Journal for ImmunoTherapy of Cancer

Interleukin-2 (IL-2) remains an attractive cytokine for enhancing antigen-specific CD8 T cell responses in cancer immunotherapy, but systemic toxicity hinders its broad clinical application. To address this, various IL-2-based therapeutics have been engineered with altered IL-2 receptor bias or targeted delivery to tumors, the tumor microenvironment, or immune cell populations. Ideally, IL-2 signals should be selectively delivered to antigen-specific CD8 T cells, boosting their responses and promoting effector differentiation while sparing non-targeted populations. Immuno-STATTM (Selective Targeting and Alteration of T cells) is a fusion protein platform comprising a bivalent peptide-MHC class I complex and an affinity-attenuated IL-2 mutein that co-stimulates TCR and IL-2 signaling in epitope-specific CD8 T cells. Here, we investigated whether a DbGP33-41-targeted Immuno-STAT enhances DbGP33-specific CD8 T cell responses in a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Immuno-STAT treatment selectively expanded DbGP33-specific CD8 T cells with an effector-like phenotype. Non-targeted DbGP276-specific CD8 T cells showed little to no expansion in response to DbGP33-41-targeted Immuno-STAT therapy, underscoring the selectivity of this approach. However, minor changes in phenotypic markers, including increased expression of CD25 and CX3CR1, were observed in non-targeted CD8 T cells, likely reflecting bystander IL-2 signaling. Combining Immuno-STAT with PD-1 blockade augmented DbGP33-specific CD8 T cell responses more effectively than PD-1 blockade alone, with minor effects on the non-targeted DbGP276-specific population. These findings inform the clinical development of Immuno-STAT and other IL-2 therapeutics and highlight the value of coordinated TCR and IL-2 stimulation during chronic antigen exposure, alone or in combination with PD-1 blockade. IMPORTANCEInterleukin-2 (IL-2) is a key cytokine for promoting effector differentiation of antigen-specific CD8 T cells and remains an attractive agent in cancer immunotherapy, but systemic toxicity limits its clinical use. This study addresses a central challenge in IL-2-based immunotherapy: delivering IL-2 to cognate antigen-specific CD8 T cells while minimizing activation of non-targeted populations. Using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection, we show that the Immuno-STAT (Selective Targeting and Alteration of T cells) platform selectively expands targeted virus-specific CD8 T cells and enhances their function while limiting effects on non-targeted populations. We also show that combining Immuno-STAT with PD-1 blockade further enhances targeted virus-specific CD8 T cell responses during chronic LCMV infection. These findings provide mechanistic and preclinical support for integrating T cell receptor (TCR) specificity with IL-2 signaling to advance cancer immunotherapy and guide next-generation IL-2 therapeutics for cancer and chronic infection.

BackgroundCross-presentation of tumor antigens by antigen-presenting cells (APCs) is essential for initiating effective anti-tumor T cell immunity. The presence of cross-presenting immune cells across multiple solid tumors correlates with improved clinical outcomes. Despite the importance of this process, the identities and characteristics of tumor-derived MHC-I antigens that are cross-presented by APCs remain largely undefined, limiting rational design of targeted immunotherapies. MethodsWe performed an immunopeptidomic analysis of cross-presented glioblastoma (GBM) antigens on APCs, including bone marrow-derived macrophages, bone marrow-derived dendritic cells, and splenic dendritic cells, using SILAC labeling and in vitro co-culture systems. Additionally, we also profiled endogenous APC and tumor antigen repertoires. We made selected cross-presented antigen targets into mRNA vaccines and evaluated their immunogenicity in comparison to tumor endogenous antigens in vivo. ResultsWe identified over one thousand putative cross-presented GBM antigens. Comparative analysis of endogenous APC and tumor antigen repertoires revealed that cross-presented antigens possess distinct features and are predominantly shaped by intrinsic antigen processing and presentation pathways within APCs, resulting in limited cross-presentation of tumor-specific epitopes. Two doses of mRNA encoding cross-presented tumor-specific epitopes delayed tumor growth and elicited robust antigen-specific T cell responses. ConclusionOur findings define the landscape and constraints of tumor antigen cross-presentation in GBM and establish a framework for improved antigen selection in the development of next-generation GBM immunotherapies.

BackgroundPancreatic ductal adenocarcinoma (PDAC), a condition representing 90% of pancreatic cancers, shows one of the lowest 5-year survival rates across all cancer types. Current therapeutic approaches remain largely inefficient, in part due to the presence of a hostile tumor microenvironment (TME), impeding immune cells infiltration and function. Specifically, Natural Killer (NK) cells from PDAC patients exhibit impaired phenotype and cytotoxic functions. NK cell immunotherapy represents a safe and promising approach to restore NK cell cytotoxicity against PDAC. MethodsWe developed a dual strategy based on i) the re-activation of NK cells through Natural Killer activating multimeric immunotherapeutic complexes (NaMiX) composed of IL-15/IL-15R dimers coupled to anti-NKp46 single-chain variable fragments (scFvs) and ii) the crosslinking of activated NK cells to PDAC cells with a Trispecific Killer Engager (TriKE) targeting NKG2D, NKp30 and the tumor-associated antigen CEA. We evaluated the ability of these constructs to stimulate NK cell functions across BxPC-3 PDAC cell line and patient-derived organoid models and in humanized NSG mice bearing PDAC xenografts. ResultsNaMiX stimulated the activation and cytotoxic functions of NK cells towards pancreatic BxPC-3 cells in vitro while TriKE cross-linked NK cells to BxPC-3 cells. The cytotoxic effects of NaMiX were further enhanced when combined with the crosslinking abilities of TriKE for the killing of NK cell-mediated BxPC-3 spheroid and PDAC patient-derived organoids. In humanized mice bearing BxPC-3 xenografts, NaMiX induced cytotoxic lymphocyte expansion, and increased tumor infiltration of NK cells, while TriKE tended to slow tumor progression. ConclusionsThis proof-of-concept study reports for the first time that activating and engaging NK cells with immunoconjugates are a promising therapeutic avenue for PDAC treatment. Efforts should now focus on the optimization of NK cell therapeutic modalities to favor the infiltration of a high number of NK cells into the tumor.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer, with limited therapeutic options and extremely high mortality rates. While immune checkpoint blockade (ICB) therapy is effective in many types of human cancers, responses in PDAC patients remain poor, partly due to the weak immunogenicity of PDAC tumors. We hypothesized that a whole-cell PDAC vaccine could improve anti-tumor responses if optimized to expose a more stimulatory repertoire of tumor antigens. To test this, we used murine Panc02 pancreatic cancer cells to screen several stress-inducing treatments (UV, hypoxia, heat shock, and hydrogen peroxide [H2O2]), among which low-dose oxidative stress (0.05% H2O2 for 2h) was identified as the optimal inducer of immunogenic cell death (including increased surface calreticulin, ERp57 exposure, HMGB1 release and MHC class I expression). We then prepared a whole-cell vaccine of fixed H2O2-treated Panc02 cells, which induced robust tumor-specific immunity in C57BL/6 mice bearing syngeneic Panc02 tumors. Vaccine-treated mice displayed a significant increase in tumor-reactive IFN{gamma}+ T cells, as well as extensive tumor infiltration by CD4 + and CD8 + T cells and NCR1+ NK cells. When used prophylactically, the vaccine significantly delayed tumor growth and extended survival, whereas therapeutic application markedly slowed tumor progression. Importantly, combining the whole-cell Panc02 vaccine with anti-PD-1 therapy induced complete tumor regression in a subset of animals. Together, these data demonstrate that controlled oxidative stress can convert autologous tumor cells into an effective whole-cell vaccine without the need for genetic modification or prior neoantigen identification, offering a scalable strategy for personalized immunotherapy in PDAC. STATEMENT OF SIGNIFICANCEThis study demonstrated that oxidative stress-induced immunogenic cell death reprograms pancreatic tumor cells to induce danger signaling and enhance antigen presentation, thereby promoting immune infiltration and sensitizing tumors to PD-1 blockade.

BackgroundThe role of intratumoral plasma cells in immune checkpoint blockade (ICB) therapy has never been tested although their presence is linked with improved patient response and survival. Malignant peripheral nerve sheath tumors (MPNSTs) are deadly sarcomas with minimal responsiveness to ICB therapies. Strikingly, drugs inhibiting cyclin-dependent kinases 4/6 (CDK4/6) and MEK sensitize de novo MPNSTs to immunotherapy targeting programmed death-ligand 1 (PD-L1), which correlates with increased intratumoral plasma cells. Here, we tested if plasma cells mediate the MPNST response to anti-PD-L1 therapy. MethodsAnti-tumor activity of PD-L1 inhibition, with or without CDK4/6-MEK inhibition, was measured in de novo MPNSTs within wild-type versus plasma cell-deficient mice. Plasma cell-dependent effects of CDK4/6-MEK inhibition on priming the MPNST immune environment were determined by single cell transcriptomics and immunostaining. FindingsMPNSTs lacking plasma cells failed to respond to anti-PD-L1 monotherapy and were no longer sensitized to immunotherapy by CDK4/6-MEK inhibition. Plasma cell-deficient MPNSTs exposed to CDK4/6-MEK inhibitors had impaired antigen presentation on major histocompatibility class I (MHC-I) and decreased CD8+ T cell infiltration and activation. Complementary analyses of human sarcomas showed increased intratumoral plasma cell signatures prognose better patient survival. InterpretationPlasma cells favorably remodel the tumor immune environment by increasing CD8+ T cell infiltration and are critical for successful ICB therapy in MPNSTs. This work may help inform ICB treatment strategies and cancer patient stratification for many different tumor types. FundingThis research was supported by University of Iowa Sarcoma Research Program awards and NIH grants T34-GM141143, T32-GM067795, F31-CA281312, P30-CA086862, and R01-NS119322. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSFor many types of cancer, intratumoral plasma cells have been correlated with better patient survival and improved response to immune checkpoint blockade (ICB) therapies. However, the biology underlying those associations is not understood and no study has examined the requirement of plasma cells in immunotherapy response. Compelling data in malignant peripheral nerve sheath tumors (MPNSTs) showed that dual kinase inhibition of oncogenic CDK4/6 and MEK induced intratumoral plasma cell accumulation and sensitized tumors to ICB therapy. While CDK4/6-MEK inhibition is known to enhance antitumor immunity in other tumor types by CD8+ T cells or natural killer (NK) cells, a role for plasma cells has never been explored. Added value of this studyStudies were performed in MPNSTs, an under-researched cancer that normally responds poorly to ICB monotherapies. This is the first investigation to show that intratumoral plasma cells are essential for successful ICB therapy and they support anti-tumor immunity by promoting a pro-inflammatory, CD8+ T cell state involving MHC-I antigen presentation. Findings provide new insight into immunomodulatory effects of CDK4/6-MEK inhibitor therapies, revealing plasma cells are needed for those drugs to activate CD8+ T cell mediated antitumor immunity. Implications of all the available evidenceThe fundamental advance in understanding how plasma cells promote successful ICB immunotherapy is likely applicable to other solid tumors and may guide novel therapeutic strategies in which plasma cell-inducing agents are combined with ICB antibodies. Moreover, an increased presence of intratumoral plasma cells in tumor specimens may streamline clinical decisions regarding which patients are most likely to benefit from ICB therapy.

1IntroductionTumor infiltrating lymphocytes (TIL) drive the anti-tumor activity of a broad class of immunotherapies. In situ TIL are composed of T cells that recognize tumor antigens (Tumor Reactive T cells, or TRTs) as well as bystander T cells with specificity for other antigens. TRT clonotypes are associated with a unique and tumor-driven exhausted transcriptional state, enabling single-cell RNA sequencing (scRNA-seq)-based predictive models for TRTs using experimentally validated clone labels. MethodsIn this study, a clonotype-level CD8+ TRT classifier (TRACE) was built using an aggregated dataset of validated tumor reactive clonotypes and associated scRNA-seq data from multiple publications that overcomes the limitations of training on a single dataset, donor, or indication. TRACE does not require dataset manipulation for training or prediction, enabling it to be easily applied to new test datasets as they emerge. ResultsTRACE exhibited robust performance on held-out TIL and PBMC clones - achieving a mean Matthews correlation coefficient of 0.84 and F1-score of 0.85 - comparable to or outperforming other TRT prediction methods. We experimentally confirmed the reactivity of TRACE-identified TRT clones by co-culturing engineered, ex vivo expanded TIL with autologous melanoma tumor cell lines. Finally, we applied TRACE to evaluate the frequency of TRT across hundreds of patient samples from multiple tumor atlases spanning lung, colorectal, and pancreatic cancer. TRACE scores were observed to be significantly higher in exhausted CD8 T cells in tumors but not in exhausted cells in normal adjacent or non-cancer samples, suggesting specificity towards identifying tumor-antigen experienced T cells. ConclusionTRACE is a tumor reactivity scoring algorithm released with open model weights that can be applied to tissue or blood single-cell RNAseq datasets. Its application should be of general interest for characterizing the fraction of TRTs in TIL and for establishing correlations with clinical response to immunotherapies.

Cytotoxic CD8+ T cell responses targeting tumor neoantigens are critical for immunotherapy efficacy and are widely studied across different preclinical mouse tumor models. Defined neoantigens are commonly introduced to enable tracking of tumor-specific T cells; however, variation in neoantigen choice may yield immune phenotypes attributable to differences in neoantigen immunogenicity, complicating interpretation of tumor-intrinsic mechanisms. Here, we determined the relative immunogenicity of a set of 25 commonly used mouse tumor-derived and model neoantigens to facilitate comparison of neoantigens across studies. We found that in silico predicted major histocompatibility complex (MHC) binding affinity poorly stratified in vivo immunogenicity. In contrast, experimental measurement of peptide-MHC complex stability (Koff), more so than measured affinity (KD), closely correlated with the relative magnitude of neoantigen-targeted vaccine responses in vivo. Thus, we report the relative stability of a known set of commonly used neoantigens as a reference and provide a simple method to benchmark novel neoantigens against this library. This framework will allow contextualization of the level of immunogenicity of newly identified neoantigens and aid in comparative interpretation of tumor-immune phenotypes across studies.

Cytotoxic T cells (CTL) are crucial for adaptive immunity that leads to prolonged survival and potential cures for cancer. Recent clinical data has shown that pharmacological inhibition of SUMOylation (SUMOi) profoundly modifies tumor microenvironment (TME) and activates CTL, although the mechanism is not well described. In this study, we found that T cell specific knock out (KO) of the most dominant SUMO paralog, Sumo2/SUMO2, in both mouse and human CD8+ T cells significantly enhanced CD8+ T cell activation that is independent of the known mechanism - inducing type I IFN (IFN-I) expression by myeloid cells. Sumo2/SUMO2 KO in CD8+ T cells increased chromatin accessibility for transcription factors BATF, JunB, ATF3, FRA1, FRA2, and AP1 that are known to promote T cell activation and proliferation. Using antigen-specific T cell models, OT1 and Chimeric Antigen Receptor (CAR)-T cells, we found that Sumo2 KO CD8+ T cells had significantly higher tumor infiltration as revealed by flow cytometry, immuno-fluorescence (IF) staining, and single nuclei RNA-sequencing (snRNA-seq) and conferred greater tumor growth inhibition than wildtype (WT) control T cells. snRNA-seq also revealed Sumo2 KO CD8+ T cells increased the expression of Tumor Necrosis Factor-Related Apoptosis-inducing Ligand (TRAIL), induced apoptosis genes in tumor cells and activated IFN-I and IFN-{gamma} responsive genes in all cell types in the TME. These findings elucidate a novel mechanism regarding how SUMOylation can directly control CTL activation and tumor infiltration that activate anti-tumor immunity in the TME. SUMO2 KO can also be a potential strategy to enhance adoptive T cell therapies of solid tumors by enhancing their activity, tumor infiltration and their ability to after the TME.

PURPOSE: Newly-diagnosed glioblastoma (nGBM) is a devastating tumor with median survival of only 14-18 months despite aggressive standard of care (SOC). Dendritic cell (DC) homologous antigenic double-loading provides a powerful pattern-based signal that initiates cDC1-like skewing of monocytic precursors, inducing downstream development of CD8+ memory effectors. Here we report phase I results for DOC1021 (dubodencel), a novel DC vaccine regimen integrated with SOC. METHODS: In this dose-escalating study, DC prepared from mobilized peripheral blood were doubly loaded with autologous tumor lysate and amplified tumor mRNA and administered bilaterally near the deep cervical node chains in three biweekly courses given with weekly peg-IFN after conclusion of chemoradiation. Four dose levels from 3.5x106 to 3.6x107 total cells were tested. Patients with subtotal resection or tumor progression prior to vaccination were not excluded. RESULTS: Eighteen patients (median age 61 years (range 47-73), 94% MGMT unmethylated, 25% subtotal/partial resected) completed vaccination (16 nGBM, 2 recurrent) with no dose-limiting toxicities. Attributable AE were mostly mild and flu-like or injection-site reactions. Twelve-month OS among the newly-diagnosed cohort was 88% compared to an expected ~60% for SOC alone. Patients who received observation rather than reoperation in response to worsening MRI contrast-enhancement demonstrated gradual lesional resolution and improved OS. Immunophenotyping revealed post-vaccination elevations in CD4 and CD8 memory T-cells in peripheral blood, and spatial transcriptomic analysis revealed foci of activated inflammatory complexes at the primary tumor site. CONCLUSIONS: DOC1021 was safe, feasibly integrated within SOC, and associated with more favorable outcomes in this challenging patient population. Patients who received observation rather than reoperation for worsening MRI contrast-enhancement exhibited superior survival, suggesting an immune-reactive tumor microenvironment manifesting as pseudo-progression. These data supported initiation of a randomized Phase II trial (NCT06805305) for nGBM.

CAR-T immunotherapy has achieved remarkable efficacy in hematologic malignancies. However, the widespread clinical adoption of autologous CAR-T products remains constrained by high costs, lengthy manufacturing process, and limited accessibility. Universal or off the shelf CAR-T (UCAR-T) cells derived from healthy donors offer a promising alternative, enabling immediate treatment at a lower cost. However, the allogeneic nature of UCAR-T cells triggers immune rejection by the host immune system after infusion, thereby compromising their persistence and therapeutic efficacy. Current strategies to circumvent this rejection focus on disrupting HLA class I expression. Although this modification allows UCAR-T cells to successfully evade T cell mediated elimination, the loss of HLA class I molecules renders them vulnerable to attack by host natural killer (NK) cells. In contrast to previous approaches that attempt to retain certain non-classical HLA molecules (such as HLA-E or HLA-G) to inhibit NK cells, we directly focused on editing the ligands that mediate NK cell rejection. Through transcriptomic and in vitro validation analyses, we found that UL16 binding proteins (ULBP) 2/5/6 were substantially upregulated in UCAR-T cells compared with nontransduced donor T cells. Elevated ULBP expression effectively activates the NKG2D receptor on allogeneic NK cells and leads to killing of UCAR-T cells, thereby impairing UCAR-T function. To test whether abrogating this NK activating signal could improve UCAR-T persistence and antitumor efficacy, we generated ULBP knockout UCAR-T cells using CRISPR-Cas9 editing. Deletion of ULBP2/5/6 significantly reduced NK cell mediated killing in vitro without affecting CAR expression or T cell effector function. Compared with wild type UCAR-T cells, ULBP deficient UCAR-T cells exhibited enhanced tumor killing efficacy in the presence of NK cells. Collectively, our findings identify ULBP upregulation as one of the mechanisms underlying NK cell mediated rejection of HLA deficient UCAR-T cells. Targeted ablation of ULBP molecules provides a novel strategy to confer resistance to host NK cells, thereby improving the therapeutic potential of universal CAR T products.

ObjectivesHomologous recombination repair (HRR) deficiency is associated with improved immunotherapy responses in non-small cell lung cancer (NSCLC) patients. The HRR genes BRCA1/2 are key regulators of DNA repair, yet their impact on the tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unclear. MethodsUsing single-cell sequencing and multi-omics data, we characterized BRCA1/2 mutation-associated transcriptional programs, immune cell composition, and functional alterations in T cells, investigating the molecular and immune architecture of BRCA-mutant LUAD patients. ResultsBRCA1/2 mutations were associated with increased genomic instability and poor prognosis in LUAD patients, but predicted better clinical outcomes following immune checkpoint blockade (ICB) treatment. BRCA1 mutations correlated with an upregulated type I IFN/IFN-{gamma} signature and CD8+ T cell activation. BRCA2 mutations were associated with alveolar/stress/inflammatory responses and enhanced MHC-II antigen presentation, linked to CD4+ T cell differentiation. Both alterations coincided with reduced CD28 co-stimulation and CTL activity, hinting of immune evasion. We identified two tissue-resident memory T cell (Trm) subsets as predictors of clinical outcomes and ICB response. BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity. Furthermore, a cancer-promoting program activated by BRCA1 mutation was vulnerable to histone deacetylase inhibitors, which inhibited LUAD tumor growth. ConclusionsThis study provides a preliminary characterization of the BRCA-mutant TME in LUAD patients, revealing distinct transcriptional and immune patterns that highlight differences in BRCA1/2-associated molecular architecture and offer a framework for improving therapy efficacy in LUAD.

BackgroundImmune checkpoint inhibition has transformed cancer therapy; however, many patients fail to respond to single-agent blockade, and combination strategies are often limited by toxicity. Central nervous system tumors exploit multiple immunosuppressive pathways, including the CD200 and PD-1/PD-L1 axis to evade anti-tumor immunity and support tumor aggressiveness. MethodsWe investigated ARL200, a peptide ligand targeting the CD200 activation receptor (CD200AR) using in vitro immune assays, murine syngeneic tumor models, phosphoproteomics, and correlative studies from a first-in-human trial in recurrent glioblastoma. ResultsARL200 exposure activated DAP10/12-dependent signaling and downregulated multiple inhibitory immune checkpoint receptors, including CD200R1, PD-1, and CTLA-4, and checkpoint ligands, CD200 protein and PD-L1, through suppression of the JAK1/3-SHP-STAT-IKK/{beta}-NF{kappa}B pathway. Distinct ARL200 variant peptides elicited unique immune responses. In patients with recurrent glioblastoma, ARL200 treatment was associated with immune activation, reduced inhibitory checkpoint expression, and evidence of antigen-specific memory responses without treatment-related toxicity. ConclusionsTargeting CD200AR enables coordinated modulation of multiple immune checkpoints with a single agent, representing a next-generation immunotherapeutic strategy opening a new pathway for treating aggressive malignancies. Key PointsO_LIARL200 elicits an active immune response for the development of a potent and durable anti-tumor response C_LIO_LIARL200 abolishes the suppressive effects of multiple immune checkpoint blockades C_LIO_LIDifferent ARL200 sequences drive alternative immune responses. C_LI Importance of the StudyTumors exploit multiple immune checkpoint pathways to suppress antitumor immunity, particularly within the immunosuppressive microenvironment of the central nervous system. Current immune checkpoint inhibitors often require combination therapy to achieve clinical efficacy, frequently at the cost of increased toxicity. In this study, we demonstrate that targeting the CD200 activation receptor (CD200AR) with a peptide ligand provides a novel strategy to simultaneously downregulate multiple inhibitory immune checkpoints, including CD200R1, PD-1, PD-L1, and CTLA-4, through a shared intracellular signaling pathway. ARL200 engagement activates DAP10/12-dependent signaling while suppressing the JAK1/3-SHP-STAT-IKK/{beta}-NF{kappa}B axis, thereby overriding tumor-mediated immunosuppression. Importantly, this multi-checkpoint modulation is achieved with a single therapeutic agent and translates to immune activation and clinical responses in patients with recurrent glioblastoma, with minimal treatment-related toxicity. These findings establish CD200AR targeting as a next-generation immunotherapeutic approach with the potential to improve the safety and efficacy of immune-based therapies for aggressive CNS malignancies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/26345679v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@17a5010org.highwire.dtl.DTLVardef@11e67eborg.highwire.dtl.DTLVardef@1387c07org.highwire.dtl.DTLVardef@156d418_HPS_FORMAT_FIGEXP M_FIG C_FIG

Immune checkpoint inhibitors, particularly T cell targeting anti-PD(L)1 therapies, have revolutionized the treatment landscape for solid malignancies, but challenges related to non-responsiveness and the development of treatment resistance continue to be observed. An additional immunosuppressive axis relates to prostaglandin signaling downstream of cyclooxygenase-2 (COX2), where COX2 inhibitors have shown clinical promise in re-engaging both T and non-T cell immune compartments, yet have suffered from toxicity concerns. We report here the preclinical characterization of OKN4395, a highly potent and specific first-in-class triple antagonist of EP2, EP4, and DP1, major tumor immunosuppressive receptors downstream of COX2. OKN4395 restores immune function on both T cells and NK cells in vitro. Additionally, OKN4395 acts synergistically with anti-PD1 to increase speed and depth of antitumor activity. Overall, these findings robustly support the clinical investigation of OKN4395 in an ongoing Phase 1 trial (NCT06789172) as an innovative cancer immunotherapy for solid tumors, as a single agent and in combination with anti-PD1 therapy. Statement of significanceOKN4395, a first-in-class oral EP2/EP4/DP1 antagonist, reverses prostanoid-driven immunosuppression to restore antitumor immunity. Integrated pharmacology defines mechanism, translational biomarkers as well as both monotherapy and anti-PD1 combination strategies. These data position prostanoid tri-receptor antagonism as a translatable strategy in solid tumors. A global Phase 1 study is underway (NCT06789172).

PurposeCirculating tumor DNA (ctDNA) analyses are informative as an early indicator of immunotherapy response in advanced non-small cell lung cancer (NSCLC); however, the clinical value of ctDNA molecular response requires further validation. Patients and MethodsAs part of a prospective clinical protocol (NCT05995821), we conducted targeted error-correction sequencing of ctDNA (n=328) and matched WBC DNA (n=109) from 109 patients with metastatic NSCLC who received anti-PD-(L)1 either as monotherapy or in combination. Following cellular origin resolution of 2,818 variants, landmark molecular response (mR) was defined as undetectable ctDNA within 3-9 weeks of treatment initiation. ResultsPre-treatment ctDNA burden, but not blood tumor mutation burden, predicted survival. Implementing a tumor-naive WBC DNA-informed approach increased the number of evaluable cases without compromising the overall accuracy of landmark ctDNA molecular responses. A direct comparison of single-timepoint on-therapy ctDNA assessment with ctDNA dynamics from baseline to the 3-9-week interval, along with an analysis of heterogeneity in molecular response within the 3-9-week window, showed that undetectable ctDNA at the landmark timepoint can effectively predict survival outcomes. A significant enrichment in landmark ctDNA mR was noted among patients with progression-free survival (PFS) [≥]6 months with immunotherapy (p=2.5e-05) and chemo-immunotherapy (p=0.02). Patients in the landmark mR group had longer progression-free (p=1.6e-06) and overall survival (p=2.5e-05) than those with molecular progression. ConclusionsLandmark ctDNA molecular response provides a real-time, accurate approach for monitoring immunotherapy clinical outcomes. Although not currently validated for regulatory use, these findings demonstrate the potential utility of ctDNA as an early endpoint in clinical trials. Translational RelevanceEmploying circulating tumor DNA (ctDNA) dynamics as an early indicator of immunotherapy response requires a roadmap for the next-generation sequencing approach, definition of molecular response and establishment of its clinical sensitivity. In this study, we introduce the concept of a landmark ctDNA molecular response, determined 3-9 weeks after initiation of immunotherapy, that maximizes the number of evaluable patients without sacrificing the specificity of the approach. Notably, when evaluating heterogeneity in ctDNA detection within the landmark 3-9-week window and assessing the impact of landmark interval dynamics on survival, we found that a single ctDNA assessment performed similarly to multiple ctDNA measurements within the landmark window (most notably, regardless of whether the timepoints were concordant or discordant). Our findings demonstrate that a single assessment of early on-therapy landmark ctDNA molecular response, can identify patients at risk of disease progression and enable future intervention and therapy optimization.

PurposeBispecific T cell engagers (BiTEs) have recently been approved as a locoregional immunotherapy for malignant ascites. Although ascites is recognised as a lipid-rich, immunosuppressive environment, the mechanisms by which ascites, particularly its lipid components, suppress antitumour immunity remain poorly understood. Here, we investigated the impact of ascites-associated lipids on T cell immunosuppression and assessed whether lipid modulation could enhance the efficacy of BiTE therapy. Experimental DesignTranscriptomic profiling was performed on T cells treated with acellular ascites fluid to identify gene expression signatures associated with ascites exposure. Functional assays were conducted to evaluate the effects of ascites-associated lipids on T cell activation and cytotoxicity. In parallel, T cells were cocultured with ovarian cancer cells and EpCAM-targeting BiTEs in the presence or absence of a lipid-removal agent to assess how lipid depletion affected BiTE efficacy. ResultsT cells exposed to acellular ascites fluid exhibited an enriched transcriptomic signature associated with cholesterol efflux and incomplete fatty acid oxidation, which are metabolic features often found in exhausted T cells. These alterations converged on a metabolically imbalanced state linked to impaired plasma membrane signalling. Lipid removal from ascites selectively rescued CD137 expression but not CD25, and restored BiTE-mediated cytotoxicity, suggesting a differential impact of lipid metabolism on TCR complex-dependent versus cytokine-driven activation pathways. ConclusionsThese findings identified lipid as a driver for T cell dysfunction in ovarian cancer ascites. Removal of ascites lipids restored T cell activation and augmented BiTE-mediated cytotoxicity, supporting a combination approach to potentiate BiTE therapy in malignant ascites. Translational RelevanceMalignant ascites represents a lipid-rich, immunosuppressive tumour microenvironment that is increasingly targeted by emerging T cell-based therapies. Although EpCAM-targeting bispecific T cell engagers (BiTEs) have recently been approved for malignant ascites and multiple similar BiTEs are in clinical development, the mechanisms by which ascites impairs T cell function and potentially limits therapeutic efficacy remain poorly understood. Using patient-derived ascites throughout, this study demonstrated that lipid metabolic reprogramming, rather than immune checkpoint upregulation, was a driver of T cell dysfunction. Importantly, we demonstrated that lipid removal from ascites rescued T cell function and restored BiTE efficacy, identifying a targetable metabolic barrier to immunotherapy. While EpCAM was used as a proof-of-concept target, we anticipate the metabolic insights and therapeutic strategies identified here will be equally applicable to other BiTE and CAR-T platforms, supporting a new combination approach for the treatment of malignant ascites.

Background and AimsPancreatic ductal adenocarcinoma (PDAC) is characterized by profound immune exclusion and resistance to immunotherapy. Although WNT signaling has been implicated in PDAC, its cellular source within the tumor microenvironment and its contribution to immune suppression remain poorly defined. This study investigated whether myeloid-derived WNT signaling promotes PDAC progression. MethodsTranscriptomic data from human PDAC cohorts, including The Cancer Genome Atlas (TCGA), and published single-cell RNA sequencing datasets were analyzed. Macrophage-associated WNT5A expression in human PDAC biopsies was assessed using in situ hybridization and immunofluorescence. Macrophage-derived WNT secretion was genetically disrupted using macrophage-specific Porcn knockout mice in orthotopic and subcutaneous KPC tumor models. Lineage-resolved spatial organization of macrophage subsets was characterized using Ms4a3 fate-mapping double-reporter mice with immunofluorescence and imaging mass cytometry. Macrophages-CD8 T cells interactions were assessed using tumor-educated macrophage conditioned media, pharmacologic ARG1 inhibition, and in vivo CD8 T cell depletion. ResultsPDAC tumors with high macrophage signatures showed enrichment of noncanonical WNT signaling, and macrophage-associated WNT5A was detected in human biopsies. Disruption of macrophage-derived WNT secretion suppressed tumor growth, reversed immune exclusion, and enhanced cytotoxic CD8 T cell infiltration. Spatial lineage-resolved analysis demonstrated progressive accumulation of Hexb tissue-resident macrophages that dominated advanced lesions and formed a WNT-rich niche closely associated with Trem2Arg1 monocyte-derived macrophages. Mechanistically, macrophage-derived noncanonical WNT activated a JNK/c-Jun-ARG1 axis that inhibited CD8 T cell proliferation, an effect abolished by myeloid WNT loss. ConclusionsMyeloid-derived noncanonical WNT establishes a lineage-structured macrophage niche that enforces immune exclusion in PDAC. Targeting macrophage-restricted WNT signaling represents a promising strategy to reprogram the PDAC immune microenvironment.

BackgroundImmune checkpoint blockade (ICB) therapies demonstrate low efficacy in microsatellite stable (MSS) colorectal cancer (CRC) due to an immune-desert tumor microenvironment (TME) characterized by low antigen presentation and limited tumor-infiltrating lymphocytes (TILs). Harmine, a natural small-molecule and its promising derivatives ACB1801 have shown anti-tumor potential in preclinical models; however, their potential to reprogram the TME and overcome ICB resistance in MSS CRC remains unexplored. This study investigates whether and how ACB1801 can reshape TME to sensitize MSS CRC to ICB therapies. MethodsWe used the CT26 MSS colorectal cancer mouse model to evaluate the ability of the harmine derivative ACB1801 to enhance the efficacy of anti-PD-1 therapy. To characterize its mode of action, we performed immune landscape analysis and transcriptomic profiling of both CD45- and CD45+ tumor-derived cells. In parallel, mechanistic studies were conducted in vitro using mouse and human MSS CRC cell lines. ResultsWe demonstrate that the harmine derivative ACB1801 enhances the effectiveness of anti-PD-1 therapy in an MSS CRC mouse model. Combination therapy significantly increased CD8+ T cell infiltration and reduced regulatory T-cell (Treg) density in the TME. Transcriptomic profiling of CRC cells isolated from tumors treated with either anti-PD-1 alone or in combination with ACB1801 revealed significant enrichment of metabolic pathways in the combination group, characterized by reduced glycolysis and enhanced ferroptosis signatures. These findings were supported by in vitro data showing that ACB1801 reduces tumor cell glycolytic activity and promotes ferroptotic vulnerability. Mechanistically, ACB1801 induced STAT1 signaling, promoted CXCL10 release, and enhanced major histocompatibility complex class I (MHC-I)-dependent antigen presentation on tumor cells, thereby increasing tumor susceptibility to anti-PD-1 therapy. ConclusionCollectively, our findings indicate that combination therapy with harmine derivatives and ICBs represents a promising strategy for treating MSS CRC patients.

Terminally differentiated CD56negCD16pos NK cells have been described after chronic viral and malaria infections, and in children diagnosed with Burkitt lymphoma (BL). Despite CD56neg NK cells appearing to be poor at direct cytotoxicity, they express high levels of cytotoxic granules (i.e. granzymes, perforin), activation markers, and Fc-{gamma} receptors (CD32 and CD16) that are typically engaged in antibody-dependent cell cytotoxicity (ADCC). In addition, the abundance of CD56neg NK cells strongly correlates with IgG1 and IgG3 plasma levels, which are essential subclasses for ADCC. To determine whether CD56neg NK cells have superior ADCC capacity relative to CD56dim NK cells, we performed ADCC assays using effector cells from pediatric cancer patients and healthy children from malaria endemic regions of Kenya, targeting in vitro rituximab-treated commercial and newly established BL cell lines. We found that CD56neg NK cells were indeed capable of in vitro ADCC, showing a significant increase of CD107a-mediated degranulation in the presence of rituximab; however, they were not as efficient as CD56dim NK cells. Moreover, we found that the ADCC magnitude was significantly lower against EBV-Type 2 (EBV-T2) BL lines compared to EBV-Type 1 (EBV-T1). EBV-T2 tumor cell lines expressed significantly more lytic viral proteins than EBV-T1, making them more sensitive to direct cytotoxicity. Results from this study highlight the importance of assessing inter-patient variation in NK cell profiles in conjunction with ADCC sensitivity and EBV type within tumor cells when evaluating clinical outcomes for NK-mediated immunotherapies. SignificanceEBV type dictates NK cytotoxicity: EBV-T1 BL cells require rituximab for NK killing, while EBV-T2 BL cells are eliminated without antibody assistance, highlighting target-specific immune response to EBV-associated cancers.

Colorectal cancer (CRC) is the leading cause of cancer mortality in young adults, with chemoresistance and metastatic progression severely limiting treatment options. MUC4, a membrane-bound mucin normally confined to the apical surface of epithelial cells, becomes aberrantly overexpressed across the tumor cell membrane in CRC and other cancer types, promoting cancer survival, immune evasion, and metastasis. Therefore, MUC4 is a promising candidate for the development of targeted therapies. We detected MUC4 cell surface expression in a panel of human CRC cell lines. We engineered MUC4-specific chimeric antigen receptor (CAR) T cells and evaluated their activity against methotrexate-resistant MUC4-expressing human CRC cell lines HT29-MTX and T84. MUC4 CAR-T cells efficiently eliminated HT29-MTX and T84 cells in vitro and delayed HT29-MTX subcutaneous tumor growth in vivo. Importantly, MUC4 CAR-T cell treatment significantly reduced tumor burden and improved survival in a highly aggressive and lethal CRC model, intraperitoneal HT29-MTX CRC metastatic dissemination in NSG mice. Mouse necropsies revealed no off-target in vivo toxicities associated with MUC4 CAR-T cell therapy. Altogether, our findings establish MUC4 as an important and clinically relevant CAR-T cell target and demonstrate therapeutic efficacy of MUC4 CAR-T cell therapy for invasive CRC, a setting where chemotherapy typically fails. MUC4-targeted CAR-T cell therapy has the potential to fill a critical treatment gap for patients with chemoresistant and invasive CRC and may extend to other MUC4-expressing solid tumors. Statement of PriorityColorectal cancer is increasingly diagnosed in younger individuals, a trend accompanied by a higher prevalence of aggressive and chemotherapy-resistant disease. Despite advances in systemic therapy, outcomes for patients with refractory or metastatic colorectal cancer remain poor, underscoring a critical unmet clinical need. CAR-T cell therapy offers a mechanism to selectively target tumor cells that evade conventional treatments and may thus be particularly valuable in mucinous, chemotherapy-resistant subsets. Developing effective CAR-T strategies for new targets in colorectal carcinoma, such as MUC4, is an urgent priority in this era of rising early-onset disease.

BackgroundTumor-associated macrophages (TAMs) display context-dependent functional polarization, but whether their prognostic impact is consistent across tumor types remains unclear. MethodsWe analyzed RNA-sequencing and clinical data from The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD; n=648), lung squamous carcinoma (LUSC; n=623), and melanoma (SKCM; n=466). Cox proportional hazards models adjusted for age and AJCC stage evaluated per-standard deviation (SD) expression of TAM markers (FOLR2, TREM2) and T-cell markers (CD8A, CXCL9). Cross-histology interaction terms tested divergence between LUAD and LUSC. ResultsIn melanoma, higher FOLR2 (HR 0.87), TREM2 (HR 0.83), CD8A (HR 0.69), and CXCL9 (HR 0.67) independently predicted improved survival. LUAD showed largely neutral macrophage effects. In contrast, LUSC demonstrated an adverse association for FOLR2 (HR 1.28). Interaction analysis confirmed significant divergence for FOLR2 and TREM2 between LUAD and LUSC. ConclusionsTAM-associated prognostic effects reverse by tumor histology, supporting tumor-context-dependent macrophage polarization and informing macrophage-targeted therapeutic strategies.