Supplementary Table 3. Demographic and clinical characteristics of patients from the HCRN 16-260 trial included and not included in the study.

Supplementary Table 5. Association between density of terminally exhausted CD8+ TILs measured as continuous variables and clinical outcomes (ORR, PFS).

Serous endometrial cancer (SEC) is an aggressive subtype of endometrial cancer (EC) with poor prognosis and limited treatment options. Here, we developed a clinically relevant, immunocompetent serous-like mouse model incorporating oncogenic PIK3CA mutation, Trp53 loss, and MYC overexpression. Using this model together with human EC cell lines, patient-derived organoids (PDOs), xenografts, and patient datasets, we investigated mechanisms underlying resistance to PI3Kα-targeted therapy. Single-cell profiling reveals that FGFR1/2 upregulation associates with intrinsic resistance, whereas FGFR3 characterizes acquired resistance. Dual FGFR and PI3Kα inhibition produced superior tumor control compared with either agent alone. Mechanistically, FGFR signaling promotes immune evasion by downregulating MHC-I/HLA-mediated antigen presentation and enriching M2-type tumor-associated macrophages. FGFR inhibition reversed these changes and synergized with anti-PD-1 therapy to enhance antitumor immune responses and establish durable immune memory. Collectively, these findings identify FGFR signaling as a key driver of therapeutic resistance and immune escape in SEC and support FGFR-targeted combination strategies.

UV radiation (UVR) drives high mutational burdens, yet precursor melanocytes accumulate these mutations without triggering immune clearance. Here, we investigated whether melanocyte-intrinsic transcriptional program(s) underlie immune tolerance to mutations resulting from UVR exposure. In primary human melanocytes, expression of PD-L1 (CD274) was dependent on microphthalmia-associated transcription factor (MITF), a crucial regulator of melanocyte development and an intermediate in the UV-tanning pathway. MITF directly activated PD-L1 transcription by binding a conserved upstream enhancer containing functional E-box elements. MITF determined both baseline melanocytic PD-L1 expression in healthy skin and its induction following UVR, independent of interferon signaling. Melanocyte-restricted Pd-l1 deletion in mice triggered CD8+ T cell infiltration and depigmentation after long-term UVB exposure, recapitulating features of human vitiligo. PD-L1-deficient human induced pluripotent stem cell (iPSC)-derived melanocytes underwent increased apoptosis and were more susceptible than PD-L1-intact melanocytes to gp100-specific CD8+ T cell killing. Thus, a melanocyte-intrinsic MITF-PD-L1 tolerance program protects melanocytes from autoimmune destruction, potentially facilitating early immune evasion during melanoma development and conversely underlying the responsiveness of melanoma to PD-1/PD-L1 blockade.

Abstract Solid tumors hijack immune system through tumor-immune cycle. Here, we tackle multiple key steps to achieve a great therapeutic effect for clear cell renal cell carcinoma (ccRCC) treatment. Using direct stochastic optical reconstruction microscopy (dSTORM) and immunohistochemistry (IHC), we first quantified the expression densities of carbonic anhydrase IX (CAIX) and CD70, two tumor associated antigens (TAAs) that are highly expressed in ccRCC despite of stages. We found that targeting both CAIX and CD70 could significantly increase the target tumor population thus addressing tumor heterogeneity. The affinity/avidity fine-tuned anti-CAIX CAR G9 only kills CAIX high tumor cells but not CAIX low normal tissues, successfully mitigating on-target off-tumor (OTOT) toxicities. To combat immunosuppressive tumor microenvironment (TME), we armed CAR-T cells with immune checkpoint inhibitor (ICI) as a payload to prevent CAR-T cells from being exhausted as well as reverse the exhaustion of other tumor infiltrating leukocytes (TILs). In previous clinical trials, the combination of anti-PD-1 nivolumab and anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) ipilimumab demonstrated greater benefits for intermediate- and poor-risk RCC patients. Through phage display and antibody engineering, we obtained a novel tetravalent bispecific antibody (BsAb) targeting PD-1 and CTLA-4, which showed superior efficacy compared to nivolumab and ipilimumab combination therapy. This dual-targeted fine-tuned immune restoring (DFIR) CAR-T showed a great level of cytotoxic activity and maintained an active antitumor immunity in a multi-challenge assay compared to CAR-T cells with irrelevant payload. In a humanized ccRCC orthotopic mouse model, we demonstrated that the CAR-T cells with BsAb payload exhibited enhanced tumor control. In summary, anti-CAIX/CD70 DFIR CAR-T cell therapy holds the promise to achieve cures of advanced RCC by addressing tumor heterogeneity, mitigating OTOT toxicity and rewiring immunosuppressive TME. Citation Format: Yufei Wang, Nithyassree Murugan, Gabriella Kastrunes, Hsien-Chi Yuan, Alicia Buck, Matthew Chang, Christian Coherd, Yasmin Nabil Laimon, Marion Grimaud, Atef Fayed, Oanh Pham, George Talbott, Wonyeong Kang, Jiwon Yang, James Keck, James Lim, David Barbie, Wenxin Xu, Jon Wee, Toni K. Choueiri, Sabina Signoretti, Gordon J. Freeman, Wayne A. Marasco. Design of dual-targeted fine-tuned immune restoring (DFIR) CAR-T cell therapy to treat advanced renal cell carcinoma (RCC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr PR002.

Supplementary Table 1. List of the antibodies and dyes used for the Multiplex IF assay (panel 1).

Supplementary Table 4. Association between density or percentage of candidate biomarkers measured as continuous variables and clinical outcomes (ORR, PFS) controlled for IMDC risk groups (favorable vs. poor/intermediate).

Supplementary Figure 1. A representative image of a ccRCC sample from the HCRN GU16-260 trial analyzed by multiplex immunofluorescence for expression of CD8, PD-1, TIM-3, LAG-3 and CD163. The insets highlight a CD8+PD-1+TIM-3-LAG-3- TIL and a CD163+ TAM

Abstract Determining how antibodies that target immune checkpoints alter lymphocyte functions can improve our understanding of immune checkpoint inhibitors responses in patients. HERV-H LTR-associating 2 (HHLA2, B7-H7) is an immune checkpoint expressed by many tumors. Our group previously found that B7-H7 has an inhibitory receptor, KIR3DL3, in addition to having an activating receptor, TMIGD2. Both B7-H7 receptors are expressed by subsets of T cells and NK cells. However, little is known about how different subsets of lymphocytes are affected by B7-H7 engagement of these receptors, and multiple B7-H7:KIR3DL3 blocking antibodies are currently enrolling cancer patients in Phase I clinical trials (NCT05824663, NCT06240728).In this study, we explored the effect of tumors expressing B7-H7 on NK cells in vitro by single cell RNA sequencing (scRNAseq). NK cells were isolated from peripheral blood and co-cultured with K562 cells or K562 cells overexpressing B7-H7 for 2hr. Co-cultures were performed in the presence of different B7-H7 blocking antibodies or isotype controls. 133,017 cells passed quality control, with 9 distinct NK cell populations being identified. Primary NK cells not co-cultured with tumor cells were mainly composed of two TXNIP+ CD96 hi NK cell clusters that had low expression of cytotoxic and inflammatory genes. Additionally, these two clusters had the highest expression of TMIGD2, suggesting that these populations represent resting NK cells. Co-culture with K562 or K562-B7-H7 cells increased the proportion of highly cytotoxic and inflammatory NK cell clusters. However, XCL2, CSF2, IFNγ and IL3 were higher in NK cells co-cultured with K562-B7-H7 than with K562, suggesting B7-H7 on K562 cells is proinflammatory in this early activation model. Notably, only a small subset of peripheral blood NK cells expressed the inhibitory receptor KIR3DL3, as expected in early NK activation. Furthermore, all antibody treatments regardless of the Fc isotype were associated with increased proportions of CRTAM+ IL2RA+ TNFRSF9+ NK cell clusters, compared to NK cells cultured without antibody. Differential gene expression analysis between B7-H7 antibody clones 6F10 and 2C4 by respective Fc (IgG1 or IgG4) suggested blocking TMIGD2 with the dual receptor blocking B7-H7 antibody (6F10) reduces the extent of activation of the NK cells compared to the B7-H7 antibody (2C4) that blocks KIR3DL3 but not TMIGD2.Our work suggests the importance of selecting B7-H7 therapeutics that spare the interaction of B7-H7:TMIDG2 to maintain any NK-dependent antitumor benefit of B7-H7 expression. Citation Format: Michael Gomez, Deepthi Chowbene, Nahuel Perrot, Nikolaos Kalavros, Shuoshuo Wang, Antonella Arruda de amaral, David F. McDermott, Gordon J. Freeman, Ioannis S. Vlachos, Kathleen M. Mahoney. Investigating the transcriptomic signature of B7-H7-mediated activation of NK cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6559.

Abstract Purpose: We investigated components of the immune tumor microenvironment as determinants of clinical outcome to anti–PD-1 therapy in patients with metastatic clear-cell renal cell carcinoma (ccRCC) treated with first-line nivolumab in the phase II, nonrandomized HCRN GU16-260 trial. Experimental Design: Pretreatment primary ccRCCs from 72 patients were analyzed via multiplex immunofluorescence and image analysis to assess nonterminally exhausted CD8+ (CD8+PD-1+TIM-3−LAG-3−) tumor-infiltrating lymphocytes (TIL), PD-1+ regulatory T cells (Treg), total and peritumoral tertiary lymphoid structures (TLS), and CD163+ tumor-associated macrophages (TAM). Clinical endpoints included objective response rate (ORR) and progression-free survival (PFS). Results: The densities of CD8+PD-1+TIM-3−LAG-3− TILs, total and peritumoral TLSs, and CD163+ TAMs, as continuous variables, were associated with improved ORR [odds ratio: 1.54 (1.10–2.16) for TILs; 1.18 (1.02–1.37) for total TLSs; 1.10 (1.02–1.18) for peritumoral TLSs; and 2.21 (1.33–3.69) for TAMs] and longer PFS [hazard ratio: 0.79 (0.67–0.95) for TILs; 0.92 (0.85–0.99) for total TLSs; 0.94 (0.90–0.98) for peritumoral TLSs; and 0.77 (0.61–0.97) for TAMs]. Although the percentage of PD-1+ Tregs as a continuous variable was not associated with outcomes, at an optimal cutoff, a high percentage of PD-1+ Tregs tended to be associated with lower ORR (12.5% vs. 43.6%, P = 0.093) and was associated with shorter PFS (3.4 vs. 10.9 months, P < 0.001). The biomarkers were not strongly correlated with each other, and their integration in multibiomarker models further stratified outcomes. Conclusions: Individual immune cell populations within the ccRCC microenvironment are associated with response/resistance to first-line anti–PD-1 therapy. Our findings support the development of combined immune marker models to identify patients with divergent outcomes.

Supplementary Table 11. ORR and PFS per density of total TLS (high vs low) combined with % of PD-1+ Tregs (high vs low).

In cancer and chronic infection, CD8 T cell exhaustion is hallmarked by expression of inhibitory receptors such as PD1, TIM3, LAG3 and others1-3. Thus, inhibitory molecule focus has been limited to cell-surface proteins. Here we evaluate the surface lipid metabolite phosphatidylserine (PS) as a regulator of exhaustion. PS primarily localizes to the inner plasma membrane of live cells but is well known to be externalized to the outer membrane during cell death. The role of exposed PS on live immune cells is less clear. We show that viable, antigen-specific CD8 T cells externalize PS during lymphocytic choriomeningitis virus (LCMV) infection. T cell activation induced initial PS exposure, and chronic antigen stimulation sustained externalization. Transcriptomic and lipidomic analyses also identified PS accumulation in exhausted CD8 T cells. To evaluate a role for exposed PS in exhaustion, we treated LCMV chronically infected mice with a PS-targeting antibody (mch1N11)4 and found that it expanded LCMV-specific CD8 responses. PD1+TCF1+ stem-like CD8 T cells downregulated quiescence-associated gene modules and increased proliferation after antibody treatment, highlighting an inhibitory role for PS. Mechanistically, exposed PS on T cells functioned extrinsically to suppress dendritic cell immunostimulatory phenotypes, in turn limiting CD8 T cell responses. PS-targeting antibody with anti-PDL1 synergized to increase CD8 responses and improve viral control. Finally, we show that PD1+ CD8 T cells from human tumours can also expose PS. In summary, we detail CD8 T cell PS biology and provide insight into a mechanism by which exposed PS functions as a 'non-classical' extrinsic inhibitory molecule in exhaustion.

Estrogen receptor (ER) positive breast cancer is the most prevalent subtype, commonly responsive to endocrine therapies. Immune checkpoint inhibitors (ICIs) have limited efficacy in ER-positive disease, highlighting the need for the development of combination immunotherapies for these patients. We previously established that nitroso-N-methylurea-induced mammary tumors in outbred Sprague-Dawley rats mimic immune evasive mechanisms and the heterogeneity of ICI response observed in patients. We identified a “luminal growing” gene signature in ER-positive tumors, which correlated with tumor growth and immune-related differences. Here, we evaluated targeting candidates from this signature KMT5B/C and IKBKE using inhibitors A-196 and IKBKEi respectively, alongside anti-estrogen (fulvestrant) and a TGFβ blocking antibody (NIS793), both individually and in combination with αPD-L1, within this rat model. Fulvestrant emerged as the most effective treatment, inducing regression of most existing tumors and reducing on-treatment tumor burden when combined with αPD-L1. A-196, while ineffective as a monotherapy, demonstrated enhanced response when combined with αPD-L1. Comprehensive tumor profiling through polychromatic flow cytometry and single-cell RNA sequencing revealed that A-196 induced a luminal-to-basal shift in tumor epithelial cells, enhancing antigen presentation, whereas epithelial-to-mesenchymal transition was linked to fulvestrant resistance. Our findings underscore the value of the rat mammary tumor model for preclinical studies in ER-positive breast cancer and advocate for the further validation and potential clinical development of KMT5B/C inhibitors to enhance the efficacy and broaden the applicability of ICI therapy in cancer patients.

Oncolytic virotherapy represents a promising yet under-explored approach for precision cancer treatment, particularly when tailored to tumor-specific molecular profiles. Patients with high-grade isocitrate dehydrogenase (IDH) mutant astrocytomas have limited treatment options and poor prognoses. Here, we investigate the therapeutic efficacy of rQNestin34.5 v.2 (CAN-3110), an engineered oncolytic herpes simplex virus 1 (oHSV-1), in IDH1-R132H-mutant diffuse gliomas. We demonstrate that the IDH1-R132H mutation enhances glioma susceptibility to viral infection through upregulation of Nectin-1, the main HSV-1 entry receptor. Concurrently, IDH1-R132H-driven DNA hypermethylation suppresses interferon (IFN) signaling, creating a permissive microenvironment that facilitates viral replication and tumor cell apoptosis. In immunocompetent murine glioma models, intratumoral administration of rQNestin34.5 v.2 induces robust antitumor immune activation, including increased immune infiltration and systemic IFN-γ release. However, elevated expression of poliovirus receptor (PVR) and the immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on tumor-infiltrating leukocytes suggests a potential resistance mechanism to virotherapy. Combining rQNestin34.5 v.2 with TIGIT blockade enhances therapeutic efficacy compared to monotherapy, identifying IDH1-R132H as a potential predictive biomarker for oncolytic virotherapy response.