Journal for Immunotherapy of Cancer最新文献

Background: ERBB2/HER2 is overexpressed or mutated in ~15% of gastric cancers due to amplification or mutation of the ERBB2 locus. While the tumor cell-intrinsic consequences of ERBB2 overexpression are well understood, much less is known about its effects on the tumor microenvironment.

Methods: We have developed genetically engineered ectopic and orthotopic syngeneic models of organoid-based gastric cancer that have allowed us to study the tumor microenvironment of ErbB2-overexpressing tumors, using spectral flow cytometry, single cell RNA sequencing, and TCR repertoire sequencing. Interventions such as anti-Erbb2 and anti-PD1 antibody treatments were used as well.

Results: We find that ErbB2 drives the infiltration of CD4⁺ and CD8⁺ T-cells, which express granzymes, FasL, and surface markers indicating chronic activation, and in the case of CD8⁺ T-cells, have undergone clonal expansion. The adoptive transfer of T-cells sorted from ErbB2-overexpressing tumors reduces the growth of ErbB2-expressing, but not control tumors in T-cell-deficient recipients. PD-1-specific checkpoint blockade synergizes with an ErbB2-targeting antibody to reduce the growth of ErbB2-expressing, but not control tumors. Mechanistically, ErbB2 overexpression results in micronuclei formation and the transcriptional activation of numerous interferon-responsive genes in vitro and in vivo; mice lacking the type I interferon receptor show higher engraftment rates and lower T-cell infiltration than wild-type controls.

Conclusions: The combined data indicate that ErbB2, perhaps by driving micronuclei formation, has immunogenic properties that manifest in the form of increased T-cell infiltration and expansion, which can be exploited therapeutically by combining PD1-directed checkpoint blockade with ErbB2-targeted therapy.

Background: The acidic tumor microenvironment (TME) in solid tumors, driven by abnormal metabolism and lactic acid accumulation, suppresses chimeric antigen receptor-T (CAR-T) cell efficacy while posing safety risks from on-target, off-tumor toxicity (OTOT). This study aims to develop a novel CAR-T technology that leverages lactic acid as a tumor-specific trigger to achieve precise control of CAR activity. The objective is to enable adaptation to the acidic TME while maintaining robust anti-tumor efficacy and mitigating OTOT.

Methods: We engineered a lactic acid-responsive promoter (LARP) using RNA sequencing-identified lactic acid-sensitive genes. This promoter was integrated into HER2-targeting CAR to construct LAR CAR-T cells. CAR expression dynamics under acidic vs neutral conditions were quantified via flow cytometry. Phenotypic profiling (memory markers), in vitro cytotoxicity, and cytokine secretion were assessed. In vivo OTOT was evaluated in our previously constructed humanized HER2 mice, while anti-tumor efficacy and OTOT were further tested in this mouse model bearing tumors.

Results: Our findings demonstrate that the LARP responds to lactic acid, leading to increased CAR expression in acidic conditions. The ex vivo-expanded LAR CAR-T cells exhibited an enhanced memory phenotype and superior tumor-killing capacity in vitro under acidity. In vivo, LAR CAR-T cells achieved tumor eradication comparable to conventional CAR-T cells and exhibited significantly enhanced safety profiles, characterized by the absence of acute hepatotoxicity and minimal off-target organ toxicity.

Conclusions: Our LARP strategy exploits tumor acidity as a precise low/high switch for CAR-T cells. By restricting potent CAR expression to the acidic TME while minimizing activity in normal tissues, LAR CAR-T overcomes key barriers of efficacy and OTOT in solid tumors. This lactic acid-sensing paradigm offers a clinically translatable platform for precise immunotherapy.

Background: Biliary tract cancer (BTC) is an aggressive malignancy with limited treatment options and a poor prognosis. Although immune checkpoint inhibitors combined with chemotherapy have improved patient outcomes, their toxicity remains concerning. This phase II multicenter trial evaluated the efficacy and safety of pembrolizumab plus lenvatinib with a reduced-dose gemcitabine and oxaliplatin (GEMOX) regimen as a first-line therapy for advanced BTC.

Methods: 60 patients with unresectable or metastatic BTC were enrolled from five centers in China. Patients received pembrolizumab (200 mg, every 3 weeks), lenvatinib (8 or 12 mg daily), and modified GEMOX (gemcitabine 1000 mg/m² and oxaliplatin 85 mg/m² on day 1 of each 3-week cycle) for 6-8 cycles, followed by maintenance with pembrolizumab and lenvatinib. The primary endpoint was objective response rate (ORR), and the secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety.

Results: At a median follow-up of 16.0 months, the ORR (complete response 5.0%, partial response (PR) 50.0%) and disease control rate were 55.0% and 93.3%, respectively. The median PFS and OS were 12.5 months (95% CI 7.93 to 16.3), and 19.5 months (95% CI 17.97 to not estimable), respectively. Elevated baseline CA19-9 (>37 U/mL) and carcinoembryonic antigen levels (>5 ng/mL) were independently associated with poor OS and PFS, respectively. The regimen showed manageable toxicity, with 95% of patients experiencing treatment-emergent adverse events (AEs), mostly grades 1-2; grade 3-4 AEs occurred in 65% of patients, with no treatment-related deaths. Immune-related AEs occurred in 11.7% of the patients and were predominantly mild.

Conclusions: Pembrolizumab plus lenvatinib with reduced-dose GEMOX demonstrated promising efficacy and a favorable safety profile in advanced BTC, suggesting that chemotherapy de-escalation may optimize the efficacy-toxicity balance. Further randomized studies are warranted to confirm these findings and refine biomarker-based treatment selections.

Background: The local effect of radiotherapy (RT) is enhanced by CD8⁺ T-cell responses elicited through dendritic cell (DC)-mediated cross-presentation of tumor antigens, facilitated by RT-induced damage-associated molecular patterns. The abscopal effect-regression of non-irradiated tumors-has been observed clinically, particularly in combination with immune checkpoint blockade, although it remains uncommon. To better understand how to enhance this effect, we investigated two RT/α-programmed death 1 (PD-1)-based triple combinations incorporating either α-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or CD122-targeted interleukin (IL)-2 complexes (IL-2c).

Methods: We tested these regimens in B16 melanoma and C51 colon carcinoma models in mice with one irradiated and one non-irradiated tumor on opposite flanks.

Results: In both models, RT/αPD-1/αCTLA-4 elicited a stronger abscopal response than RT/αPD-1/IL-2c. In the C51 model, RT/αPD-1/αCTLA-4 achieved a 61.5% abscopal cure rate, dependent on both CD8⁺ and CD4⁺ T cells. In contrast, the less effective RT/αPD-1/IL-2c response required only CD8⁺ T cells. The enhanced abscopal effect with RT/αPD-1/αCTLA-4 was associated with increased numbers, effector function, and reduced exhaustion of tumor-specific CD8⁺ tumor-infiltrating lymphocytes (TILs) and of CD4⁺ TILs, along with elevated CD80⁺CD86⁺ DCs in abscopal tumors, as shown by flow cytometry; immunofluorescence confirmed increased T-cell infiltration. CD4⁺ T-cell depletion during RT/αPD-1/αCTLA-4 treatment impaired abscopal but not irradiated tumor control, reducing infiltration of tumor-specific CD8⁺ T cells and conventional (c) DC1s, and diminishing cDC1-mediated cross-presentation in abscopal tumors. Activated CD4⁺ T cells upregulated CD80/CD86 on cDC1s and enhanced cross-presentation, partly via interferon-γ and tumor necrosis factor. Adoptively transferred tumor-specific CD8⁺ T cells from tumor-irradiated donors localized to unirradiated tumors and draining lymph nodes in αPD-1/αCTLA-4-treated recipients, but not in untreated or CD4⁺ T cell-depleted mice.

Conclusions: These results demonstrate that an RT-based combination therapy that robustly induces CD4⁺ T cells alongside CD8⁺ T cells can elicit a strong abscopal response and suggest that CD4⁺ effector T cells act at abscopal sites by promoting DC-mediated cross-presentation of tumor antigens to CD8⁺ T cells originating from the irradiated tumor.

Background: NUT carcinoma is a rare but highly lethal solid tumor without an effective standard of care. NUT carcinoma is caused by bromodomain-containing NUTM1 fusion oncogenes, most commonly BRD4::NUTM1. BRD4::NUTM1 recruits p300 to acetylate H3K27 forming expansive stretches of hyperacetylated chromatin called "megadomains" with the overexpression of corresponding oncogenes, including MYC. We hypothesized that transcriptional dysregulation caused by BRD4::NUTM1 would lead to the generation of cancer-specific antigens that could be therapeutically actionable.

Methods: We integrated genomics, computational antigen prediction software, targeted immunopeptidomics using single-labeled and double-labeled peptide standards, and gain/loss-of-function genetic experiments on a panel of cell lines (N=5), a patient-derived xenograft, a tissue microarray (N=77), and patient samples from the Tempus AI Sequencing Database harboring evidence of NUTM1 fusions (N=165). We created an αPRAME₄₂₅ T-cell receptor (TCR) × SP34 αCD3 bispecific molecule modeled after brenetafusp, an αPRAME₄₂₅ TCR bispecific T-cell engager, as well as αPRAME₄₂₅ TCR T-cells based on anzutresgene autoleucel and we applied these products to NUT carcinoma cells in vitro.

Results: We identified PRAME as the most commonly expressed cancer/testis antigen in patient samples harboring the three canonical NUT carcinoma fusions (BRD4::NUTM1, BRD3::NUTM1, and NSD3::NUTM1). Additionally, 56% (43/77) of NUT carcinoma tissue microarray samples stained positive for PRAME. BRD4::NUTM1 expression in HEK 293T cells enhanced PRAME levels and BRD4::NUTM1 knockout in NUT carcinoma cells reduced PRAME levels. Immunopeptidomics detected more PRAME-derived human leukocyte antigen (HLA) ligands (N=9) than all other cancer/testis antigens combined (N=5). Targeted mass spectrometry detected the HLA-A*02:01/SLLQHLIGL (PRAME₄₂₅) epitope in 100% (4/4) of HLA-A*02+, PRAME+ NUT carcinoma samples at higher levels (>0.01 fM) than HLA-A*02:01/RLDQLLRHV (PRAME₃₁₂) or HLA-A*02:01/YLHARLREL (PRAME₄₆₂). The αPRAME₄₂₅ TCR × SP34 αCD3 bispecific molecule and αPRAME₄₂₅ TCR T-cells each exhibited potent, T-cell mediated cytotoxicity against PRAME+ NUT carcinoma cells.

Conclusions: PRAME is highly and frequently expressed in NUT carcinoma, and the most common oncoprotein causing NUT carcinoma, BRD4::NUTM1, contributes to these high PRAME levels. PRAME epitopes presented by HLA class I are a previously unrecognized therapeutic vulnerability for NUT carcinoma that warrants clinical trials testing PRAME-targeted immunotherapies in this neglected patient population.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related death and remains a significant global health challenge. Cancer vaccines have emerged as a promising immunotherapy for long-term tumor control. While Listeria monocytogenes (Lm)-based intravenous vaccines can generate tumor-reactive CD8 T cells, clinical trial success has been limited. Here, we sought to determine whether in vivo targeting of gastrointestinal tissues with foodborne delivery of Lm-based cancer vaccines controlled tumor growth in murine models of CRC.

Methods: The ActA and InlB virulence genes were deleted from a mouse-adapted Lm strain expressing ovalbumin and containing an internalin A mutation (InlA^M Lm-ova) that allows epithelial cell invasion of mice to generate an oral vaccine administered via consumption of inoculated bread. Immunogenicity and safety were tested in C57Bl/6 mice. Vaccine efficacy was evaluated with CRC tumors delivered by colonoscopy-guided orthotopic transplantation into the colon submucosa. Microsatellite instability high MC38 cell line expressing ovalbumin or genetically engineered microsatellite stable AKPS (Apc ^KO Kras ^G12D Trp53 ^KO Smad4 ^KO) organoids expressing low levels of ovalbumin (lo^SIIN) were used. Vaccines were tested in prophylactic and therapeutic settings and in the context of immune checkpoint inhibitors (ICI).

Results: Oral immunization induced a robust CD8 T cell response that was similar in magnitude and phenotype to the fully virulent Lm. Immunized mice did not lose weight, and Lm was contained to intestinal tissues. Mice prophylactically immunized with the vaccine were protected from CRC tumors. Therapeutic immunization of mice bearing lo^SIIN AKPS tumors revealed curtailed growth of the local tumor but did not improve survival. Immunization with anti-programmed cell death protein-1 and anti-cytotoxic T-lymphocyte-associated protein 4 controlled tumors when coupled with therapeutic immunization. Protection correlated with accumulation of ova-specific CD8 T cells within the tumor.

Conclusions: Oral Lm-based cancer vaccines targeting CRC elicit robust, widely disseminated, and persistent tumor-specific immune responses in mice. These vaccines limit CRC development when administered prophylactically and provide tumor control when administered therapeutically with ICI. Thus, oral delivery of Lm-based cancer vaccines coupled with ICI may provide improved control of CRC progression in clinical application.

Background: Glioblastoma, one of the most aggressive brain tumors, has been largely resistant to conventional immunotherapies, underscoring the need for novel treatment approaches. A promising strategy involves simultaneously inhibiting immunosuppressive pathways in the tumor microenvironment, as these pathways play pivotal roles in immune evasion. However, the therapeutic potential of combined targeting of these key immunosuppressive pathways in glioblastoma remains underexplored. We hypothesized that co-targeting the transforming growth factor (TGF)-β and PD-1 pathways could enhance immune responses against glioblastoma.

Methods: Human glioblastoma datasets were interrogated for the expression of PD-L1, TGF-β, and TGF-β target genes. Bintrafusp alfa, a first-in-class bifunctional fusion protein that blocks PD-L1 while sequestering TGF-β in the tumor microenvironment, was used to simultaneously inhibit both pathways. Its effects were assessed in vitro using human and mouse glioma cells and in vivo in immunocompetent, syngeneic mouse glioma models. High-dimensional flow cytometry was used to analyze treatment-induced changes in the tumor microenvironment.

Results: We observed a strong correlation between TGF-β and PD-L1 co-regulation, suggesting interconnected immunosuppressive mechanisms as part of a gene expression network. In vitro, bintrafusp alfa inhibited TGF-β-induced Smad2 phosphorylation, a bona fide response marker of TGF-β pathway activation, and enhanced immune cell-mediated killing of glioma cells. In vivo, combined targeting of both immunosuppressive pathways significantly improved survival of glioma-bearing mice, with long-term survivors exhibiting protection from tumor re-challenge. This survival benefit was not seen in T cell-deficient mice, confirming the necessity of adaptive immunity. High-dimensional flow cytometry of single-cell suspensions from tumor-bearing hemispheres revealed a distinct remodeling of immune subsets in the bintrafusp alfa-treated group compared with control-treated mice.

Conclusions: Our findings provide strong support for the combined targeting of TGF-β and PD-L1 as a promising immunotherapeutic strategy to overcome immunosuppressive barriers in glioblastoma and induce potent antitumor responses.

Neoadjuvant chemoimmunotherapy (chemo-IO) has fundamentally reshaped the treatment paradigm for resectable non-small cell lung cancer (NSCLC), challenging long-held surgical boundaries and redefining what constitutes "resectable" disease. Trials such as CheckMate-816, KEYNOTE-671, and AEGEAN have demonstrated that integrating immune checkpoint blockade with chemotherapy yields unprecedented rates of pathological response and event-free survival, positioning chemo-IO as the new global standard for stage IB-IIIA NSCLC. Yet these advances bring new complexities; how do we define resectability in an era of immunotherapeutic downstaging, and how should multidisciplinary teams adapt to evolving biology? Traditional radiological and anatomic criteria now sit alongside immune-mediated regression and circulating tumor DNA (ctDNA) kinetics as measures of treatment success. ctDNA clearance and pathological response serve as powerful surrogates for long-term survival, with ongoing studies such as MERMAID-1/2 exploring their potential to guide adjuvant therapy and spare overtreatment. The modern challenge lies in integrating these biomarkers into surgical decision-making and developing standardized, biology-informed resectability frameworks. Future progress will depend on close collaboration between surgeons, oncologists, and translational scientists to expand surgical candidacy safely and define the next generation of curative strategies in lung cancer.

Background: The CDKN2A gene encodes two canonical tumor suppressors, p16INK4A and p14ARF, which safeguard cells from malignant transformation by inducing cell cycle arrest and apoptosis in response to aberrant growth signals. Paradoxically, many cancers overexpress these proteins when downstream effectors that enforce negative feedback regulation are lost or inactivated. For example, p14ARF, which regulates p53 activation, is aberrantly expressed in more than 50% of tumors with inactivating p53 mutations. Here, we evaluated the feasibility of targeting dysregulated p16INK4A and p14ARF expression using TCR-T cell therapeutics.

Methods: We analyzed a panel of p16INK4A- and p14ARF-derived peptides for HLA-A*02:01-associated presentation and recognition by CD8⁺ T cells. Antigen-specific T cell receptors were isolated from healthy donor repertoires and expressed in primary T cells to assess specificity, functional avidity, tumor recognition, and safety using in vitro T cell functional assays, in vivo tumor models, and an in vivo safety model.

Results: We identified a unique and well-presented p14ARF epitope that was consistently detected in the HLA-A*02:01-associated immunopeptidome of cancer biopsies but not in normal tissues. High-avidity ARF-specific TCRs were isolated from the peripheral repertoire of healthy donors, and TCR-transduced T cells mediated potent tumor cell killing in vitro and in vivo in preclinical models. Furthermore, targeting p14ARF-expressing cells did not result in detectable on-target toxicity in an in vivo safety model.

Conclusions: These findings demonstrate the feasibility of targeting dysregulated tumor suppressor proteins with TCR-T cell therapeutics and identify p14ARF as a promising target for future therapies.

Background: Immunotherapy has had limited success in pancreatic cancer, largely due to a low mutational burden and immunosuppressive microenvironment. Here we hypothesized that systemic delivery of viral antigens can redirect pre-existing antiviral immunity against pancreatic tumors.

Methods: Cytomegalovirus (CMV, a β-herpesvirus) was chosen, as the majority of the population is infected and it induces an extremely large/broad memory T-cell response. Mice latently infected with murine CMV (MCMV) were orthotopically implanted with pancreatic cancer cells and treated with systemic injections of MCMV T-cell epitopes. Tumor growth was monitored by ultrasound two times a week, and immune cell infiltration was analyzed by histology, flow cytometry and single-cell RNA sequencing (scRNA-seq). Statistical analysis was performed by two-way analysis of variance with Sidak correction.

Results: MCMV peptide-epitope therapy (MCMVp) promoted preferential accumulation of MCMV-specific T cells within pancreatic tumors, delaying tumor growth and increasing survival. Immunophenotyping and scRNA-seq analyses showed these T cells were highly activated and cytotoxic, leading to increased tumor necrosis and caspase-3 activation. Depletion of CD4 and CD8 T cells abolished the impact of MCMVp therapy, indicating the antitumor response is T-cell dependent. Together, these results show that CMV-specific T cells can be repurposed to combat pancreatic cancer.

Conclusions: Our studies reveal that CMV-specific viral memory T cells can be re-directed to control a solid tumor normally refractory to immunotherapy via a simple, intravenous injection of T-cell peptide epitopes. This mutation-agnostic approach has significant potential for the development of "off-the-shelf" therapeutics by stimulating pre-existing antiviral memory, and it is widely applicable due to the high prevalence of CMV.