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Table S1

GH-secreting PAs starburst plot gene details. The top 50 most significantly differentially expressed genes contained within Figure 5 for GH-secreting PAs as compared to endocrine-inactive PAs. The table has five columns; 1) gene symbol, 2) Entrez ID number, 3) the log fold change value from the RNA-seq differential expression analysis comparing GH-secreting PAs to endocrine-inactive PAs, 4) the change in methylation beta values between GH-secreting PAs and endocrine-inactive PAs, and 5) the genomic feature for the methylation data.

PDF file - 44K, Irradiated whole cell sarcoma-pulsed APCs do not protect mice from a challenge with non-homologous sarcomas. Kaplan-Meier survival curves of mice vaccinated with irradiated whole cell sarcoma-pulsed APCs or autophagosome-pulsed APCs. Autophagosome vaccination was performed as in Figure 2. Subcutaneous whole cell vaccinations consisted of 3x106 antigen-presenting cells pulsed with 5x106 irradiated tumor cells injected into the lower right flank of the mouse. Mice were challenged with 30,000 viable MCA-304 sarcomas. Tumor growth was monitored at least twice weekly. Each survival plot represents 2 independent experiments with 5 mice per group (n=10). Survival curves denoted with � represent statistically significant (p>0.05) protection from a tumor challenge compared to no vaccine.

Figure S1 shows combination treatment induces splenomegaly and T cells Apoptosis Figure S2 shows PD-L1 is increased on splenic T cells with combination treatment Figure S3 shows combination treatment increases costimulatory receptors on T cells Figure S4 shows delayed anti-PD-1 and anti-PD-L1 are ineffective at tumor control Figure S5 shows sequential combination provides anti-tumor immunity in the 4T1 model. Figure S6 shows sequential combination does not induce inflammatory cytokines Figure S7 shows sequential combination does not increase costimulatory receptors Figure S8 shows combination treatment induces functional PyMT-specific T cells

Table of significant SCNAs among PAs. This table highlights the top 10 most significant GISTIC2 calls for all PAs in this study, each PA subtype, and invasive versus non-invasive PAs.

ACTH-secreting PAs starburst plot gene details. The top 50 most significantly differentially expressed genes contained within Figure 5 for ACTH-secreting PAs as compared to endocrine-inactive PAs. The table has five columns; 1) gene symbol, 2) Entrez ID number, 3) the log fold change value from the RNA-seq differential expression analysis comparing ACTH-secreting PAs to endocrine-inactive PAs, 4) the change in methylation beta values between ACTH-secreting PAs and endocrine-inactive PAs, and 5) the genomic feature for the methylation data.

Supplementary Figure Legends

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Supplementary Data from Partial CD4 Depletion Reduces Regulatory T Cells Induced by Multiple Vaccinations and Restores Therapeutic Efficacy

Supplementary methods

Purpose: Antibodies specific for inhibitory checkpoints PD-1 and CTLA-4 have shown impressive results against solid tumors. This has fueled interest in novel immunotherapy combinations to affect patients who remain refractory to checkpoint blockade monotherapy. However, how to optimally combine checkpoint blockade with agents targeting T-cell costimulatory receptors, such as OX40, remains a critical question.Experimental Design: We utilized an anti-PD-1–refractory, orthotopically transplanted MMTV-PyMT mammary cancer model to investigate the antitumor effect of an agonist anti-OX40 antibody combined with anti-PD-1. As PD-1 naturally aids in immune contraction after T-cell activation, we treated mice with concurrent combination treatment versus sequentially administering anti-OX40 followed by anti-PD-1.Results: The concurrent addition of anti-PD-1 significantly attenuated the therapeutic effect of anti-OX40 alone. Combination-treated mice had considerable increases in type I and type II serum cytokines and significantly augmented expression of inhibitory receptors or exhaustion markers CTLA-4 and TIM-3 on T cells. Combination treatment increased intratumoral CD4+ T-cell proliferation at day 13, but at day 19, both CD4+ and CD8+ T-cell proliferation was significantly reduced compared with untreated mice. In two tumor models, sequential combination of anti-OX40 followed by anti-PD-1 (but not the reverse order) resulted in significant increases in therapeutic efficacy. Against MMTV-PyMT tumors, sequential combination was dependent on both CD4+ and CD8+ T cells and completely regressed tumors in approximately 30% of treated animals.Conclusions: These results highlight the importance of timing for optimized therapeutic effect with combination immunotherapies and suggest the testing of sequencing in combination immunotherapy clinical trials. Clin Cancer Res; 23(20); 6165–77. ©2017 AACR.See related commentary by Colombo, p. 5999

Purpose: Pituitary adenomas are one of the most common benign neoplasms of the central nervous system. Although emerging evidence suggests roles for both genetic and epigenetic factors in tumorigenesis, the degree to which these factors contribute to disease remains poorly understood.Experimental Design: A multiplatform analysis was performed to identify the genomic and epigenomic underpinnings of disease among the three major subtypes of surgically resected pituitary adenomas in 48 patients: growth hormone (GH)–secreting (n = 17), adrenocorticotropic hormone (ACTH)–secreting (n = 13, including 3 silent-ACTH adenomas), and endocrine-inactive (n = 18). Whole-exome sequencing was used to profile the somatic mutational landscape, whole-transcriptome sequencing was used to identify disease-specific patterns of gene expression, and array-based DNA methylation profiling was used to examine genome-wide patterns of DNA methylation.Results: Recurrent single-nucleotide and small indel somatic mutations were infrequent among the three adenoma subtypes. However, somatic copy-number alterations (SCNA) were identified in all three pituitary adenoma subtypes. Methylation analysis revealed adenoma subtype-specific DNA methylation profiles, with GH-secreting adenomas being dominated by hypomethylated sites. Likewise, gene-expression patterns revealed adenoma subtype-specific profiles. Integrating DNA methylation and gene-expression data revealed that hypomethylation of promoter regions are related with increased expression of GH1 and SSTR5 genes in GH-secreting adenomas and POMC gene in ACTH-secreting adenomas. Finally, multispectral IHC staining of immune-related proteins showed abundant expression of PD-L1 among all three adenoma subtypes.Conclusions: Taken together, these data stress the contribution of epigenomic alterations to disease-specific etiology among adenoma subtypes and highlight potential targets for future immunotherapy-based treatments. This article reveals novel insights into the epigenomics underlying pituitary adenomas and highlights how differences in epigenomic states are related to important transcriptome alterations that define adenoma subtypes. Clin Cancer Res; 24(17); 4126–36. ©2018 AACR.

PCA analysis of genome-wide methylation profiles. PCA analysis using all probes in the methylation data set. Points are colored by the percent of the genome altered by SCNAs. The three PA subtypes are indicated by the shape of the points, with GH-secreting PAs represented by dots, ACTH-secreting PAs represented by triangles, and endocrine-inactive PAs represented by squares. Ellipses indicate 95% confidence intervals. Overall, PAs cluster by PA subtype and not by the percent of the genome disrupted by SCNAs.

Purpose: A single vaccination of intact or reconstituted-lymphopenic mice (RLM) with a granulocyte macrophage colony-stimulating factorsecreting B16BL6-D5 melanoma cell line induces protective antitumor immunity and T cells that mediate the regression of established melanoma in adoptive immunotherapy studies. We wanted to study if multiple vaccinations during immune reconstitution of the lymphopenic host would maintain a potent antitumor immune response.Experimental Design: RLM were vaccinated multiple times over a 40-day period. Spleens were isolated from these mice, activated in vitro, and adoptively transferred into mice bearing 3-day experimental pulmonary metastases.Results: Multiple vaccinations, rather than boosting the immune response, significantly reduced therapeutic efficacy of adoptive immunotherapy and were associated with an increased frequency and absolute number of CD3+CD4+Foxp3+ T regulatory (Treg) cells. Anti-CD4 administration reduced the absolute number of Treg cells 9-fold. Effector T-cells generated from anti-CD4treated mice were significantly (P < 0.0001) more therapeutic in adoptive transfer studies than T cells from multiply vaccinated animals with a full complement of CD4+ cells.Conclusion: These results suggest that CD4+ Treg cells limit the efficacy of multiple vaccinations and that timed partial depletion of CD4+ T cells may reduce suppression and tip-the-balance in favor of therapeutic antitumor immunity. The recent failure of large phase III cancer vaccine clinical trials, wherein patients received multiple vaccines, underscores the potential clinical relevance of these findings. (Clin Cancer Res 2009;15(22):688190)

Purpose:Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed.Patients and Methods:Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets.Results:Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti–PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti–PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response.Conclusions:These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

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PCA analysis of genome-wide gene expression profiles. PCA analysis using all genes in the gene expression data set. Points are colored by the percent of the genome altered by SCNAs. The three PA subtypes are indicated by the shape of the points, with GH-secreting PAs represented by dots, ACTH-secreting PAs represented by triangles, and endocrine-inactive PAs represented by squares. Ellipses indicate 95% confidence intervals. Overall, PAs cluster by PA subtype and not by the percent of the genome disrupted by SCNAs.

Table of clinical parameters for each patient sample.

Supplementary Figure 1. IL-15 availability promotes proliferation and maintenance of transferred CD8+ T in the spleen of recipient mice; Supplementary Figure 2. Gating strategy for the identification of adoptively transferred Pmel-1 cells and endogenous CD8+ T cells infiltrating the tumor; Supplementary Figure 3. Absolute counts of splenic Pmel-1 and CD8+ T cells are profoundly affected by hetIL-15 treatment; Supplementary Figure 4. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating Pmel-1 cells; Supplementary Figure 5. Evaluation of PD-1, Ki67 and GzmB expression by tumorinfiltrating CD8+ T cells; Supplementary Table 1. Hematological parameters during ACT treatments.

Supplementary Data from Phenotype and Functional Characterization of Long-term gp100-Specific Memory CD8+ T Cells in Disease-Free Melanoma Patients Before and After Boosting Immunization

CCR Translation for the Article from Partial CD4 Depletion Reduces Regulatory T Cells Induced by Multiple Vaccinations and Restores Therapeutic Efficacy

PDF file - 198K, Figure 1. Characterization of the anti-OX40 mAb (9B12). Figure 2. Total peripheral lymphocyte counts. Figure 3. Pharmacokinetics of CD134 (anti-OX40) mAb in patients. Figure 4. Direct ex vivo detection of the murine anti-OX40 mAb bound to T cells in treated patients. Figure 5. Regression of a pulmonary metastasis in a patient with renal carcinoma enrolled in cohort 1. Figure 6. Changes in Ki-67 expression within CD4+ and CD8+ T cell subsets examined over time after anti-OX40 from patients in cohorts 1 and 2. Figure 7. Increased proliferation of CD4+ Foxp3- T cells and CD8+ T cells correlates with a decrease or stabilization of tumor burden. Figure 8. Ki-67 expression by monkey CD4+ and CD8+ memory T cells after the administration of anti-OX40 , mouse Immunoglobulin or monkey OX40L:Ig. Figure 9. Determination of the Endpoint Titer for anti-KLH Ab.

PDF file - 68K, Details clinical trial, flow cytometry and monkey experiments referred to in the manuscript.

Accurate quantitation of antibody is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

SummaryDespite pre-clinical murine data supporting T regulatory (Treg) cell depletion as a major mechanism by which anti-CTLA-4 antibodies function in vivo, the two main antibodies tested in patients (ipilimumab and tremelimumab) have failed to demonstrate similar effects. We report analogous findings in an immunocompetent murine model humanized for CTLA-4 and Fcy receptors (hCTLA-4/hFcyR mice), where both ipilimumab and tremelimumab fail to show appreciable Treg depletion. Immune profiling of the tumor microenvironment (TME) in both mice and human samples revealed upregulation of the inhibitory Fcy receptor, FcyRIIB, which limits the ability of the antibody Fc fragment of human anti-CTLA-4 antibodies to induce effective antibody dependent cellular cytotoxicty/phagocytosis (ADCC/ADCP). Blocking FcyRIIB in humanized mice rescues Treg depleting capacity and anti-tumor activity of ipilimumab. For another target, CC motif chemokine receptor 8 (CCR8), which is selectively expressed on tumor infiltrating Tregs, we show that Fc engineering to enhance binding to activating Fc receptors, while limiting binding to the inhibitory Fc receptor, leads to consistent Treg depletion and single-agent activity across multiple tumor models, including B16, MC38 and MB49. These data reveal the importance of reducing engagement to the inhibitory Fc receptor to optimize Treg depletion by TME targeting antibodies. Our results define the inhibitory FcyRIIB receptor as a novel immune checkpoint limiting antibody-mediated Treg depletion in tumors, and demonstrate Fc variant engineering as a means to overcome this limitation and augment efficacy for a repertoire of antibodies currently in use or under clinical evaluation in oncology.Highlights-Fully human anti-CTLA-4 antibodies are limited in their capacity to deplete T regulatory cells and drive durable anti-tumor immunity in humanized FcyR/hCTLA-4 mice-The inhibitory Fcy receptor, FcyRIIB, is upregulated in the tumor microenvironment in patients and in humanized FcyR/hCTLA-4 mice-Blocking FcyRIIB leads to rescue of Treg depletion in humanized murine models-Fc engineering can improve the depleting capacity and in vivo anti-tumor activity of anti-CTLA and anti-CCR8 antibodies targeting tumor infiltrating Tregs

Adoptive cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) or TCR gene-modified T cells (TCR-T) that recognize mutant KRAS neo-antigens can mediate tumor regression in patients with advanced pancreatic ductal adenocarcinoma (PDAC) (Tran et al in N Engl J Med, 375:2255-2262, 2016; Leidner et al in N Engl J Med, 386:2112-2119, 2022). The mutant KRAS-targeted ACT holds great potential to achieve durable clinical responses for PDAC, which has had no meaningful improvement over 40 years. However, the wide application of mutant KRAS-centric ACT is currently limited by the rarity of TIL that recognize the mutant KRAS. In addition, PDAC is generally recognized as a poorly immunogenic tumor, and TILs in PDAC are less abundant than in immunogenic tumors such as melanoma. To increase the success rate of TIL production, we adopted a well-utilized K562-based artificial APC (aAPC) that expresses 4-1BBL as the costimulatory molecules to enhance the TIL production from PDCA. However, stimulation with K562-based aAPC led to a rapid loss of specificity to mutant KRAS. To selectively expand neo-antigen-specific T cells, particularly mKRAS, from the TILs, we used tandem mini gene-modified autologous T cells (TMG-T) as the novel aAPC. Using this modified IVS protocol, we successfully generated TIL cultures specifically reactive to mKRAS (G12V). We believe that autologous TMG-T cells provide a reliable source of autologous APC to expand a rare population of neoantigen-specific T cells in TILs.

Background CAPRA (NCT02565992) evaluated Coxsackievirus A21 (V937) + pembrolizumab for metastatic/unresectable stage IIIB–IV melanoma. Methods Patients received intratumoral V937 on days 1, 3, 5, and 8 (then every 3 weeks [Q3W]) and intravenous pembrolizumab 2 mg/kg Q3W from day 8. Primary endpoint was safety. Results Median time from first dose to data cutoff was 32.0 months. No dose-limiting toxicities occurred; 14% (5/36) of patients experienced grade 3‒5 treatment-related adverse events. Objective response rate was 47% (complete response, 22%). Among 17 responders, 14 (82%) had responses ≥ 6 months. Among 8 patients previously treated with immunotherapy, 3 responded (1 complete, 2 partial). Responses were associated with increased serum CXCL10 and CCL22, suggesting viral replication contributes to antitumor immunity. For responders versus nonresponders, there was no difference in baseline tumor PD-L1 expression, ICAM1 expression, or CD3+ infiltrates. Surprisingly, the baseline cell density of CD3+CD8− T cells in the tumor microenvironment was significantly lower in responders compared with nonresponders (P = 0.0179). Conclusions These findings suggest responses to this combination may be seen even in patients without a typical “immune-active” microenvironment. Trial registration number NCT02565992.

Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells., Graphical Abstract, Nishimura and colleagues demonstrate that transgene expression in gene-modified anti-tumor T cells is reversibly downregulated over time in vivo and in vitro. Treatment with IL-15 or with HDAC inhibitors (sodium butyrate or vorinostat) reduced this downregulation and maintained T cell functional responses to tumor cells.

Background: Glucocorticoid-Induced Tumor Necrosis Factor Receptor-related protein (GITR) is a co-stimulatory pathway that when triggered has potent effects on T-cell memory, proliferation and anti-tumor activity. Preclinical models identified significant synergy between anti-GITR agonist therapy and cancer vaccines to generate stronger tumor specific CD8 T cell responses. DPV-001 is an “off-the-shelf” multivalent autophagosome vaccine generated by in vitro manipulation of the autophagy pathway in human cancer cell lines. The vaccine delivers short-lived proteins (SLiPs) and defective ribosomal products (DRiPs) which are likely the dominant epitopes directly presented by MHC class I of tumor cells; but because of proteosomal degradation, are normally unavailable for cross-presentation, hence the delivery via vaccine. We hypothesize that addition of aGITR to DPV-001 vaccine will augment expansion of reactive CD4 and CD8 T cells, attenuate contraction of this response, and improve the therapeutic effect of treatment, and will result in the development of a coordinated T and B cell response to some of the same proteins, detectable using a cutting-edge seromics approach, as a window to TCR target identification for immunodynamic tracking of induced anti-cancer responses at an advanced level. Methods: Patient recruitment began in August 2022, for this first-in-human immunotherapy-trio study of DPV-001, with sequenced checkpoint inhibition (aPD-1 mAb; retifanlimab), with or without aGITR agonist mAb (INCAGN-1949), in recurrent or metastatic HNSCC (NCT04470024). Patient population to include HPV-positive or HPV-negative, ECOG 0-2, with therapy continued until confirmed progression (RECIST 1.1), up to 2 years. Primary objective is safety, DLT ≤ 33%, with secondary efficacy objectives of ORR (PR+CR) and 2 year OS. Initial safety lead-in (n = 3+3 per arm), will be followed by phase Ib expansion of one/both arms if immunologically promising, 28 patients per arm, futility if Study Drugs Cyclophosphamide 300mg/m2 IV, priming Day (-2) onlyVaccine (DPV-001)- Day 1 intranodal US bilateral inguinal- Days 8,15 intradermal, then q2wks to week 22- Thereafter q4wks until progression, up to 2 yearsaPD-1 (retifanlimab) 500mg IV q4wks, start Day 15 (Arms 1 & 2)aGITR (INCAGN01876) 300mg IV q2wks, start Day 1 (Arm 2 only) Response (RECIST 1.1) CT weeks 8 and 12, then q3mos Immunologic Monitoring PBL and sera are collected regularly and PBL are evaluated by flow cytometry. Biopsies obtained at baseline, Day 15 and Day 57, analyzed by mIF and 10x scRNA-Seq. Sera analyzed by phage immunoprecipitation (PhIP) sequencing for reactivity against the human proteome. Immune monitoring modifications that allow for improved characterization of immune cell subsets will be presented. Citation Format: Marcus A. Couey, Matthew Taylor, Tarsem Moudgil, Yoshinobu Koguchi, Anne Stadum, Tanisha Christie, William L. Redmond, Christopher Paustian, Ryan Meng, Venkatesh Rajamanickam, Lessli Rushforth, Abigail Peterson, Brady Bernard, Richard B. Bell, Carlo B. Bifulco, Traci L. Hilton, Shawn M. Jensen, Hong-Ming Hu, Brian Piening, Walter J. Urba, Bernard A. Fox, Rom S. Leidner. Trial in progress: First-in-human immunotherapy-trio for advanced head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT123.

Single cell RNA-seq (scRNA-seq) performed on cell suspensions isolated from tumor biopsies provides substantial insights into the transcriptional state of the cells in the tumor microenvironment (TME). Complementing this work with multi-omic Cellular Indexing of Transcriptomes and Epitopes (CITE-seq), which adds barcoded antibodies to label the surface of isolated cells, allows us to additionally characterize surface proteins. While information gleaned from these studies is dramatically expanding our understanding of the evolving immune response and tumor-immune interactions, it lacks an appreciation of the many spatial relationships that are present within the TME. Spatial phenotyping with the power of single cell resolution and whole slide imaging provides a valuable tool for identifying the cellular architecture and organization of spatial neighborhoods that perform critical roles in tumor progression, resistance, and clinical responses. Here, we explore bioinformatic strategies aimed at combining scRNA-seq and CITE-seq data with single-cell spatial protein data obtained with the PhenoCycler-Fusion (PCF) imaging platform. To this end, we performed whole-slide spatial phenotyping with an ultrahigh-plex PCF panel on FFPE human head and neck tumors. The same biopsies were also enzymatically digested and subjected to CITE-seq and scRNA-seq analysis. The combined multi-omic, single cell dataset provides a uniquely comprehensive account of the cellular states, functional significance and spatial neighborhoods that govern the TME. This study represents a novel approach to integrate high dimensional data from bulk tissue and single-cell spatial technologies to provide deeper insights into tumor biology and, in turn, to develop more informed strategies for combination immunotherapy trials and improved patient outcomes. Citation Format: Aditya Pratapa, Shawn M. Jensen, Niyati Jhaveri, Yoshinobu Koguchi, Venkatesh Rajamanickam, Brady Bernard, Tanisha Christie, Brian Piening, Rom S. Leidner, Oliver Braubach, Bernard A. Fox. Multi-parametric comparison of scRNA-seq with CITE-seq and ultrahigh-plex spatial phenotyping of proteins in FFPE head and neck tumor biopsies: An opportunity to generate uniquely comprehensive multi-omic single cell datasets to investigate the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6764.

The melanoma treatment landscape changed in 2011 with the approval of the first anti-cytotoxic T-lymphocyte-associated protein (CTLA)-4 checkpoint inhibitor and of the first BRAF-targeted monoclonal antibody, both of which significantly improved overall survival (OS). Since then, improved understanding of the tumor microenvironment (TME) and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. The approval of new immune and targeted therapies has further improved outcomes for patients with advanced melanoma and other combination modalities are also being explored such as chemotherapy, radiotherapy, electrochemotherapy and surgery. In addition, different strategies of drugs administration including sequential or combination treatment are being tested. Approaches to overcome resistance and to potentiate the immune response are being developed. Increasing evidence emerges that tissue and blood-based biomarkers can predict the response to a therapy. The latest findings in melanoma research, including insights into the tumor microenvironment and new biomarkers, improved understanding of tumor immune response and resistance, novel approaches for combination strategies and the role of neoadjuvant and adjuvant therapy, were the focus of discussions at the Melanoma Bridge meeting (5–7 December, 2019, Naples, Italy), which are summarized in this report.

Background The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has infected over 127 million people worldwide, with almost 2.8 million deaths at the time of writing. Since no lactating individuals were included in initial trials of vaccine safety and efficacy, research on SARS-CoV-2 vaccination in lactating women and the potential transmission of passive immunity to the infant through mother’s milk is needed to guide patients, clinicians, and policy makers on whether to recommend immunization during the worldwide effort to curb the spread of this virus. Research Aims (1) To determine whether SARS-CoV-2 specific immunoglobins are found in human milk after vaccination, and (2) to characterize the time course and types of immunoglobulins present. Methods A longitudinal cohort study of lactating women ( N = 7) who planned to receive both doses of the Pfizer-BioNTech or Moderna SARS-CoV-2 vaccine between December 2020 and January 2021 provided milk samples. These were collected pre-vaccination and at 11 additional timepoints, with the last sample at 14 days after the second dose of vaccine. Samples were analyzed for levels of SARS-CoV-2 specific immunoglobulins A and G (IgA and IgG). Results We observed significantly elevated levels of SARS-CoV-2 specific IgG and IgA antibodies in human milk beginning approximately 7 days after the initial vaccine dose, with an IgG-dominant response. Conclusions Maternal vaccination results in SARS-CoV-2 specific immunoglobulins in human milk that may be protective for infants.

Advances in immune checkpoint therapy and targeted therapy have led to improvement in overall survival for patients with advanced melanoma. Single agent checkpoint PD-1 blockade and combination with BRAF/MEK targeted therapy demonstrated benefit in overall survival (OS). Superior response rates have been demonstrated with combined PD-1/CTLA-4 blockade, with a significant OS benefit compared with single-agent PD-1 blockade. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic therapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers but they have yet to be fully characterized and implemented clinically. Overall, the progress in melanoma therapeutics and translational research will help to optimize treatment regimens to overcome resistance and develop robust biomarkers to guide clinical decision-making. During the Melanoma Bridge meeting (December 3rd–5th, 2020, Italy) we reviewed the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine.

Immunotherapy represents the third important wave in the history of the systemic treatment of cancer after chemotherapy and targeted therapy and is now established as a potent and effective treatment option across several cancer types. The clinical success of anti-cytotoxic T-lymphocyte-associated antigen (CTLA)-4, first, and anti-programmed death (PD)-1/PD-ligand (L)1 agents in melanoma and other cancers a few years later, has encouraged increasing focus on the development of other immunotherapies (e.g. monoclonal antibodies with other immune targets, adoptive cell transfer, and vaccines), with over 3000 immuno-oncology trials ongoing, involving hundreds of research institutes across the globe. The potential use of these different immunotherapeutic options in various combinations with one another and with other treatment modalities is an area of particular promise. The third Immunotherapy Bridge meeting (29-30 November, 2017, Naples, Italy) focused on recent advances in immunotherapy across various cancer types and is summarised in this report.

In our opinion the most urgent needs to improve patient outcomes are: 1) a deeper ability to measure cancer immunobiology, and 2) increased availability of agents that, coupled with predictive biomarkers, will be used to tailor anti-cancer immunity. Tailoring effective immunotherapy will entail combinations of immunotherapeutics that augment priming of anti-cancer immunity, boost expansion of effector and memory cells of the T, B and NK lineage, amplify innate immunity and relieve checkpoint inhibition. Alternatives to inducing adaptive immunity to cancer include synthetic immunology that incorporate bi-specifics that target T cells to cancer or adoptive immunotherapy with gene-modified immune cells.

Background: Preclinical studies document that complex cancer vaccines, combined with T cell agonists and anti-PD-1, can augment therapeutic efficacy. Here we report preliminary immunological analyses of patients enrolled in a first-in-human immunotherapy-trio study of multivalent autophagosome vaccine (DPV-001), with sequenced checkpoint inhibition (anti-PD-1; retifanlimab), with/without anti-GITR agonist (INCAGN-1949), in recurrent or metastatic HNSCC (NCT04470024). Methods: Peripheral blood (PB) and sera are collected regularly and PB are evaluated by flow cytometry. Biopsies obtained at baseline, D15 and D45 are analyzed by multiplex IF and 10x Genomics scRNA-Seq. Sera are being analyzed by phage immunoprecipitation (PhIP) sequencing for reactivity against the human proteome. Results: Preliminary results document increases in activated CD4 and CD8 effector memory T cells (TEM) with vaccine alone. Changes in tumor microenvironment (TEM) were also observed with increased infiltration of immune cells. Evaluation of changes to humoral immunity, T cell function and TCR analyses are ongoing. Conclusions: We previously reported immunological monitoring of a phase I/II trial of an autophagosome cancer vaccine (DPV-001) containing more than 300 shared cancer antigens, as adjuvant therapy for NSCLC. Vaccination induced or augmented immune responses to more than 50 cancer antigens shared with head and neck squamous cell carcinoma (HNSCC). Preclinical studies combining this cancer vaccine with αGITR agonist and αPD-1 augmented therapeutic efficacy [PMID: 31747946], and provided the rationale for the current study. This is the first clinical trial to perform such a study with αGITR agonist (INCAGN-1949), in humans. Support: Incyte, Providence Medical Foundation, The Harder Family, Nancy Lematta. Citation Format: Rom S. Leidner, Matthew H. Taylor, Tarsem L. Moudgil, Tanisha L. Christie, Yoshinobu Koguchi, Alexa Dowdell, William L. Redmond, Shawn M. Jensen, Carmen Ballesteros-Merino, Jake A. Vancampen, Venkatesh Rajamanickam, Brady M. Bernard, Christopher Paustian, Traci L. Hilton, Hong-Ming Hu, Richard B. Bell, Walter J. Urba, Carlo B. Bifulco, Brian Piening, Bernard A. Fox. Preliminary immunological monitoring of first-in-human immunotherapy-trio for advanced head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT502.

A collaborative think tank involving panellists from immuno-oncology networks, clinical/translational investigators and the pharmaceutical industry was held in Siena, Italy, in October 2017 to discuss the evolving immune-oncology landscape, identify selected key challenges, and provide a perspective on the next steps required in the translation of current research and knowledge to clinical reality. While there is a trend of combining new agents (e.g., co-stimulator agonists) with a PD-1/PD-L1 treatment backbone, use of alternative combination therapy approaches should also be considered. While the rapid evolution in systems biology provides a deeper understanding of tumor and tumor microenvironment heterogeneity, there remains the need to identify and define genuinely predictive biomarkers to guide treatment and patient selection. Cross-specialty and cross-sector collaboration, along with a broader collective data-sharing approach are key to optimizing immuno-oncology therapy in clinical practice. Continued support of younger research-clinicians is essential for future success in clinical, translational and basic science investigations.

Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.

ImportanceThe SARS-CoV-2 pandemic has infected over a hundred million people worldwide, with almost 2.5 million deaths at the date of this publication. In the United States, Pfizer-BioNTech and Moderna vaccines were first administered to the public starting in December 2020, and no lactating women were included in the initial trials of safety/efficacy. Research on SARS-CoV-2 vaccination in lactating women and the potential transmission of passive immunity to the infant through breast milk is needed to guide patients, clinicians and policy makers during the worldwide effort to curb the spread of this virus.ObjectiveTo determine whether SARS-CoV-2 specific immunoglobins are found in breast milk post-vaccination, and to characterize the time course and types of immunoglobulins present.DesignProspective cohort studySettingProvidence Portland Medical Center, Oregon, USAParticipantsSix lactating women who planned to receive both doses of the Pfizer-BioNTech or Moderna vaccine between December 2020 and January 2021. Breast milk samples were collected pre-vaccination and at 11 additional timepoints, with last sample at 14 days post 2nd dose of vaccine.ExposureTwo doses of Pfizer-BioNTech or Moderna SARS-CoV-2 vaccine.Main Outcome(s) and Measure(s)Levels of SARS-CoV-2 specific IgA and IgG immunoglobulins in breast milk.ResultsIn this cohort of 6 lactating women who received 2 doses of SARS-CoV-2 vaccine, we observed significantly elevated levels of SARS-CoV-2 specific IgG and IgA antibodies in breast milk beginning at Day 7 after the initial vaccine dose, with an IgG-dominant response.Conclusions and RelevanceWe are the first to show that maternal vaccination results in SARS-CoV-2 specific immunoglobulins in breast milk that may be protective for infants.

Despite the success of checkpoint blockade in some cancer patients, there is an unmet need to improve outcomes. Targeting alternative pathways, such as costimulatory molecules (e.g. OX40, GITR, and 4-1BB), can enhance T cell immunity in tumor-bearing hosts. Here we describe the results from a phase Ib clinical trial (NCT02274155) in which 17 patients with locally advanced head and neck squamous cell carcinoma (HNSCC) received a murine anti-human OX40 agonist antibody (MEDI6469) prior to definitive surgical resection. The primary endpoint was to determine safety and feasibility of the anti-OX40 neoadjuvant treatment. The secondary objective was to assess the effect of anti-OX40 on lymphocyte subsets in the tumor and blood. Neoadjuvant anti-OX40 was well tolerated and did not delay surgery, thus meeting the primary endpoint. Peripheral blood phenotyping data show increases in CD4+ and CD8+ T cell proliferation two weeks after anti-OX40 administration. Comparison of tumor biopsies before and after treatment reveals an increase of activated, conventional CD4+ tumor-infiltrating lymphocytes (TIL) in most patients and higher clonality by TCRβ sequencing. Analyses of CD8+ TIL show increases in tumor-antigen reactive, proliferating CD103+ CD39+ cells in 25% of patients with evaluable tumor tissue (N = 4/16), all of whom remain disease-free. These data provide evidence that anti-OX40 prior to surgery is safe and can increase activation and proliferation of CD4+ and CD8+ T cells in blood and tumor. Our work suggests that increases in the tumor-reactive CD103+ CD39+ CD8+ TIL could serve as a potential biomarker of anti-OX40 clinical activity., Different neoadjuvant therapies have been proposed to improve immunotherapy for cancer treatment. Here, the authors perform a phase Ib clinical trial where an agonist OX40 antibody provided prior to surgery is well tolerated and increases proliferation and activation of tumor antigen-specific T cells in head and neck cancer patients.

Purpose: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed. Patients and Methods: Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets. Results: Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti–PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti–PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response. Conclusions: These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

Five years ago, the Melanoma Research Foundation (MRF) conducted an assessment of the challenges and opportunities facing the melanoma research community and patients with melanoma. Since then, remarkable progress has been made on both the basic and clinical research fronts. However, the incidence, recurrence, and death rates for melanoma remain unacceptably high and significant challenges remain. Hence, the MRF Scientific Advisory Council and Breakthrough Consortium, a group that includes clinicians and scientists, reconvened to facilitate intensive discussions on thematic areas essential to melanoma researchers and patients alike, prevention, detection, diagnosis, metastatic dormancy and progression, response and resistance to targeted and immune-based therapy, and the clinical consequences of COVID-19 for patients with melanoma and providers. These extensive discussions helped to crystalize our understanding of the challenges and opportunities facing the broader melanoma community today. In this report, we discuss the progress made since the last MRF assessment, comment on what remains to be overcome, and offer recommendations for the best path forward.

Simple Summary Oral squamous cell carcinoma has developed different strategies to escape from T-cell-mediated immune surveillance, which is mediated by changes in the composition of cellular and soluble components of the tumor microenvironment as well as an impaired expression of molecules of the antigen processing machinery leading to a downregulation of HLA class I surface antigens. In depth characterization of these escape mechanisms might help to develop strategies to overcome this tolerance. In this study, human papilloma virus negative oral squamous cell carcinoma lesions were analyzed regarding the protein expression of major components of the HLA class I antigen processing/presentation pathway in correlation to the intra-tumoral immune cell composition, IFN-γ signaling and clinical parameters, which was further confirmed by bioinformatics analyses of datasets obtained from The Cancer Genome Atlas. This novel knowledge could be used for optimizing the design of immunotherapeutic approaches of this disease. Abstract Progression of oral squamous cell carcinoma (OSCC) has been associated with an escape of tumor cells from the host immune surveillance due to an increased knowledge of its underlying molecular mechanisms and its modulation by the tumor microenvironment and immune cell repertoire. In this study, the expression of HLA class I (HLA-I) antigens and of components of the antigen processing machinery (APM) was analyzed in 160 pathologically classified human papilloma virus (HPV)-negative OSCC lesions and correlated to the intra-tumoral immune cell response, IFN-γ signaling and to the patient’s outcome. A heterogeneous but predominantly lower constitutive protein expression of HLA-I APM components was found in OSCC sections when compared to non-neoplastic cells. Tumoral HLA-I APM component expression was further categorized into the three major phenotypes HLA-Ihigh/APMhigh, HLA-Ilow/APMlow and HLA-Idiscordant high/low/APMhigh. In the HLA-Ihigh/APMhigh group, the highest frequency of intra-tumoral CD8+ T cells and lowest number of CD8+ T cells close to FoxP3+ cells were found. Patients within this group presented the most unfavorable survival, which was significantly evident in stage T2 tumors. Despite a correlation with the number of intra-tumoral CD8+ T cells, tumoral JAK1 expression as a surrogate marker for IFN-γ signaling was not associated with HLA-I/APM expression. Thus, the presented findings strongly indicate the presence of additional factors involved in the immunomodulatory process of HPV-negative OSCC with a possible tumor-burden-dependent complex network of immune escape mechanisms beyond HLA-I/APM components and T cell infiltration in this tumor entity.

The Italian Network for Tumor Biotherapy (Network Italiano per la Bioterapia dei Tumori [NIBIT]) Foundation hosted its annual 2020 Think Tank meeting virtually, at which representatives from academic, clinical, industry, philanthropic, and regulatory organisations discussed the role of neoadjuvant immunotherapy for the treatment of cancer. Although the number of neoadjuvant immunotherapeutic trials is increasing across all malignancies, the Think Tank focused its discussion on the status of neoadjuvant trials in cutaneous melanoma (CM), muscle-invasive urothelial bladder cancer (MIBC), head and neck squamous cell carcinoma (HNSCC), and pancreatic adenocarcinoma (PDAC). Neoadjuvant developments in CM are nothing short of trailblazing. Pathologic Complete Response (pCR), pathologic near Complete Response, and partial Pathologic Responses reduce 90–100% of recurrences. This is in sharp contrast to targeted therapies in neoadjuvant CM trials, where only a pCR seems to reduce recurrence. The pCR rate after neoadjuvant immunotherapy varies among the different malignancies of CM, MIBC, HNSCC, and PDAC and may be associated with different reductions of recurrence rates. In CM, emerging evidence suggests that neoadjuvant immunotherapy with anti-CTLA-4 plus anti-PD1 is a game changer in patients with palpable nodal Stage III or resectable Stage IV disease by curing more patients, reducing recurrences and the need for surgical interventions, such as lymph node dissections and metastasectomies. The Think Tank panel discussed future approaches on how to optimise results across different tumour types. Future approaches should include reducing monocyte-mediated (tumour-associated macrophages) and fibroblast-mediated (cancer-associated fibroblasts) barriers in the tumour microenvironment to facilitate the recruitment of immune cells to the tumour site, to reduce immune-suppressive mediators, and to increase antigen presentation at the site of the tumour.

Microsatellite-stable (MSS) colorectal cancers are characterized by low mutation burden and limited immune-cell infiltration and thereby respond poorly to immunotherapy. Here, we report a case of metastatic MSS colorectal cancer with a robust anticancer immune response. The primary tumor was resected in 2012, and the patient received several cycles of chemotherapy until 2017. In 2018, the patient underwent a left hepatectomy to remove a new metastasis. Analysis of the metastatic tumor revealed a strong CD8+ T-cell response. A high frequency of CD8+ T cells coexpressed CD39 and CD103, a phenotype characteristic of tumor-reactive cells. Using whole-exome sequencing, we identified somatic mutations that generated peptides recognized by CD39+CD103+CD8+ T cells. The observed reactivity against the tumor was dominated by the response to a single mutation that emerged in the metastasis. Somatic mutations that were not immunogenic in the primary tumor led to robust CD8+ T-cell expansion later during disease progression. Our data suggest that the cytotoxic treatment regimen received by the patient might be responsible for this effect. Hence, the capacity of cytotoxic regimens to prime the immune system in colorectal cancer patients should be investigated further and might provide a rationale for combination with immunotherapy.

The global SARS-CoV-2 (CoV2) pandemic has focused efforts to generate effective vaccines that induce potent and persistent immunity. Recently, we described a novel vaccine approach using electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax) plus plasmid Interleukin-12 (IL-12). IL-12 is an effective vaccine adjuvant that increases Th1 and Th2 antibodies in the serum. Here we examine the persistence of anti-Spike antibodies present in the serum of mice one year following CORVax vaccination. C57BL/6 and BALB/c were vaccinated intramuscular (IM) and/or intradermal (ID) with a plasmid encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12. Mice received plasmid EP immediately following injection. Splenocytes and serum were harvested at various time points to interrogate virus-specific cellular responses as well anti-spike antibody titers. Anti-Spike IgG antibodies were elicited by EP of CORVax (IC50 = 1/2112), as well as EP of CORVax combined with IL-12 (IC50 = 1/4214) approximately 40 days after the booster vaccination. These anti-Spike IgG titers decayed over time but were still present 1 year after vaccination: CORVax (IC50 = 1/351 day 146, IC50 = 1/208 day 383); CORVax + IL12 (IC50 = 1/590 day 146, IC50 = 1/266 day 383). Our data shows that EP of CORVax induces IgG responses to CoV2 Spike and the CoV2 Spike receptor binding domain. At one year following vaccination the anti-Spike IgG titers were higher in mice that received CORVax plus IL-12, however the rate at which the titers waned from their initial peak was comparable whether the mice received IL-12 or not. Additional studies are ongoing to determine whether the addition of IL-12 will enhance an anti-Spike memory response. Generous support from The Chiles Foundation, Nancy Lematta, and the Providence Medical Foundation.

To prevent SARS-CoV-2 infections and generate long-lasting immunity, vaccines need to generate both, a strong humoral immune response as well as viral-specific CD4 and CD8 T cells. Previous results from our lab have shown that immunization in the presence of an agonist antibody targeting OX40 led to higher antibody titers and increased numbers of antigen-specific CD4 and CD8 T cells. Using the same strategy, we explored the effect of OX40 co-stimulation in the prime and boost together with the SARS-CoV-2 spike protein + adjuvant in C57Bl/6 mice. Our results show that OX40 engagement led to a significant increase in long-lived antibody responses when compared to mice that did not receive additional co-stimulation. In addition, spike protein and peptide-specific proliferation were greatly increased for both CD4 and CD8 T cells, with augmented secretion of IFN-γ, TNF-α and IL-2. Booster immunizations (7 months post prime) did not lead to anergy, but instead further increased the antibody and T cell responses. In initial experiments, the self-amplifying RNA (saRNA) vaccination generated lower antibody titers, independent of OX40 co-stimulation. However, the saRNA vaccine did induce a robust expansion of antigen-specific CD8 T cells, that expressed high levels of GrzmB, which was further increased by OX40 administration. The strong immune responses and the differential effects of the protein and saRNA vaccines suggest that heterologous prime-boost approaches, as currently approved in adults, might be beneficial in boosting different arms of the anti-viral immune response against SARS-CoV2, with OX40 agonists enhancing both approaches. Further studies in animals will aid defining the effects and benefits of this approach.

Background Tumor Infiltrating Lymphocyte (TIL) therapy has proven effective for patients with stage IV melanoma, however there are critical issues that can limit the efficacy of standard TIL therapy across a wide range of different malignancies. We and others have shown that some tumor types contain a low percentage of tumor-specific T cells. We hypothesize that most of the patients that do not respond to TIL therapy are likely receiving a low percentage of tumor-reactive T cells and therefore a high percentage of non-therapeutic bystander TIL. We have developed a streamlined method that expands a highly enriched fraction of tumor-reactive T cells contained within the CD39+CD103+CD8+ TIL in greater than 90% of patient samples from a wide variety of malignancies (melanoma, colon cancer, head and neck cancer, etc.). This TIL product displays a broad repertoire of tumor-specific TCRs. The expanded CD39/CD103 TIL can kill autologous tumors in vitro, but the possibility remains that they could revert to a suppressed or exhausted state when they reach the tumor microenvironment upon transfer back into patients. To mitigate the suppressive effects of the tumor microenvironment we have evaluated Phio Pharmaceutical’s self-delivering RNAi INTASYL(TM) platform to silence PD-1 in the expanded TIL product. Methods The TIL product was treated during the rapid expansion phase of the protocol with either nontargeting control compounds or PD-1 targeting INTASYL™ compounds. PD-1 protein levels and TIL functionality were assessed via flow cytometry and cytokine bead array. Results Silencing of PD-1 expression in the expanded TIL product was obtained by adding the self-delivering RNAi compounds to the cell culture media, without needing transfection media, delivery formulations or electroporation. The RNAi-treated TIL product showed increased IFN-?? TNF-α and Granzyme B expression. Conclusions These data highlight a promising combination to improve the activity of tumor-reactive TIL in future human clinical trials.