targeting of mCALR with CAR-T cells as a therapeutic strategy for MPNs. The chosen construct shows strong preclinical efficacy against established cell lines and patient-derived cells. Additionally, transcriptomic profiling uncovered apoptosis resistance mechanisms and supports a combination strategy with BH3 mimetics, such as venetoclax. These findings provide a compelling rationale for ongoing preclinical development and future clinical application of anti-mCALR CAR-T cells for the treatment of MPNs.

Background: While adipose tissue constitutes a substantial proportion of breast composition, the functional characteristics and pathological relevance of the adipocyte microenvironment in breast carcinogenesis remain undercharacterized. This study employs single-nucleus RNA sequencing (snRNA-seq) to establish a comprehensive cellular atlas of adipocyte heterogeneity across molecular subtypes of breast cancer, aiming to elucidate subtype-specific adipocyte contributions to tumor microenvironment modulation.

Methods: snRNA-seq was performed on breast adipose tissues isolated from individuals without cancer and treatment-naïve breast cancer. Various adipocyte and pre-adipocyte subclusters were identified. Comparative analyses of cellular distribution and transcriptional profiles were performed across disease states and molecular subtypes. Pseudotime, cell communication, and immunofluorescence analyses were further implemented to investigate cellular dynamics and microenvironment interactions.

Results: snRNA-seq data of 86,529 nuclei were obtained. Three adipocyte and seven pre-adipocyte subclusters were identified, of which Adi_LDLR, Pre_Adi_LDLR, and Pre_Adi_LGR4_TGFBR1 were restricted to cancer-associated adipose (CAAs). Adi_LDLR and Pre_Adi_LDLR were enriched in estrogen receptor (ER)-positive CAAs and related to cell senescence and immunosuppression. Pre_Adi_LGR4_TGFBR1 was predominantly present in triple-negative breast cancer, functionally pro-proliferative, immunosuppressive, and lacked normal adipose function. The immunofluorescence intensity of LDLR (p=0.031) and TGFBR1 (p=0.038) was positively associated with disease recurrence, suggesting the formation of immunosuppressive niches by these cancer-specific adipose subsets in both subtypes. Cell communication analyses revealed a specific (pre-) adipocyte-macrophage interaction via ligand-receptor pairs involved in stromal remodeling and tumor migration for ER-positive tumors, whereas tumor proliferation and metastasis for triple-negative ones likely contribute to tumor progression.

Conclusions: This study delineated a distinct adipocyte landscape in breast cancer and subtype-specific immunosuppressive niches fostered by CAAs and (pre-) adipocyte-macrophage interactions. These findings provide novel therapeutic targets for microenvironment-directed interventions in breast oncology.

Sarcomas are rare malignancies of mesenchymal origin, characterized by significant biological and clinical heterogeneity. Many subtypes demonstrate limited sensitivity to standard systemic treatments, including immune checkpoint inhibitors. Cell therapy has emerged as a promising strategy, with the potential of durable clinical responses seen with genetically-engineered T-cell receptor T-cell therapies (TCR-T) such as those targeting the cancer-testis antigen MAGE-A4 in synovial sarcoma, leading to the US Food and Drug Administration approval of afamitresgene autoleucel in 2024. This constituted only the second approval of a cell therapy in a solid tumor following lifileucel in melanoma and demonstrated the potential of cell therapies in sarcomas. This review provides the current landscape and growing potential of cell therapies in sarcomas, including TCR-T, chimeric antigen receptor-T cells, tumor-infiltrating lymphocytes, natural killer (NK) cells, and mesenchymal stromal cells. However, the broader application of these therapies is hindered by the lack of targetable sarcoma-restricted immunogenic epitopes, spatiotemporal intratumoral heterogeneity, and a profoundly immunosuppressive tumor microenvironment that impedes effector-cell trafficking, expansion and persistence. While cell therapies hold promise for integration into precision medicine approaches for sarcomas, their successful implementation will require careful evaluation of clinical feasibility, logistical considerations and cost-effectiveness to optimize patient outcomes.

Purpose: Chemoradiotherapy (CRT) combined with anti-PD-1 for locally advanced esophageal squamous cell carcinoma (ESCC) has shown promising efficacy but lack the predictive biomarkers to identify patients who could benefit from this therapy. The predictive value of serum cytokines in ESCC patients remains unclear. We aimed to identify cytokine-based biomarkers for treatment response and survival in this setting.

Experimental design: Exploratory analyses were conducted on 81 ESCC patients from two phase II trials treated with CRT plus toripalimab, with validation in an independent prospective cohort (n=61). Nineteen serum cytokines were assessed at baseline, during, and post-CRT plus anti-PD-1 antibody. A cytokine-based risk score model (CYTOscore) was constructed. Multi-omics profiling including RNA-seq, WES, and spatial transcriptomics were performed to explore potential differences in tumor microenvironments.

Results: Cox analyses identified Interleukin-8 (IL-8), C-C motif chemokine ligand 3 (CCL3), and C-C motif chemokine ligand 4 (CCL4) as potential biomarkers and were used to constructed the CYTOscore. Patients stratified by baseline CYTOscore showed significantly longer OS (HR, 0.31; 95%CI, 0.16-0.62; p= 0.00045) and PFS (HR, 0.33; 95%CI, 0.17-0.62; p= 0.00036) in the low-risk group, which also had higher complete response (CR) rates (66% vs 35%, p=0.014). These finding were next validated in the external cohort, with the low-risk group demonstrating higher CR rates (66% vs 27%, p=0.039) and longer OS (HR 0.30, 95% CI 0.09-0.99, p=0.045). A nomogram incorporating baseline CYTOscore and clinical characteristics showed promising predictive accuracy in 1-, 2-, and 3-year OS (AUC=0.77, 0.78, and 0.76). Multi-omics analysis revealed enriched interferon-γ/α signaling in B cells within low-risk patients.

Conclusions: The CYTOscore based on IL-8, CCL3, and CCL4 effectively predicts treatment response and survival in ESCC patients receiving CRT plus anti-PD-1 antibody.

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.