Abstract Background: Immune responses to shared cancer antigens have correlated with durable clinical responses. DPV-001 is a novel microvesicle vaccine that contains >300 proteins for genes commonly overexpressed by cancer and is enriched for short-lived proteins (SLiPs), and defective ribosomal products (DRiPs), including non-canonical peptides, representing potential shared non-mutated alternative cancer neoantigens (dark matter). Preclinical studies identified increased therapeutic efficacy when this vaccine strategy was combined with anti-PD-1 and anti-GITR, leading to this clinical trial for patients with advanced or metastatic HNSCC. Methods: Following safety run-in, eligible pts were randomly assigned 1:1 to receive DPV-001 +/- GITR agonist mAb (INCAGN-1949; q2wks). All received sequenced PD-1 mAb (retifanlimab; q4wks) starting D15. Safety was evaluated as the primary endpoint and efficacy was evaluated as a secondary endpoint. Tumor biopsies were taken pre-treatment, Wk 2 and 8, and assessed by CITE-seq, scRNA-seq, BCR-seq, and TCR-seq. Multiplex immunofluoresence was performed on biopsies. Blood samples were taken pre-treatment and at multiple timepoints and analyzed by flow cytometry and seromics. Results: 18 pts received backbone therapy of DPV-001 + sequenced PD-1; of these, 9 pts also received GITR starting D1 (Arm 2). G3 irAE's included: hypothyroidism & mucositis (Arm 1, same pt); pneumonitis and adrenal insufficiency (Arm 2, separate pts). No G4 toxicity was observed. Combining both arms, the response rate (RR) for PD-1 naïve pts was 55% (5/9), and 33% (3/9) for PD-1 experienced pts. Two CR's have been observed in Arm 2. Durability of response (DOR) and progression-free survival (PFS) will be reported at the meeting. DPV-001 induced increased Ki67+ CD4 EM (p<0.042) by D15, prior to first dose of PD-1. Addition of GITR at D1 further increased Ki67+ CD4 (p=0.008) & CD8 EM cells (P=0.0006) by D15. Activated or proliferating CD4 and CD8 EM were significantly increased in both arms by D30 (p<0.025). Across above specified analyses, pts receiving GITR had the greatest increase. Evaluable pts had significantly (all p<0.032) increased numbers of TIL expressing IFN-γ and/or GZMB, LAG-3, and TIM3 in on-treatment biopsies. TCR, BCR, and seromic analyses, as well as studies to identify targets of anti-cancer immunity, are ongoing. Conclusions: This 18 pt trial of DPV-001 and sequenced PD-1 +/- GITR shows a promising RR and evidence of increased activation and expansion of effector T cells in PBL and tumor. Upregulation of LAG-3 and TIM3 by T cells that infiltrate the tumor and have increased in number, provide a rationale for including inhibitors for both in this treatment strategy. Current efforts include evaluating whether immune responses target shared non-canonical alternative neoantigens, or Dark Matter, contained in DPV-001, and whether synchronized antibody and cellular response is evidenced. Acknowledgements: Incyte, Providence Portland Medical Foundation, Steve and Cindy Harder, Trial Registry: NCT04470024, Ethics Approval: PSJH IRB# 2020000480 Citation Format: Rom S. Leidner, Christopher Paustian, Shawn M. Jensen, Tarsem Moudgil, Yoshinobu Koguchi, Noah Simons, Venkatesh Rajamanickam, Ryan Meng, Marcus A. Couey, Quyen Vu, Matthew H. Taylor, Anne Long, Abigail J. Staeck, Lakhvir Kaur, Tijana Jovanovic, Brady Bernard, Tanisha Christie, Noriko Iwamoto, Yuriko Minegishi, Koji Ueda, William Redmond, Lessli M. Rushforth, Takashi Shimada, Patrick Rethwisch, Ashish Patel, Richard B. Bell, Carlo B. Bifulco, Traci L. Hilton, Hong-Ming Hu, Brian Piening, Walter J. Urba, Bernard A. Fox. An off-the-shelf multivalent vaccine containing cancer’s dark matter, DPV-001, combined with PD-1 +/- GITR in head & neck cancer: safety, efficacy, and immunodynamics from the phase 1 GITRVax trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT112.

Background Our group developed a strategy for generating an 'off-the-shelf' multivalent proteasome-blocked autophagosome vaccine that contains proteins for many genes commonly overexpressed in adenocarcinoma and squamous cell cancers. This strategy exploits in vitro manipulation of the antigen presentation pathway to concentrate the dominant epitopes presented by MHC, including short-lived proteins (SLiPs), defective ribosomal products (DRiPs), and Dark Matter, the short-lived non-canonical peptides that are not expressed in the thymus and represent potential shared alternative cancer neoantigens.1 In preclinical models this vaccine strategy provides significant protection as a single agent,2 and significantly increased therapeutic efficacy when combined with anti-GITR and anti-PD-1.3 Based on these studies we hypothesize that addition of anti-GITR to the human vaccine, DPV-001, and anti-PD-1, will augment expansion and limit contraction of the anti-cancer immune response. A phase I clinical trial was initiated, and preliminary immunological monitoring data will be presented. Methods Patients received DPV-001, with sequenced checkpoint inhibition (aPD-1 mAb; retifanlimab), with or without aGITR agonist mAb (INCAGN1876), in recurrent or metastatic HNSCC (NCT04470024). Tumor biopsies were taken pre-treatment, week 2 and 8. Blood samples were taken pre-treatment and at multiple timepoints and analyzed by flow cytometry and seromics. Tumor biopsies and blood were assayed by CITE-seq, scRNA-seq, BCR-seq, and TCR-seq. Multiplex immunofluorescence (mIF) was performed on biopsies. Results In the first 4 patients evaluated, tumor-infiltrating T cells at week 8 increased from pre-treatment levels by an average of 4.3 fold (range 2.9 – 6.7, p=0.032). The density of CD39/CD103 double positive cells, previously shown to identify tumor-reactive T cells,4 also increased in all week 8 biopsies (mean 14.7 fold, range 5–40). All patients showed increased numbers of cells expressing IFN-γ and GZMB and increased numbers of T cells expressing LAG3+ in week 8 biopsies. Preliminary TCR evaluation of the tumor identified proliferation of clones previously undetected in PBL, including αβ T cells, iNKT and MAIT cells. Expansion of clones that predated treatment was also identified. Conclusions An increase in intra-tumoral T cells expressing activation and effector molecules is encouraging and studies are underway to expand the number of patients analyzed. Increased expression of LAG3 by T cells that infiltrate and have expanded in the tumor, provide a rationale for including anti-LAG-3 in this treatment strategy. Future plans include evaluating whether immune responses target shared non-canonical alternative neoantigens, or Dark Matter, contained in DPV-001 and whether antibody responses identify targets of cellular immunity. Acknowledgements Support: Incyte, UbiVac, Providence Medical Foundation, The Chiles Foundation, The Harder Family, Nancy Lematta, Robert W. Franz and Elsie Franz Finley, The Murdoch Memorial Trust Trial Registration NCT04470024 References Fox BA, Urba WJ, Jensen SM, Page DB, Curti BD, Sanborn RE, Leidner RS. Cancer's Dark Matter: Lighting the Abyss Unveils Universe of New Therapies. Clinical Cancer Research 2023;29:2173–2175. 10.1158/1078–0432.CCR-23–0422. Twitty CG, Jensen SM, Hu H-M, Fox BA. Tumor-Derived Autophagosome Vaccine: Induction of Cross-Protective Immune Responses against Short-lived Proteins through a p62-Dependent Mechanism. Clinical Cancer Research 2011;17:6467–6481. 10.1158/1078–0432.CCR-11–0812. Patel JM, Cui Z, Wen Z-F, Dinh CT, Hu H-M. Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism. J Immunother Cancer 2019;7:311. 10.1186/s40425–019-0786–7. Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724. 10.1038/s41467–018-05072–0. Ethics Approval PSJH IRB# 2020000480

Background By integrating genomics, transcriptomics, proteomics, and metabolomics, researchers have gained unprecedented insights into the complex interplay between tumors and the immune system. This wealth of data has fueled the discovery of novel biomarkers for early cancer detection, prognostic stratification, and prediction of treatment response. Ultrahigh-plex multispectral immunohistochemistry enables the ability to analyze a large set of biomarkers simultaneously while maintaining the context of tissue architecture. By obtaining spatial information, researchers can understand the contact interactions and proximity between different cell types, leading to a deeper understanding of cellular dynamics and disease progression. This approach enables the identification of complex cellular patterns, such as immune cell infiltration, cell-cell interactions, or tumor heterogeneity. Ultimately, these findings may contribute to the development of more effective therapies and diagnostics for cancer patients. Methods We performed whole-slide spatial phenotyping with an ultrahigh-plex PhenoCycler-Fusion panel on FFPE human head and neck tumor biopsies (pre-treatment and post-treatment) from a patient receiving immunotherapy. Enumeration of cell phenotypes and spatial location analysis was performed using QuPath,1 R, and proprietary software. Results Within the invasive margin of the tumor (+/- 20mm of the tumor edge) we measured baseline densities of CD8+ T cells (264 cells/mm2) and CD4+ T cells (458 cells/mm2) at the pretreatment timepoint, with the densities substantially increased following treatment (1042 cells/mm2 CD8; 652 cells/mm2 CD4). The CD8+ T cells present within the invasive margin also displayed functionality as evidenced by their expression of GranzymeB (109 vs 397 cells/mm2, pre vs post). In addition, differential enrichment was observed within the invasive margin compared to the entire biopsy (41% vs 58%, pre vs post). In conjunction tumor cells at the post-treatment timepoint showed higher expression of the pro-apoptotic BAX protein (34% vs 58%, pre vs post) with reduced expression of the anti-apoptotic Bcl-2 protein (32% vs 12%, pre vs post). Conclusions Data from this ultrahigh-plex multispectral panel has provided a number of potential biomarkers for investigation in patients with head and neck cancer receiving immunotherapy. These findings inform development of smaller, more efficient multispectral panels to be designed and run on larger cohorts of patients. Additionally, examining cell populations residing within specific spatial tumor microenvironments (ie., invasive margin) uncovers additional metrics that should be examined in association with a patient's response to treatment. Acknowledgements Support: Providence Medical Foundation, The Chiles Foundation, The Harder Family, Nancy Lematta, Robert W. Franz and Elsie Franz Finley, The Murdoch Memorial Trust Reference Bankhead P, et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports 2017. Ethics Approval PSJH IRB# 06–108