CD19-directed chimeric antigen receptor (CAR) T-cell therapy has significantly advanced the treatment landscape for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). However, up to 60% of patients do not achieve a complete response. To uncover determinants of therapeutic efficacy, we analyzed the infusion products of eight r/r DLBCL patients with distinct clinical responses to axicabtagene ciloleucel using single-cell transcriptomics. Compared to patients who exhibited progressive disease, infusion products of complete responders demonstrated enriched signatures of type I interferon (IFN-I) signaling. Based on these findings, we developed a novel strategy to improve CD19-directed CAR T-cell treatment efficacy by incorporating IFN-I as an enhancer during the ex vivo manufacturing process, with IFN-I removal before CAR T-cell infusion to avoid in vivo toxicities. For both CD28- and 4-1BB-costimulated second-generation CARs, we found that low-strength IFN-I signaling enhanced CAR T-cell cytotoxicity and treatment efficacy against B-cell lymphoma and leukemia. Our low-strength IFN-I-enhanced CAR T-cell ex vivo manufacturing approach leverages an existing FDA-approved pharmacologic agent, circumvents in vivo interferon-associated toxicities, and remains fully compatible with current CAR constructs and manufacturing workflows. Together, our results establish IFN-I as a potent and costimulation-independent enhancer of CAR T-cell efficacy and provide a translationally feasible approach to enhance CAR T-cell therapies.

The influence B cells have in the tumor microenvironment has historically remained unresolved across solid tumors. At the same time, numerous findings of ectopic germinal centers in inflamed tumors containing tertiary lymphoid structures have been the impetus for redefining what effective antitumor immunity looks like. In this issue, Zeng and colleagues and Rodriguez Chevez and colleagues enrich our perspective on the roles of intratumoral B cells and their cross-talk with T cells intratumorally to augment adaptive immune responses targeting tumor antigens. See related article by Zeng et al., p. 771 See related article by Rodriguez Chevez et al., p. 792.

Loss-of-function STK11 mutations occur in 15-20% of lung adenocarcinomas (LUAD) and correlate with immunotherapy failure and worse survival. By integrating analysis of human tumor samples, a human LUAD cell line panel, and CCLE and TCGA datasets, we found that C3 production was increased in human STK11-mutant versus STK11-wildtype LUAD, modulated by LKB1 loss, while high C3 expression in STK11-mutant LUAD was associated with worse survival. STK11-KO in syngeneic murine LUAD tumors resulted in increased neutrophil and reduced T cell infiltration and anti-PD-1 resistance. STK11-KO tumor growth was similar in C3-/- versus wildtype mice. In contrast, C3 deletion in STK11-KO tumors resulted in dramatic inhibition of tumor growth and enhanced sensitivity to anti-PD-1 in immunocompetent mice but had no significant effect in CD8-depleted wildtype mice or in nude mice, pointing to tumor-derived C3 promoting immune evasion. Mechanistically, STK11 loss drove tumor-derived C3 production and downstream CXCL2 and complement factor H (CFH) production that promoted immune evasion and impaired anti-PD-1 efficacy. Our results show a C3-driven signaling axis for STK11-mutant LUAD promoting immune evasion and identifies therapeutic targets to render these tumors sensitive to anti-PD-1.

Patients with bulky, unresectable stage III non-small cell lung cancer (NSCLC) face poor outcomes with standard concurrent chemoradiotherapy (cCRT) due to large radiation fields and toxicity risks. We evaluated the feasibility and efficacy of induction immunochemotherapy to downstage tumors prior to cCRT. In this single-arm, phase II study, eligible patients received two cycles of induction serplulimab plus chemotherapy. Patients without progression underwent multidisciplinary re-staging to receive either cCRT with serplulimab consolidation or surgery. The primary endpoints were objective response rate (ORR) post-cCRT and changes in lung volume receiving ≥20 Gy (V20). Forty-nine patients were enrolled (75.5% stage IIIB/IIIC). Following induction, the V20 was significantly reduced by 11.41% (p < 0.001). The ORR following cCRT was 77.14% (95% CI: 59.86-89.58%). The 12-month progression-free survival (PFS) and overall survival (OS) rates were 76.15% and 90.54%, respectively. Notably, six patients successfully underwent R0 resection, with five achieving a pathological complete response. Grade 3 adverse events occurred in 14.29% of patients, and toxicities were manageable. Induction serplulimab-chemotherapy prior to cCRT is a promising strategy for bulky stage III NSCLC. This approach significantly reduced radiation volumes, achieved high response rates, and enabled curative surgery in selected patients with manageable toxicity.

Abstract Background: Patients with advanced thyroid cancer have few treatment options and poor survival [e.g. 10% at 10 years for radioactive iodine-refractory differentiated thyroid cancer (DTC)] and immune checkpoint inhibitors (ICI) have not shown benefit in these patients. The goal of this project is to identify novel strategies to improve the efficacy of ICI therapy in thyroid cancer. Prior work showed that suppressive myeloid cells are important contributors to immune escape and ICI resistance in thyroid cancer. In addition, clinical studies have shown that the presence of MDSCs in the peripheral blood of thyroid cancer patients correlates with more advanced disease and recurrence. In melanoma, colon, and lung tumor models, the TLR9 agonist CpG can act on myeloid cells to reduce suppressive function, increase antigen presentation and augment T cell proliferation, activation, and ingress into the tumor. We hypothesized that combined ICI therapy with TLR9 agonist CpG reverse of MDSC suppression to improve anti-tumor efficacy of ICI in DTC. Methods: Mice bearing poorly differentiated BRAFV600E+ papillary thyroid cancer tumors were randomized to treatment with isotype control or ICI (anti-PD1 or anti-PDL1; 250mg/dose i.p., 2x weekly) antibodies, with or without CpG-1826 peritumorally. In addition, we tested ICI with directly conjugated CpG given intraperitoneally compared to ICI alone as a potential systemic therapy. Immune populations in tumor and spleen were evaluated using immunofluorescence, flow cytometry, and in vitro functional assays. Tumor volume was measured thrice weekly. Results: ICI treatment alone (anti-PD1 or anti-PDL1) had no effect on tumor growth compared to isotype control. There were no differences in CD44+ IFNγ+CD8+ effector T cells and CD11b+Gr1hi MDSCs in tumor-bearing mice. Interestingly, peritumoral injection of CpG with concurrent ICI treatment resulted in significantly slower tumor growth (p&amp;lt;0.05) and increased tumor-infiltrating cytotoxic IFNγ+CD8+ T cells. Ex vivo assessment of tumor-associated immune cells showed that CpG treatment abrogated MDSC-mediated suppression of CD8+ T cell proliferation and IFNg production. CpG showed modest but not significant direct effects on CD8+ T cells. Furthermore, PD1+CD8+ T cells and PDL1+ MDSCs were enriched in the TME and wondered whether this checkpoint protein enrichment could be leveraged for targeting of CpG therapy to the tumor. Systemic delivery of CpG is limited by systemic toxicity, whereas intratumoral CpG delivery has limited use for metastatic disease and short intratumoral half-life. allowing for it to be specific targets for Cpg conjugates to deliver directly to the TME. ICI-conjugated CpG (anti-PD1 or anti-PDL1) administered effectively reduced tumor growth and increased population of cytotoxic effector CD44+ IFNγ+ CD8+ T cells in the TME (p&amp;lt;0.05 for anti-PDL1-CpG, trend for anti-PD1-CpG). Conclusion: Our study underscores the role of MDSCs in ICI-resistance seen in advanced thyroid cancer and suggests that TLR9 agonists may improve clinical outcomes for patients. Citation Format: Joah E. Lee, Jaden Nguyen, Jarod Olay, Kyleigh Kimbrell, Amber Lu, Aime T. Franco, Alan L. Epstein, Trevor E. Angell, Melissa G. Lechner. Reversing immune checkpoint inhibitor resistance rherapy in advanced thyroid cancer [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-A008.

Abstract Chemotherapy induces senescence rather than apoptosis in breast cancer cells that have wild type p53, leading to relapse and worse patient outcomes. Senescent tumor cells evade immune-mediated cell death by upregulating several hundred interferon gamma (IFNγ)-dependent genes that suppress T cell activity, including Cd274 (PD-L1), but also sensitize to immune checkpoint inhibitors through expression of antigen presentation and MHC-I/II genes. The expression of IFNγ in the residual disease of treated tumors is entirely dependent on tiny, potent population of CD4+ T cells. Syngeneic mammary tumors transplanted orthotopically into Cd4-KO mice have no expression of IFNγ or associated target genes during senescence, while tumors in Cd8-KO mice have an intermediate phenotype. To investigate the mechanism of IFNγ expression by the CD4+ T cells in the residual disease of chemotherapy-treated, senescent tumors, we first examined the role of T cell receptor (TCR) engagement. We transplanted MMTV-Wnt1 and MMTV-PyMT tumor models into WT or SMARTA1 mice that have a specific CD4+ TCR that recognizes the LCMV GP61-80 epitope. After treatment, we found that isolated tumor cells from SMARTA1 mice expressed p53 target genes, such as Cdkn1a, but failed to express IFNγ target genes despite having an abundant CD4+ T cell population. To determine if tumor cell expression of the LCMV GP61-80 epitope could rescue IFNγ-dependent gene expression induced by CD4+ cells in SMARTA1 mice, we transduced our two tumor models with a vector containing mCherry or mCherry-LCMV GP61-80. Following transplant and treatment, we found that senescent tumor cells expressing mCherry-LCMV GP61-80 from treated SMARTA1 host mice fully restored expression of IFNγ-dependent genes to comparable levels observed in WT hosts. Senescent tumor cells expressing mCherry alone in SMARTA1 hosts, however, were dramatically diminished in expression of IFNγ target genes. Flow cytometry on isolated CD45+ cells from treated and untreated tumors showed that immune populations remained relatively stable between host genotypes. In sum, our data demonstrate that, in the residual disease of treated tumors, the IFNγ that induces tumor expression of critically important immune modulatory and antigen presentation genes is derived entirely from CD4+ TCRs that recognize specific tumor antigens. Citation Format: Calvin WT. Adam, Raegan M. Kvadas, Fang-Yen Chiu, David A. Gervasio, Timothy Kayes, Dorota D. Wyczechowska, James G. Jackson. Recognition of senescent breast tumor antigen by CD4+ T cells causes IFNγ release leading to target gene expression [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-B001.

Abstract Background: High-grade neuroendocrine neoplasms (NENs), including poorly differentiated neuroendocrine carcinomas (NECs) and well-differentiated grade 3 neuroendocrine tumors (NETs), are aggressive malignancies with limited effective treatment options. Immune checkpoint inhibitors (ICIs) have demonstrated limited clinical activity in these neoplasms. Seneca Valley Virus (SVV-001) is a novel oncolytic RNA virus that has shown synergistic activity with ICIs in preclinical cancer models. Additionally, SVV-001 has been observed to reverse ICI resistance in vivo, further supporting its evaluation in combination with nivolumab and ipilimumab. Methods: This is an investigator-initiated, phase 1, dose-escalation and cohort-expansion study evaluating intratumoral SVV-001 in combination with nivolumab and ipilimumab in patients with histologically confirmed poorly differentiated NEC or well-differentiated grade 3 NET. The trial was activated in March 2025, with patient enrollment currently ongoing. A standard 3+3 dose-escalation design is being employed to determine the recommended phase 2 dose (RP2D). Following dose escalation, an expansion cohort will further evaluate safety and preliminary signals of activity. Tumor endothelial marker 8 (TEM8), a potential biomarker of SVV-001 sensitivity, will be assessed as part of correlative studies. Hereby we present the interim safety and the first efficacy results from dose escalation cohorts. Results: As of January 12, 2026, the trial has completed enrolling patients in first two cohorts – dose level 1 (DL1) and dose level 2, and 2 patients in dose level 3 (DL3) cohort. All 9 enrolled patients received one dose of SVV-001 in combination with ipilimumab and nivolumab per protocol. No DLTs were observed in DL1 and DL2. Overall two patients experienced Grade 3 serious adverse events (anorexia, weight loss; hyperbilirubinemia, increase of alanine amino transferase; and seizures) unrelated to SVV-001, and one patient reported Grade 3 adverse event (elevated creatinine, xerostomia) possibly related to SVV-001 injection. Two patients achieved a partial response, 2 had stable disease and 3 came off study due to disease progression. Updated safety and efficacy data will be presented at symposium. Conclusion: The interim data demonstrate an acceptable safety profile and encouraging signs of efficacy in a disease representing a significant unmet medical need. Clinical Trial Identifier: NCT06889493 Citation Format: Aman Chauhan, Isildinha M. Reis, Chinmay Jani, Rakhi Modak, Daniel Bilbao. Cortes, Stephanie Baboun, Alexander Rivera, Hung Trinh, Paul Hallenbeck, Jaime Merchan, Gilberto Lopes, Peter Hosein. A phase 1 trial of the oncolytic virus SVV-001 with Nivolumab and Ipilimumab in patients with high grade Neuroendocrine Neoplasms [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-B002.

Abstract Introduction CART19/20 was developed by engineering autologous naïve/memory T (TN/MEM) cells with a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR) with the goal of overcoming resistance to single-target CD19-directed CAR-T cells. We report an update of a first-in-human phase 1 clinical trial of CART19/20 for patients with relapsed/refractory non-Hodgkin lymphoma (R/R NHL) and small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) (NCT04007029) with a median follow-up of over 4 years. Methods Eligible patients were ≥18 years old with R/R NHL and SLL/CLL, with ECOG 0 or 1. Patients were included if they had 2 or more prior lines of therapy with diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBCL) and 3 or more prior lines for CLL/SLL, follicular lymphoma (FL), and mantle cell lymphoma (MCL). Autologous leukocytes were obtained by leukapheresis and sorted for CD62L+ TN/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR, and subsequent expansion of gene-modified T-cells. The primary end point was safety, and secondary endpoints were clinical response, progression free survival (PFS), overall survival (OS), and CART19/20 persistence. Results As of the data cutoff of November 25, 2025, 15 patients had received CAR-T 19/20 cells (6 DLBCL, 4 FL, 4 MCL, 1 HGBCL). Patients were treated at two dose levels, either 50x106 +/- 30% CAR+ cells (12 patients) or 200 x106 +/- 30% CAR+ cells (3 patients). The median age was 60 (range 29 to 70). Patients had received a median of 4 prior lines of therapy (range 2 to 8). All patients except one had stage IV disease, and 11 of 15 received bridging therapy. The CART19/20 cells were enriched with central-memory T cells (TCM: CD45RA–/CD45RO+/CD62L+). Maximum CRS was grade 2 which occurred in one patient. There were 9 patients that experienced max grade 1 CRS and no ICANS or neurotoxicity. There were 12 serious adverse events and two dose limiting toxicities. Fourteen of fifteen patients achieved an objective response (93% ORR), with 12 patients (80%) achieving a complete response (CR). With a median follow up of 51 months (range: 6.4 – 73.1), the median PFS was 37.5 months (95% CI 4.8 – not estimable) and the median OS was not yet reached. The median peak CAR-T19/20 cell expansion was 592,500 cells/mL whole blood, with mean persistence of CART 19/20 from date of infusion to last date of measurable CART19/20 cells of 526 days. Conclusion Overall, this phase 1 trial demonstrates that CART19/20 is a safe and effective therapy with the potential to overcome common resistance mechanisms of CAR-T cell therapy. Citation Format: Sophie Carlson, Benjamin Puliafito, Christopher M. Walthers, Brenda Ji, Jacob Naparstek, Jia Chen, Mobina Roshandell, Caitlin Harris, Mobina Khericha Gandhi, Melanie Ayala Ceja, Christy Sidhu, Karla Nawaly, Martin Allen-Auerbach, Sven De Vos, Patricia Young, Caspian Oliai, Gary Schiller, John Timmerman, Antoni Ribas, Yvonne Chen, Sarah Larson. Phase 1 Trial of Bispecific CART19/20 Cells for Relapsed or Refractory Non-Hodgkin Lymphoma: Updated Results with Over Four Years Median Follow Up [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C001.

Abstract Background: Metastatic breast cancers often derive limited benefit from immunotherapy, potentially due to impaired antigen presentation and limited cross-presenting cDC1 activity. We tested a rational triplet designed to induce immunogenic antigen release (pegylated liposomal doxorubicin; Doxil), expand dendritic cell populations including cDC1s (Flt3 ligand; Flt3L), and license antigen-presenting cells (APC) by CD40 agonism (CD40a) to increase co-stimulation T cell immunity. Methods: Female mice bearing orthotopic EO771, AT3, or 4T1 tumors (∼50 mm3) received Doxil D1, Flt3L D4-8, and an agonistic CD40 antibody D11, 14, 17, with monotherapy and doublet controls. Tumors and draining lymph nodes (TDLN) were analyzed by immunohistochemistry, flow cytometry, scRNA-seq, and TCR-seq. Mechanistic requirements were assessed using Batf3-/- mice, immune cell depletion, IL-12 neutralization, and inhibition of lymphocyte egress (FTY720). Results: Triplet therapy improved tumor control and survival across models. Flt3L expanded cDC1, cDC2, and mature DC states, while CD40a increased co-stimulation and antigen-presentation programs (Cd86/B2m) and induced Il12b. APC remodeling was associated with increased intratumoral CD8+ T cell infiltration, reduced LAG-3/TIM-3 exhaustion markers, and enrichment of stem-like features. TCR-seq demonstrated clonal expansion with addition of Flt3L and CD40a to Doxil. Tumor control required cDC1s as efficacy was lost in Batf3-/- mice, while remaining intact after depletion of the other 2 primary antigen presenting cells (macrophages and B cells). CD8 depletion partially reduced efficacy, whereas combined CD4/CD8 depletion eliminated tumor control. Benefit persisted with FTY720, supporting reinvigoration of pre-existing intratumoral T cells. We found efficacy was dependent on IL-12, as neutralization abolished tumor control. Conclusion: Anthracycline chemotherapy plus Flt3L-driven DC expansion and CD40 licensing remodels murine breast tumors toward cDC1 and IL-12 dependent T cell control. This framework is being evaluated in a phase I pilot combining Doxil with CDX-301 (recombinant Flt3L) and CDX-1140 (CD40 agonist) in metastatic or unresectable locally advanced HER2-negative breast cancer (NCT05029999). Two lead-in cohorts randomize participants 2:1 to one cycle of triplet therapy vs Doxil alone with paired biopsies at baseline and post-cycle 1 lead-in; all patients then transition to triplet therapy. The primary endpoint is recommended phase II dose for CDX-1140 based on DLTs; secondary endpoints include CD8+ T cell infiltration (triplet vs Doxil alone) and clinical activity. Citation Format: Preteesh L. Mylabathula, Riyasat Ali, Skanda Hebbale, Xinyi Qi, Shruthi Nooka, Sangeetha M. Reddy. cDC expansion and CD40 activation after immunogenic chemotherapy shift the breast tumor microenvironment toward functional T cell immunity and IL-12-dependent tumor control [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-B008.

Abstract Background: Pleural mesothelioma (PM) is an aggressive malignancy with a poor prognosis. The clinical efficacy of standard therapy with immune checkpoint inhibitors (ICI) is limited and heterogeneous across PM subtypes. Tumor-intrinsic characteristics (i.e., inflammatory phenotype, molecular features, DNA methylation) may influence immune responsiveness, but predictive biomarkers of ICI therapy efficacy in PM are still lacking. Methods: NIBIT-EPI-MESO is a retrospective, multicenter study, sponsored by the NIBIT Foundation, evaluating biological correlates of clinical outcomes in PM patients (pts) treated with ICI (i.e., anti-CTLA-4 plus anti-PD-1, anti-CTLA-4 plus anti-PD-L1, or anti-CTLA-4 monotherapy). Pre-ICI therapy FFPE tumor samples were analyzed by RRBS methylation (n=83 pts) and RNA-seq (n=82 pts), with methylation subtypes defined by consensus clustering of the top 1% most variable CpGs. Tumor microenvironment (TME) was characterized by multiplex immunofluorescence analysis of CD4, CD8, CD20, CD68, CD163 (n=35 pts). Integrated multi-omics analyses were used to associate tumor biology with clinical outcome of PM pts. Results. Unsupervised methylation profiling identified four PM subsets with increasingly global DNA methylation levels: demethylated (DEM), LOW, intermediate (INT), and CpG island methylator phenotype (CIMP). Methylation subtypes were significantly associated with response to ICI, with LOW/DEM enriched among responder (R) pts and INT/CIMP in non-R pts (p= 0.002); no association of response to ICI was found with PM histotype (p=0.33). The LOW subset exhibited the longest median overall survival (mOS) and the highest 3-year OS rate, expressed genes involved in pathways associated with innate and adaptive immune responses, and showed an “inflamed” TME (i.e., CD8+ T cells, CD20+ B cells). Conversely, the CIMP subtype had the shortest mOS and OS rate, was characterized by genes enriched in developmental, morphogenetic and cell cycle-related processes, along with a “desert” TME. Functional characterization of the identified methylation classes of PM was validated in the MESOMICS dataset. Accordingly, a PM methylation subtype classifier was developed to predict response to ICI therapy. Conclusions: Tumor DNA methylation defines biologically and clinically distinct immune phenotypes in PM and robustly predicts clinical response and long-term survival in ICI-treated PM patients, regardless of tumor histology. Citation Format: Luana Calabrò, Francesca Pia Caruso, Alessia Covre, Teresa Maria Rosaria Noviello, Maria Fortunata Lofiego, Rossella Tufano, Luigi Ferraro, Piera Grisolia, Antonio De Falco, Vincenzo Lagano, Francesco Sgambelluri, Giovanna Sabella, Giulia Rossi, Giulia Gibilisco, Francesco Marzani, Emma Bello, Elena Simonetti, Vincenzo D'Alonzo, Michele Caraglia, Sandra Coral, Antonina De Angelis, Luigi Cerbone, Sara Delfanti, Federica Grosso, Anna Maria Di Giacomo, Massimo Milione, Roberta Mortarini, Andrea Anichini, Michele Ceccarelli, Michele Maio. Tumor methylation subtypes predict the clinical outcome to immunotherapy in pleural mesothelioma patients from the NIBIT-EPI-MESO study [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C009.