Journal for Immunotherapy of Cancer最新文献

Background: IGSF9, immunoglobulin superfamily member 9, has been reported to inhibit T cell proliferation and activation, thereby promoting tumor immune escape. Tumor-associated macrophages (TAMs), the most abundant tumor-infiltrating immune cells, play a crucial role in forming the immunosuppressive tumor microenvironment. We find that IGSF9 strongly binds to TAMs, however, how it affects TAMs function remains unreported.

Methods: The spatial transcriptomics dataset (GSE189487) was analyzed to clarify the relationship among IGSF9, senescent TAMs, and T cells. RNA-seq revealed differentially expressed genes. Flow cytometry was employed to assess the binding of IGSF9-ECD proteins to macrophages. Macrophage-educated by IGSF9 were co-cultured with T cells, and the phagocytosis was observed. Membrane-system yeast two-hybrid screening, GST pull-down, and co-IP were used to identify the binding partner of IGSF9. Cellular senescence markers and the senescence-associated secretory phenotype (SASP) were assessed by flow cytometry and immunofluorescence. LL/2-control or LL/2-Igsf9 cells were injected into C57BL/6, NSG, and monocytes-depleted C57BL/6 mice. Similarly, MC38-OVA-control or MC38-OVA-Igsf9 cells were inoculated into OT-II mice. Finally, anti-IGSF9 and its LALA-PG-mutant variant were administered to C57BL/6 mice to monitor tumor growth and SASP expression.

Results: The spatial transcriptomics dataset (GSE189487) revealed that the level of IGSF9 in tumor cells was positively correlated with the senescence of TAMs, and RNA-seq revealed the differentially expressed genes which were related to senescence. Macrophage educated by IGSF9 exhibited distinct senescence phenotypes and immunosuppressive features, and IGSF9 bound to TMUB1 to activate the IL-6/STAT3 signal pathway to trigger the above phenotype. In vivo data showed that IGSF9 could induce TAM senescence, leading to inhibition of T cell activity and formation of an immunosuppressive microenvironment, then promoting tumor immune escape. Treatment with anti-IGSF9, including the LALAPG-mutant variant that reduces antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis, mitigated TAM senescence and immunosuppression, activated T cells, and suppressed tumor growth.

Conclusions: TMUB1 as a binding protein of IGSF9 is first discovered, and TAMs educated by IGSF9 exhibit senescent and suppressive phenotype to orchestrate an inhibitory tumor microenvironment, which can be reversed by anti-IGSF9 to suppress tumor progression.

Background: Given the success of checkpoint inhibitor therapy in the advanced Merkel cell carcinoma (MCC) setting, there is interest in exploring immunotherapy as a neoadjuvant approach. We report the primary results of a neoadjuvant study of lenvatinib plus pembrolizumab in resectable MCC.

Methods: In this single-center, phase II open-label trial, resectable stage II-IV MCC patients received 6 weeks of neoadjuvant therapy with lenvatinib 20 mg orally daily plus pembrolizumab 200 mg intravenous dose every 3 weeks. Following local therapy, patients received continued adjuvant pembrolizumab monotherapy to complete a total treatment duration of 1 year. Pathological complete response (pCR) rate was the primary endpoint of the study.

Results: 26 patients were enrolled, including 5 (19.2%) with clinical stage II disease, 20 (76.9%) with stage III, and 1 (3.8%) with stage IV. Following neoadjuvant treatment, 2 patients (7.7%) were unable to undergo planned surgery, one due to progressive disease and one due to toxicity. On intention to treat, 15 of the 26 patients (57.7%) achieved pCR. Among 22 radiographically evaluable patients, 16 (72.7%) achieved an objective response. At a median follow-up of 20.0 months, median progression-free survival (PFS) has not been reached. PFS significantly correlated with radiographic response to neoadjuvant therapy. pCR was associated with superior PFS, though this result was not statistically significant (p=0.22). Grade 3 treatment-related adverse events (TRAEs) occurred in 14 patients (53.8%), most commonly grade 3 hypertension in 11 patients (42.3%). No grade 4-5 TRAEs were observed.

Conclusions: Lenvatinib plus pembrolizumab demonstrated encouraging efficacy with anticipated toxicity when used as neoadjuvant therapy for MCC. Further investigation of these promising findings is warranted.

Trial registration number: NCT04869137.

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.