Background Therapeutic options for advanced esophageal squamous cell carcinoma (ESCC) after first-line failure remain limited, particularly in patients previously exposed to immune checkpoint inhibitors (ICIs). We evaluated the efficacy, safety, and exploratory biomarker correlates of camrelizumab, an ICI, combined with nimotuzumab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, as second-line therapy for ESCC. Methods In this multicenter, single-arm, phase II study, patients with advanced ESCC who progressed after first-line therapy received camrelizumab (200 mg every 2 weeks) plus nimotuzumab (400 mg weekly). The primary endpoint was the objective response rate (ORR). Results Between November 2021 and December 2024, 46 patients were enrolled. The confirmed ORR was 32.6% (15/46; 95% CI 19.5 to 48.0) and the disease control rate was 82.6% (38/46; 95% CI 68.6 to 92.2). Median progression-free survival (PFS) was 9.13 months (95% CI 5.95 to 9.76), and median overall survival (OS) was 12.62 months (95% CI 9.40 to 15.01). Clinical activity was observed across subgroups, including immunotherapy-naïve and previously treated patients (ORR 32.0% vs 33.3%). Patients with EGFR amplification demonstrated a higher ORR (47.1% vs 24.0%) and longer median OS (13.17 vs 9.99 months). Among patients with M1 disease (n=39), the ORR was 30.8% (95% CI 17.0 to 47.6), the median PFS was 8.48 months (95% CI 5.95 to 9.59), and the median OS was 12.55 months (95% CI 7.95 to 14.88). Treatment-related adverse events occurred in 80.4% of patients, with grade ≥3 events in 8.7%. Exploratory analyses suggested that MUC16 mutations were associated with lower ORR (8.3% vs 46.4%, p=0.030), NOTCH3 mutations with prolonged survival (median PFS not reached vs 8.21 months, HR 0.20, p=0.015; median OS not reached vs 10.58 months, HR 0.22, p=0.026), and MTAP deletions with shorter PFS (3.71 vs 9.49 months, HR 3.18, p=0.005). Conclusions Camrelizumab combined with nimotuzumab demonstrated encouraging antitumor activity and a manageable safety profile as second-line therapy for advanced ESCC. Trial registration number NCT03766178 .
Background Non-small cell lung cancer (NSCLC) is one of the frequently occurring cancers characterized by molecular heterogeneity and multiple immune cell infiltration patterns, which are associated with treatment sensitivity and resistance. However, the specific microenvironmental cells and their mechanisms that lead to treatment resistance in patients need to be explored in greater depth. Methods On the basis of patients receiving neoadjuvant therapy in our center, a multicenter, multicohort NSCLC spatial transcriptome, single-cell transcriptome, T-cell receptor repertoire sequencing, bulk RNA transcriptome, phosphorylated proteome, genome mutation, and clinical data were included for a comprehensive assessment of the therapeutic and prognostic impact of HIF1A + CSF3R + neutrophils in NSCLC. In vitro experiments validated the functional phenotype of HIF1A + CSF3R + neutrophils and co-localization interactions with other cellular subpopulations. Gradient boosting machine (GBM) constructed region of interest (ROI) models for evaluation. Computer-aided drug design (CADD) was used to predict targeted small molecule drugs, and in vivo mouse models were constructed to assess the effectiveness of the combination treatment regimen. Results Centered on HIF1A + CSF3R + neutrophils, recruited exhausted T cells and stromal cells form a hypoxic niche within the tumor region, which was enriched in non-response patients. ROI composed of these specific cellular subpopulations, associated with senescence and glycolysis, accurately predicting NSCLC progression, prognosis, and microenvironment composition. CADD analysis identified that platycodin-D2 specifically targeted CSF3R, reducing HIF1A expression and inhibiting neutrophil activity. Combining navitoclax, platycodin-D2 with anti-programmed cell death protein 1 (PD-1) significantly suppressed tumor proliferation and improved the immunosuppressive microenvironment. Conclusion Our study emphasized the role of HIF1A + CSF3R + neutrophils in immunotherapeutic resistance of NSCLC, constructed a microenvironmental immune dysregulation network in a hypoxic ecological niche with HIF1A + CSF3R + neutrophils as the center. Platycodin-D2 specifically targeted HIF1A + CSF3R + neutrophils, enhancing the efficacy of anti-PD-1 therapy in NSCLC.
Brain tumors are the most common solid tumors in children. Despite recent advancements in cancer survival, the prognosis for high-grade pediatric brain tumors remains poor, with treatments resulting in severe long-term side effects. Over the last decade, immune checkpoint blockade (ICB) has emerged as a promising treatment strategy, with success across various tumor types, particularly in adult malignancies. However, its efficacy in pediatric brain tumors has been limited, which can be attributed to the unique characteristics of the brain tumor microenvironment (TME), including a low mutational burden, restricted T cell infiltration, and immunosuppressive milieu. Importantly, clinically approved ICBs primarily target T cell-associated pathways, thereby neglecting other dominant immune populations within the TME. Among these, tumor-associated macrophages (TAMs) often represent the most abundant immune cell compartment within pediatric brain tumors. TAMs consist of resident microglia and infiltrating bone marrow-derived macrophages and play a central role in establishing and maintaining an immunosuppressive environment. They promote tumor progression, suppress cytotoxic T cell activity, and contribute to therapeutic resistance. However, they are highly heterogeneous, and their phenotype and functions are regulated by the surrounding TME. This intrinsic plasticity makes them promising therapeutic targets: rather than solely attempting to enhance T cell activity, reprogramming TAMs may fundamentally remodel the immune landscape of pediatric brain tumors. In this context, immune inhibitory receptors emerge as critical regulators of TAM function. While traditionally studied in T cells, inhibitory receptors expressed on macrophages influence phagocytosis, cytokine production, antigen presentation, and polarization states. Emerging preclinical evidence indicates that targeting inhibitory receptors on TAMs can shift macrophages from suppressive toward pro-inflammatory phenotypes, enhance phagocytosis, and improve survival in brain tumor models. Nevertheless, the expression patterns and functional consequences of these receptors in pediatric brain TAMs remain incompletely characterized. In this review, we examine the role of immune inhibitory receptors on TAMs in pediatric brain tumors and discuss how targeting these pathways may reprogram the TME, enhance antitumor immunity, and provide new therapeutic avenues for this challenging group of malignancies. Unlike most pediatric brain tumor immunotherapy reviews, this review specifically evaluates myeloid-targeted inhibitory receptor pathways in the pediatric brain tumor context.
Background Oncolytic virus M1 encoding a mutant IL-18 decoy (OVM18) represents a novel virotherapy that integrates selective oncolysis with localized activation of the IL-18 pathway. However, the heterogeneity of therapeutic responses suggests that host immune determinants influence its efficacy. Methods To elucidate these determinants, we performed integrated immune profiling using bulk and single-cell transcriptomics, flow cytometry, and T cell receptor (TCR) repertoire analyses. The functional contribution of specific immune subsets was validated using Batf3⁻/⁻ mice and FTY720-mediated lymphocyte trafficking blockade. Potential synergistic immunotherapies were identified through transcriptomic database screening and confirmed in tumor-bearing mice receiving combined OVM18 and immune checkpoint blockade. Results We identified intratumoral IL-18R1 + CD8 + T cells as an important effector population, whose IL-18R1 expression and intratumoral abundance positively correlate with OVM18 efficacy. Integrated single-cell RNA-sequencing and cytometry analyses revealed that IL-18R1 + CD8 + T cells are clonally expanded, tumor antigen-enriched, polyfunctional cytotoxic T cells. Their generation requires conventional type 1 dendritic cells (cDC1)-dependent priming within tumor-draining lymph nodes, followed by trafficking into tumors to mediate antitumor responses. Notably, CTLA-4 blockade enhances cDC1-derived IL-12 and promotes IL-18R1 + CD8 + T-cell expansion, thereby overcoming resistance to OVM18 therapy. Combination treatment with OVM18 and anti-CTLA-4 enhances intratumoral IL-18R1 + CD8 + T cell infiltration, delays tumor progression, and prolongs survival in poor-responsive models. Conclusions Our findings establish IL-18R1 + CD8 + T cells as an important effector population in OVM18 therapy, thereby providing a strong rationale for combining OVM18 with CTLA-4 blockade to overcome therapeutic resistance and achieve durable antitumor responses.
Background Ovarian cancer is the most lethal gynecological malignancy and lacks therapeutic options in the recurrent setting. We previously determined the safety and initial clinical activity of intraperitoneal autologous monocytes with interferon gamma and interferon alpha (AMIGA) in women with recurrent, platinum-resistant ovarian cancer, but not all enrolled patients showed benefit, warranting further analysis to understand and improve this therapy. Experimental design Bulk RNA sequencing and single-cell RNA sequencing were conducted on circulating immune cells from patients with ovarian cancer treated with AMIGA. Primary human monocytes and T cells were isolated from healthy donors and used in co-cultures with ovarian cancer cell lines to test the effects of AMIGA on T-cell recruitment. Results Comparing circulating immune cells from long-term responders to non-responders from our clinical trial revealed an increased cell-mediated immune response in long-term responders. T cells in AMIGA-treated patients also upregulated unique, specific T cell receptor-beta chain genes. Bulk RNA-seq of peripheral blood mononuclear cells revealed AMIGA-driven upregulation of CXCL10 and CCL2 mRNA. An increase in these chemokines was consistently reflected in patients’ malignant ascites. Finally, in vitro models validated increases in CXCL10 as well as CCL2, and demonstrated that these chemokines exert variable effects on T-cell chemotaxis. Conclusions The AMIGA-driven effects on T cell recruitment and upregulation of specific T cell receptor-beta chain genes support the development of future combination therapies of intraperitoneal AMIGA and T cell-based immunotherapies.
Background Immune-related adverse events (irAEs) post-immune checkpoint blockade (ICB) are a leading cause of patient morbidity. Robust peripheral biomarkers of irAEs are required to improve patient stratification to existing treatment regimens, and these are currently lacking. Seropositivity for human cytomegalovirus (CMV) is associated with protection against severe (grade 3+) irAEs post-ICB; however, the impact of infection on systemic immunity is highly variable. Here, in a prospectively recruited pan-cancer ICB-treated cohort (n=472 patients), we investigate a novel relationship between the relative baseline titer of anti-CMV IgG antibody and organ-specific protection against irAEs.Methods Peripheral blood samples were collected from 472 patients prior to and following one cycle of ICB. CMV serotyping was performed on plasma, while flow cytometry and single-cell RNA/V(D)J sequencing were performed on peripheral blood mononuclear cells. Bulk RNA-sequencing was performed on sorted CD8+ T cells. Serological and phenotyping data were integrated with long-term clinical follow-up of response and irAEs.Results In CMV seropositive patients, whereas anti-CMV IgG antibody level demonstrates stability over years, high pretreatment titer is independently associated with reduced all-organ grade 3+ irAEs. This pan-organ association subdivides into organ-specific effects; protection against non-colitis irAEs being observed only in those with an above median titer of anti-CMV IgG antibody (PHigh titer vs CMV−=2.1×10−4), whereas CMV-related protection against colitis is unrelated to titer (PLow titer=0.0012, PHigh titer=0.0031). We demonstrate that anti-CMV IgG antibody titer is robustly coupled to peripheral immune subset composition, with higher anti-CMV IgG titer associated with elevated CD4+ and CD8+ T cell cytotoxicity and effector cell expansion. Conversely, CMV seropositivity is associated with generally reduced circulating Tregs cells irrespective of titer. Furthermore, we find exacerbated T cell receptor repertoire skewing toward the largest clones in High Titer individuals, with reduced survival of these clones following ICB treatment.Conclusions This work reinforces the importance of CMV in modulating ICB-induced irAEs, revealing a complex relationship between the degree of humoral anti-CMV immunity and organ-specific protection, while further highlighting the clinical utility of CMV serology in predicting ICB-induced irAEs.
Background SAR443216 is an engineered human trispecific antibody that targets human epidermal growth factor receptor 2 (HER2)-positive (HER2+) cancer cells and activates T cells via co-engagement of cluster of differentiation (CD)3 and CD28. This first-in-human, dose-escalation study evaluated the safety, efficacy, pharmacokinetics (PK) and pharmacodynamics of SAR443216 in participants with relapsed/refractory (R/R) HER2-expressing solid tumors.Methods In this multicenter, open-label, non-randomized Phase 1 study (NCT05013554), SAR443216 was administered intravenously at dose levels (DLs) of 18–900 µg. Dose escalation occurred within participants using intraparticipant lead-in dosing (2-week and 3-week lead-in cohorts). The primary objective was to determine the maximum tolerated dose (MTD); secondary objectives included PK, immunogenicity, and preliminary clinical activity.Results 40 participants (n≥3 at each DL) were treated with SAR443216. The median treatment duration was ~8 weeks in both 2-week and 3-week lead-in cohorts. Nearly all participants (97.5%) had at least one treatment-emergent adverse event (TEAE), of which 45% were grade ≥3. Most frequent TEAEs were cytokine release syndrome (CRS, 50%), fever (35%), alanine aminotransferase elevation (32.5%), aspartate aminotransferase elevation (27.5%), and infusion-related reactions (IRRs, 27.5%). No severe CRS, IRRs, fever, or pulmonary and cardiac toxicities were observed. Disease control rates were 34.5% in the 2-week and 36.4% in the 3-week lead-in cohorts. Average duration of disease stabilization was 10.48 weeks. Median follow-up time was 3.43 weeks. No objective responses were observed. The MTD was not reached. Dose-dependent PK showed overall consistent PK profiles across DLs. SAR443216 induced serum proinflammatory cytokines and increased multiple T-cell activation markers in peripheral blood mononuclear cells, indicating T-cell activation and target engagement. However, no clear trend in T-cell abundance or activation was observed among tumor-infiltrating T cells or other immune cells.Conclusion These findings indicate that SAR443216 treatment is feasible and well tolerated in participants with R/R HER2+solid tumors. Further evaluation is warranted to fully characterize the efficacy and safety of SAR443216.Trial registration number NCT05013554.
Background Using surrogates for overall survival (OS) may expedite the development of, and patient access to, novel treatments. We assessed potential surrogates for OS in patients with metastatic melanoma treated with nivolumab-containing regimens in the first-line treatment setting.Methods We used individual-patient data from 1865 patients enrolled in four randomized controlled trials studying single-agent nivolumab or combinations of nivolumab and ipilimumab against dacarbazine or immunotherapy. Using the two-level meta-analytic framework, we evaluated three candidate surrogates: objective response rate (ORR), progression-free survival (PFS), and time to next treatment or death (TNTD). We measured the patient-level associations between candidates and OS using ORs in the case of ORR and Spearman’s correlation coefficient (ρ) in the case of time-to-event surrogate endpoints. We used R2 to measure the trial-level association between ORs or HRs for each surrogate and the HRs for OS.Results For ORR, at the individual-level, OR of survival was equal to 12.29 (95% CI 9.78 to 14.80), and at the trial-level R2 was equal to 0.62 (95% CI 0 to 1.00). For PFS, at the individual-level ρ was equal to 0.72 (95% CI 0.70 to 0.73), and at the trial-level R2 was equal to 0.73 (95% CI 0.27 to 1.00). For TNTD, at the individual-level ρ was equal to 0.77 (95% CI 0.76 to 0.78), and at the trial-level R2 was equal to 0.77 (95% CI 0.37 to 1.00). In cross-validation, the 95% prediction intervals for HRs for OS predicted by regression models always contained the observed HRs for OS, indicating the stability of the models.Conclusion At the individual-level, ORR exhibited a strong correlation with OS, whereas PFS and TNTD showed a moderate level correlation with OS. At the trial level, the key requirement for validating surrogates, all candidate surrogates demonstrated moderate predictive abilities for OS in future trials. These findings should be interpreted within the context of anti-PD-1-based therapies, with or without anti-CTLA-4 combinations, consistent with the trial evidence base included in this study.
Background Tertiary lymphoid structures (TLS) are spatially organized immune niches associated with therapeutic response and favorable outcomes in breast cancer (BC). However, TLS assessment currently relies on invasive tissue-based analyses, and the biological mechanisms underlying imaging-based TLS prediction remain poorly understood.Methods We developed and validated a spatial heterogeneity-based radiomic TLS signature (shTLS) using dynamic contrast-enhanced MRI to non-invasively predict TLS status across multicenter BC cohorts. Spatial habitat radiomics were used to capture intratumoral and peritumoral immune-related heterogeneity. Integrated multi-omics analyses, including transcriptomics, pathomics, genomics, single-cell RNA sequencing, immunohistochemistry, and multiplex immunofluorescence, were performed to biologically interpret shTLS-defined subgroups. Functional drug-sensitivity assays were conducted to assess therapeutic implications.Results The shTLS model achieved robust predictive performance across independent cohorts and molecular subtypes. High shTLS scores were associated with immune-inflamed tumors characterized by spatially clustered activated T cells and dendritic cells (DCs). In contrast, shTLS-low tumors exhibited an immunosuppressive spatial niche with peripheral accumulation of CD4+ PD-1+ T cells and plasma cells, increased immune-tumor separation, and enhanced inflammatory and immunoregulatory signaling. An indoleamine 2,3-dioxygenase 1 (IDO1)-associated immunoregulatory program was observed in the shTLS-low tumors, which appeared to be preferentially expressed by LAMP3+CCR7+ migratory DCs. Pharmacologic inhibition of IDO1 enhanced chemotherapy and CDK4/6 inhibitor sensitivity in vitro.Conclusion This study establishes spatial radiomics as a non-invasive approach to decode TLS-associated immune ecosystems and supports the presence of an IDO1-associated immunosuppressive phenotype, providing biological insight and translational rationale for patient stratification and future combination strategies.
Background A substantial amount of research has been dedicated to the mechanisms by which tumor cells evade immune system recognition and manipulate the immune microenvironment to facilitate immune escape. Recent studies have shown that viruses and tumors can protect themselves from immune cytotoxicity by remodeling the actin cytoskeleton. However, cytoskeleton-mediated immune resistance and the specific cytoskeleton-related proteins involved require further research. Methods Single-cell RNA sequencing was used to identify cytoskeleton-related genes associated with the response to anti-programmed cell death protein 1 (PD-1) therapy across four digestive tumors. Immunohistochemistry was used to detect LASP1 expression in gastric cancer and analyze its prognostic value for survival and anti-PD-1 response. The impact of LASP1 deficiency on tumor response to anti-PD-1 treatment and cytotoxic lymphocyte-mediated lysis was determined in vivo and in vitro. Live-cell imaging was used to compare actin cytoskeletal dynamics at the immunological synapse between LASP1-deficient and mock tumor cells. Molecular mechanisms underlying LASP1-mediated immune-resistance were dissected using co-immunoprecipitation, immunofluorescence, domain deletion complementation, and Laurdan staining. Results The deficiency of LASP1 in gastric cancer affected the sensitivity of tumor cells to immunotherapy and induced cytotoxic lymphocytes exhaustion. LASP1 may act as a scaffold protein to regulate the Arp2/3 complex and remodel the cytoskeleton at the immunological synapses. LASP1 deficiency in tumor cells impairs lytic immunological synapse function by disrupting cytoskeletal dynamics-mediated cell membrane lipid organization at the immunological synapse. Finally, simvastatin combined with anti-PD-1 therapy reversed immunotherapy resistance in LASP1-deficient tumors. Conclusions The deficiency of LASP1 in tumors mediates immunological synapse dysfunction by affecting cytoskeletal dynamics-mediated cell membrane lipid organization, thus enabling tumors to protect themselves from immune cytotoxicity and immunotherapy.
Background Tumor-associated macrophages (TAMs) play pivotal roles in shaping the tumor-microenvironment (TME) through functional plasticity, which is regulated by extrinsic and intrinsic signals. However, the role of vesicular trafficking in TAMs remains poorly understood. RAB31, a small GTPase enriched in myeloid cells, was proposed as a potential regulator of TAM polarization through clathrin-mediated endocytosis (CME). We hypothesized that RAB31 modulates TAM education by tumor-derived signals and thereby shapes antitumor immunity.Methods We profiled RAB31 expression in human cancers using public single-cell RNA sequencing (scRNA-seq) datasets and clinical sample immunofluorescence staining. Rab31 knockout mice were employed in subcutaneous tumor models. The TME was profiled by scRNA-seq, bulk RNA-seq, and flow cytometry. Bone marrow transplantation, adoptive cell transfer, and antibody-mediated depletion were performed to identify the effector cell populations. Co-immunoprecipitation coupled with mass spectrometry, receptor half-life assays, inhibitor intervention and lysosomal colocalization experiments dissected the molecular mechanism. Functional T-cell chemotaxis, activation, and anti-programmed death-ligand 1 (PD-L1) response assays were performed.Results RAB31 was highly expressed in TAMs across multiple cancers and correlated with poor prognosis and immunosuppressive TME. Rab31 deficiency reprogrammed TAMs to M1-like phenotype, enhanced CD8+ T-cell infiltration and activation, and suppressed tumor growth. Mechanistically, Rab31 preserves FPR2 cell surface stability through a CME-dependent mechanism; its loss redirected FPR2 to lysosomal degradation, disrupted tumor-derived ANXA1 signaling, and unleashed NF-κB activity. Rab31 deficiency synergized with anti-PD-L1 therapy in a CD8+ T cell-dependent manner.Conclusions These findings establish the CME/RAB31 pathway as an indispensable regulator of TAM polarization, underscoring the pivotal role of vesicular trafficking in the TME.
Background Metabolic reprogramming within the tumor microenvironment is a pivotal barrier to effective immune checkpoint blockade (ICB). While programmed death ligand 1 (PD-L1) is well characterized as a ligand inhibiting T-cell function, its intrinsic ‘reverse signaling’ role in regulating tumor metabolism and shaping the immune landscape remains poorly understood. Here, we investigated the metabolic determinants of resistance to anti-programmed cell death protein 1 (anti-PD-1) therapy and the underlying molecular mechanisms.Methods Integrated metabolomics and transcriptomics were performed on tumor samples from patients with non-small cell lung cancer and cell lines. Mechanisms were delineated using RNA sequencing, cleavage under targets and tagmentation assays, metabolic flux analysis, and coculture systems. The therapeutic efficacy of targeting metabolic effectors was evaluated in syngeneic mouse models and correlated with immune profiling.Results We identified a distinct metabolic signature characterized by aberrant pyruvate accumulation in patients resistant to anti-PD-1 therapy. Mechanistically, we demonstrate that antibody-mediated ligation of PD-L1 triggers an intrinsic endoplasmic reticulum (ER) stress response via the PERK–ATF4–CHOP axis. ATF4 acts as a transcriptional activator that directly upregulates pyruvate dehydrogenase kinase 4 (PDK4) (blocking pyruvate oxidation) and glutaminase (GLS) (promoting glutaminolysis), creating a ‘dual-hit’ metabolic rewiring that drives intracellular pyruvate build-up. Subsequently, tumor-secreted pyruvate is taken up by tumor-associated macrophages (TAMs) via MCT1, inducing mitochondrial reactive oxygen species accumulation and driving them into a state of cellular senescence. These senescent TAMs upregulate PD-L1 via STAT3 signaling, thereby reinforcing an immunosuppressive feedback loop. Pharmacological inhibition of PDK4 and GLS effectively abolished pyruvate accumulation, prevented macrophage senescence, and restored CD8+ T-cell cytotoxicity.Conclusions Our study identifies a novel ‘PD-L1–ER stress–pyruvate–macrophage senescence’ axis as a key mechanism underlying primary resistance to ICB. These findings highlight the non-canonical reverse-signaling function of PD-L1 in metabolic remodeling and propose that targeting the PDK4/GLS-dependent pyruvate surge offers a promising therapeutic strategy to sensitize tumors to anti-PD-1 immunotherapy.
Background Immune checkpoint inhibitors (ICIs) have limited efficacy in proficient mismatch repair/microsatellite stable (pMMR/MSS) metastatic colorectal cancer (mCRC). However, selected patients with specific metastatic patterns may derive benefit.Methods Patients with chemorefractory pMMR/MSS mCRC treated with ICI-based regimens were retrospectively identified. A comparison cohort treated with trifluridine/tipiracil±bevacizumab, regorafenib, or fruquintinib as standard of care (SOC) was generated through 1:1 propensity score matching by age, sex, Eastern Cooperative Oncology Group performance status (ECOG PS), liver metastases (present/absent), and RAS/BRAF status. Overall survival (OS) was compared using Cox regression.Results A total of 354 patients treated with ICIs and 354 treated with SOC were matched. Median age was 55 years, 52% male, 32% ECOG PS 0, 30% right-sided, and 69% RAS mutated in both groups, while 61% and 60% had liver metastases, respectively. Median OS (mOS) was 10.8 months with ICIs and 9.0 months with SOC (HR 0.76, 95% CI 0.64 to 0.92, p=0.004). In patients without liver metastases, mOS was longer with ICIs than SOC (19.1 vs 13.2 months, HR 0.59, 95% CI 0.43 to 0.80, p<0.001), whereas outcomes were similar in patients with liver metastases (6.4 vs 6.5 months, p=0.303). In univariable analyses, age, sex, primary tumor site, and RAS/BRAF status were not associated with OS. Treatment with ICIs, absence of liver metastases, one prior line of therapy, less than three metastatic sites, and ECOG PS 0 were associated with the most favorable outcomes in univariable and multivariable models.Conclusions In chemorefractory pMMR/MSS mCRC without liver metastases, ICI-based regimens yielded longer OS than SOC. Further investigation of ICIs in this patient population is warranted.
BACKGROUND:Antibody-drug conjugates (ADCs) and bispecific antibodies represent a rapidly advancing frontier in oncology, yet the abnormal tumor microenvironment (TME) hinders their delivery and reduces efficacy. Emerging immunomodulatory ADCs (IM-ADCs) demand mechanistic mathematical models that couple drug transport with immune dynamics. METHODS:Here, we present a mechanistic framework for the delivery of HE-S2 ADC, an anti-programmed cell death ligand 1 (PD-L1) antibody bearing the bifunctional immunomodulator D18. Our model integrates cancer-immune cells interactions, TME properties, such as dysfunctional vessels, elevated interstitial fluid pressure, tissue hydraulic conductivity, and vascular permeability, spatiotemporal distributions across growing tumor and adjacent host tissue, convective-diffusive transport, ADCs binding and internalization kinetics and tumor-draining lymph node biology governing antigen presentation and the generation of effector CD8+ T cells. Parameters were calibrated simultaneously with the murine MC38 and B16 tumor growth data and effector CD8+T cell data following treatment with D18, anti-PD-L1, and ADC. RESULTS:Our mechanistic spatiotemporal model captures the superior antitumor efficacy of the HE-S2 ADC relative to its individual components and provides mechanistic predictions for unmeasured variables, such as spatiotemporal dynamics of drug/immune-cell distributions. It explains reduced intratumoral D18 exposure via rapid clearance, while antibody/ADC achieves higher tumor retention through leaky tumor vasculature. The model suggests a reinforcing loop in which improved ADC exposure enhances CD8+T cell infiltration, driving tumor shrinkage that lowers fluid pressure and improves drug delivery. Parametric analyses findings support TME normalization strategies that increase functional vessel density prior to ADC administration; however, such approaches should preserve sufficient vascular permeability by maintaining vessel pore radius >~40 nm, ensuring pores remain large enough for ADC extravasation and effective intratumoral delivery. CONCLUSION:The proposed mechanistic model successfully captures how TME properties regulate the delivery and efficacy of IM-ADCs while suggesting TME normalization as a potential strategy to improve treatment outcomes.
Proteogenomic cancer antigen discovery now extends beyond somatic mutations to include unmutated, aberrantly expressed, and non-canonical sources. We distinguish two orthogonal dimensions: molecular origin (canonical or non-canonical) and normal-tissue distribution (tumor-specific antigen, tumor-associated antigen, or lineage-specific antigen). Importantly, non-canonical origin does not imply tumor specificity. For human leukocyte antigen (HLA)-restricted immunotherapy, selectivity must be established at the level of naturally presented peptide-HLA (pHLA) complexes rather than inferred from RNA expression or total protein abundance. We therefore propose a normal-immunopeptidome denominator: a context-aware atlas of benign pHLA ligands that can be used to exclude unsafe candidates before therapeutic nomination. However, this denominator is necessary but not sufficient. Candidate nomination also requires evidence of analytical confidence, natural presentation on tumor cells, and, where available, peptide abundance and tumor heterogeneity. Additional considerations include whether the target is patient-specific or shared, its functional selectivity, the potential for immune escape, receptor cross-reactivity, HLA alloreactivity, and the modality-specific therapeutic index. We present a tiered framework that distinguishes core requirements, risk-triggered evaluation criteria, and recommended enhancements. These requirements escalate as uncertainty increases. Importantly, the absence of a peptide from existing databases should not be interpreted as evidence of its absence in humans.
BACKGROUND:Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with dismal outcomes, especially in relapsed/refractory settings. Chimeric antigen receptor natural killer (CAR-NK) cell therapy holds promise but is constrained by the immunosuppressive tumor microenvironment (TME), where adenosine-mediated suppression is a key barrier. OBJECTIVE:To develop a novel CAR-NK construct cotargeting AML cells and the adenosine-rich TME to enhance antileukemia efficacy. METHODS:Ex vivo expanded primary NK cells were used to compare the effects of CD39 versus CD73 blockade on NK cell function via messenger RNA-electroporated antibodies. A CD33-CD73 dual-function CAR-NK construct (integrating CD33-specific lysis and anti-CD73scFv secretion for TME disruption) was designed and transduced into NK cells via retrovirus. Engineered NK cells were characterized for transduction efficiency, expansion, purity, viability, and CAR stability. In vitro cytotoxicity against AML cell lines and primary blasts was assessed, and in vivo efficacy was evaluated in a MOLM-13 xenograft mouse model. RESULTS:CD73 blockade more potently enhanced NK cell activity than CD39 blockade. Retroviral transduction achieved >50% efficiency, and expansion with K562-4-1BBL-mbIL-21/-15 feeder cells yielded NK cells with ≥6,000 fold expansion, >93% purity, >98% viability, and stable CAR expression. At an effector-to-target ratio of 0.5:1, CD33-CD73 CAR-NK cells mediated ~80% specific lysis, with superior cytotoxicity vs conventional CD33 CAR-NK cells. In xenografts, CD33-CD73 CAR-NK cells achieved robust tumor clearance, extended median survival by 24.5 days (59.5 vs 35 days) versus standard CD33 CAR-NK cells, and five out of six mice achieved long-term survival (>50 days). CONCLUSION:The CD33-CD73 dual-targeting CAR-NK platform synergistically targets AML cells and the adenosine-rich TME, exhibiting superior anti-leukemia efficacy. This strategy advances AML immunotherapy and provides a translational blueprint for TME-targeted therapies in other cancers.
Background Allogeneic hematopoietic stem cell transplantation (alloHSCT) can be a curative treatment for hematological diseases. After HLA-matched alloHSCT, donor T cells may recognize minor histocompatibility antigens (MiHAs), which are polymorphic HLA-binding peptides on patient cells that are absent from donor cells due to genetic differences. Donor T cells can induce beneficial anti-tumor effects if MiHAs are targeted on malignant hematopoietic cells in the patient, while graft-versus-host disease (GvHD) may develop if MiHAs are targeted on patients’ healthy non-hematopoietic tissues.Methods We previously isolated T-cell clones from patients responding to donor lymphocyte infusions (DLIs) after HLA-matched alloHSCT, and identified HLA class I-restricted MiHAs. To investigate MiHA-specific T-cell responses in patients, we here sequenced the T-cell receptors (TCRs) of MiHA-specific T-cell clones and identified 394 distinct TCRs against 122 MiHAs. We used the collection of identified TCRs to measure frequencies of matched MiHA-specific TCRs in 39 patients responding to DLI with antitumor responses accompanied with no (n=9), limited (n=8) or severe (n=22) GvHD.Results The data showed higher MiHA-specific TCR frequencies in patients with severe GvHD, which were mainly driven by clonal expansion. Moreover, within the diverse MiHA-specific TCR repertoires in these patients, we identified five public TCRs against four MiHAs with identical CDR3 regions and several TCRs targeting MiHAs with similar, but not identical, CDR3 regions.Conclusion Patients with severe GvHD have high MiHA-TCR frequencies mainly driven by clonal expansion, and that MiHA-specific TCR repertoires in patients responding to DLI after alloHSCT are highly diverse with a few public clonotypes.
Background Metastatic melanoma resistant to immune checkpoint inhibitors remains difficult to treat, and while adoptive tumor-infiltrating lymphocyte (TIL) therapy has shown durable responses, its reliance on lymphodepleting chemotherapy and high-dose interleukin (IL)-2 causes toxicity that may limit patient eligibility. The dual-cytokine-armed oncolytic adenovirus igrelimogene litadenorepvec (TILT-123) was administered with TILs in the TUNINTIL trial (trial registration: NCT04217473) in patients with metastatic melanoma resistant to immune checkpoint inhibitors, without lymphodepleting chemotherapy or IL-2 post-conditioning. This study presents a correlative immunological analysis of the phase I TUNINTIL trial evaluating TILT-123 in combination with TIL therapy.Methods The TUNINTIL trial was a first-in-human, open-label, dose-escalation, multicenter, multinational phase I trial. 17 patients with checkpoint-inhibitor-resistant metastatic melanoma received up to six intratumoral TILT-123 injections followed by TIL infusion, without lymphodepleting chemotherapy or IL-2 post-conditioning. Systemic immune profiling (serum proteomics, flow cytometry, interferon-γ ELISpot assay), intratumoral immune cell–cell profiling (multiplex immunofluorescence, H&E, adenovirus E1a immunohistochemistry), quantitative PCR, and neutralizing antibody responses were assessed at defined time points through the trial, with survival follow-up updated to March 2026. Response criteria were evaluated using Response Evaluation Criteria in Solid Tumors V.1.1 and positron emission tomography-based criteria. Statistical analyses included Kaplan-Meier survival with log-rank tests, Mann-Whitney U tests, Pearson correlation, and receiver operating characteristic/area under the curve analysis for biomarker cut-off determination.Results Tumor biopsy analyses revealed an early innate immune activation marked by natural killer-cell expansion and cytotoxic gene upregulation, followed by increased intratumoral T-cell infiltration. This occurred without lymphodepleting chemotherapy or post-conditioning IL-2. Intratumoral viral DNA was detectable in a subset of patients. The enrichment of CD27+CD28+ memory-precursor CD8+ T cell was associated with favorable clinical outcomes. Elevated monocytic myeloid-derived suppressor cells and angiogenic/inflammatory cytokines following combination treatment were associated with disease progression, highlighting the role of immunosuppressive myeloid subsets as potential mediators of therapeutic resistance. Additionally, correlative analysis in pooled TILT-123 cohorts identified serum epidermal growth factor as a candidate biomarker for stratifying and monitoring patients.Conclusions These findings provide mechanistic insights into TILT-123 combined with TIL therapy and propose future directions for biomarker-guided clinical studies.Trial registration number NCT04217473.
Background Treatment with immune checkpoint inhibitors (ICIs) has radically improved outcomes for patients with microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). Intrinsic and acquired resistance, however, remains an important concern. Pyrimidine pathway regulates the innate immune response and may offer an opportunity to enhance ICI efficacy via modulation of pyrimidine metabolism. Identification of primary, adaptive and acquired resistance mechanisms and development of actionable strategies to overcome resistance and expand the benefit of ICI is paramount. Our group is the first to show that the dihydropyrimidine dehydrogenase (DPD; gene DPYD ) metabolic pathway predicts the efficacy of ICI in MSI-H tumors. Methods Retrospective analysis of germline single nucleotide polymorphisms was done in patients with MSI-H CRC cohort (Veneto Institute of Oncology; OIOV-IRCCS, Padua, Italy) treated with immunotherapy. Dpyd knockout or overexpressed MC38 MSI-H syngeneic mice models were treated with anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) alone or in combination and their effect on tumor growth and its microenvironment was studied. Results In our real-world data analysis, genetic variants in DPYD were associated with progression-free survival and tumor response in patients with MSI-H mCRC treated with ICI. In preclinical studies, complete tumor elimination was observed in 100% of mice bearing Dpyd knockout tumors treated with a combination of anti-PD-1 and anti-CTLA-4, further validated by pharmacologically inhibiting DPD using eniluracil. Conclusion Our results showed that the pyrimidine pathway has a strong association with immunotherapy treatment response in CRC. These findings may support the development of novel treatment strategies exploiting DPD inhibition in MSI-H tumors and their quick integration into clinic.
Background Long-term survival of extensive-stage small-cell lung cancer (ES-SCLC) remains rare, with most patients experiencing disease progression during maintenance therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors have the potential to confer antitumor activity, modify tumor immunogenicity, and sensitize tumors to anti-programmed cell death protein 1/programmed death-ligand 1 therapy. We conducted this phase 2 trial to investigate the efficacy and safety of durvalumab plus olaparib as maintenance therapy in patients with ES-SCLC. Methods This was a multicenter, single-arm, phase II trial that enrolled 60 patients with previously untreated ES-SCLC ( NCT05245994 ). Patients received durvalumab (1,500 mg) combined with platinum-etoposide chemotherapy intravenously every 21 days for up to four cycles, followed by maintenance therapy with durvalumab (1,500 mg every 28 days) and oral olaparib (300 mg two times a day) until disease progression or unacceptable toxicity. Multi-omics analyses were performed to characterize molecular subtypes associated with clinical outcomes. Results The combination regimen demonstrated promising efficacy, with an alive and progression-free at 12 months rate of 25.0%, an objective response rate of 73.3%, a median progression-free survival of 6.8 months, and a median overall survival of 14.6 months. Multi-omics profiling identified a hypomethylation subgroup (cluster 1) that was associated with significantly improved survival outcomes. Further analysis revealed that this subtype exhibited enhanced antigen presentation machinery, a favorable cytokine profile, and suppression of DNA damage repair (DDR) pathways, potentially through elevated promoter methylation and transcriptional silencing of specific DDR genes, which together were associated with the favorable outcomes. Conclusions This study presents the first prospective evidence supporting durvalumab plus olaparib as maintenance therapy in ES-SCLC. Multi-omics analysis identifies that DNA hypomethylation status may enrich for patients who benefit from PARP inhibition and immunotherapy. Trial registration number NCT05245994 .
