Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors.

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103+CD39+ tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103+CD39+ CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103+CD39+ CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies

85 Detection of human angiotensin-converting enzyme 2 receptor (hACE2R) on human cancer cell lines

Background SARS-CoV-2 infections have delayed administration of treatments for some patients with cancer, increasing the number of avoidable deaths. However, we hypothesized that infection of cancer cells with SARS-CoV-2 might increase the immunogenicity of those cancer cells. Here we sought to determine whether non-small cell lung cancer (NSCLC) and head and neck squamous cell cancer (HNSCC) cell lines could be a potential target of SARS-CoV-2, which binds and infects host cells via interactions between the viral spike glycoprotein and the human angiotensin-converting enzyme 2 receptor (hACE2) receptor. Through an institutional research tissue protocol, our lab has established a panel of cancer cell lines of various histologies. Here we set out to identify whether HNSCC and NSCLC cell lines expressed the hACE2R. We also investigated the expression of neuropilin-1, a molecule reported to facilitate SARS-CoV-2 cell entry. Methods Established cell lines were phenotyped by flow cytometric analysis utilizing the anti-hACE2R antibody from Novus Biologicals. Cell lines were also stained with mIgG1 and anti-CD3 antibodies as negative staining controls. Results We identified that three of eight NSCLC cell lines expressed the hACE2R and two of these had strong expression of neuropilin-1. Evaluation of HNSCC cell lines identified seven of seven cell lines expressed detectable levels of hACE2R but only one of seven HNSCC celllines expressed substantial levels of neuropilin-1. Preliminary evaluation of a renal cell carcinoma (RCC) cell line revealed strong staining for hACE2R. Conclusions Our study found that a majority of HNSCC cell lines (100% n=7) and approximately a third of the NSCLC cell lines (37.5%, n=8) tested express the hACE2R. Some cell lines express both hACE2R and neuropilin-1, potentially increasing their susceptibility for infection with SARS-C0V-2. While these studies were performed with cultured cell lines that may have modulated expression of hACE2R, it is possible that in vivo these tumors express the ACE2R and could serve as a target and possible reservoir for SARS-CoV-2. Support The Chiles foundation, Nancy Lematta http://dx.doi.org/10.1136/jitc-2021-SITC2021.08

5 Development of a vaccine to intercept oral cancer

Background Oral cavity cancer is diagnosed in more than 300,000 people each year worldwide and approximately half of these will die within five years despite standard treatment. These cancers are often preceded by the appearance of a premalignant dysplastic lesion, which offers a unique opportunity to identify patients at high risk of developing cancer and offer them a vaccine that may prevent development of this non-viral malignancy. Lesions can be removed but because of a ‘field effect’ their entire oral mucosa is at risk – thus the vaccine. Recent studies have identified genes that are differentially expressed during progression from normal tissue to oral cancer providing a roadmap to developing a preventative vaccine [PMID: 27027432] Methods We have manufactured and performed initial characterization of a DC-targeted microvesicle vaccine, DPV-007, made specifically to intercept the progression of oral dysplasia to oral cancer. DPV-007 is manufactured using platform vaccine technology that is in the clinic. This technology captures short-lived and non-canonical proteins that make up the dominant epitopes presented by HLA and packages them in microvesicles containing DAMPs and molecular chaperones. Characterization of DPV-007 included molecular, proteogenomic, biochemical and functional assessments. Preclinical studies were performed evaluating prevention of tumor development in the 4NQO-induced tumor model. Results The DPV-007 microvesicle vaccine contains more than 200 proteins for genes that are upregulated in oral dysplasia and oral cancer. Preliminary data suggests that the vaccine may contain as many as 30 somatic variants identified as somatic mutations in the COSMIC Database. Additionally, the vaccine contains at least 11 NCI prioritized cancer antigens and has agonist activity for TLR 2, 3, and 9. In the 4NQO preclinical model, vaccination including relevant antigens and agonist activity, provided significant (p\u3c0.02) protection from lesion onset and tumor outgrowth. Conclusions The identification of genes associated with the progression of pre-cancerous lesions to cancer provides targets for active immunotherapy of this disease. In preclinical models we have shown that this vaccine strategy is effective in both protection and therapy studies [PMID: 21810919; PMID: 27874054; PMID: 31747946]. A clinical trial of a similar vaccine, DPV-001, administered as a single agent as adjuvant therapy for NSCLC, documented induction of immunity to a large number of cancer antigens contained in the vaccine and did not identify serious adverse events. Based on data summarized above, we propose to vaccinate patients with dysplastic lesions and investigate whether vaccination reduces lesion recurrence. Acknowledgements Research support was provided by Janssen Research and Development, Johnson & Johnson. Ethics Approval The Institutional Animal Care & Use Committee of the Earle A. Chiles Research Institute approved the above noted studies. Protocol 55

409 Manufacturing of a clinical scale CD8 TIL product, AGX148, with and without gene silencing of PD-1 using self-delivering RNAi INTASYLTM PH-762

Background Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a percentage of patients with melanoma, however, the efficacy of standard TIL therapy (Bulk TILs) can be limited. We hypothesize that the therapeutic efficacy of TIL therapy is dependent on the abundance of tumor-reactive T cells in the ACT product, as well as the functionality of the T cells in the product. Our approach enriches for tumor-reactive TIL by sorting CD8+ T cells from patient tumors that co-express CD103 and CD39 prior to expansion of the ACT product,1 termed AGX148. To mitigate PD-1-induced immune suppression in the Tumor Microenvironment (TME) and further enhance the therapeutic potential of AGX148 we have utilized Phio Pharmaceuticals’ self-delivering RNAi INTASYL™ PH-762 to knock down PD-1 during the expansion. Methods Surgically resected human tumor samples were provided through a collaboration with the Earle A. Chiles Research Institute’s clinical research program at the Providence Cancer Institute. We have adapted our research expansion method into an optimized clinical manufacturing process. In collaboration with the Providence Cancer Institute’s Cell Processing Facility and Phio Pharmaceuticals, we have completed three full scale IND-enabling manufacturing runs of our ACT product AGX148 w/wo PH-762 INTASYL™. Results Our data demonstrate that the AGX148 ACT product can be successfully manufactured at a clinical scale and that AGX148 treated with PH-762 during the expansion process is effective at reducing the steady-state level of PD-1 protein. We also tested AGX148 ACT product for autologous tumor recognition and killing in co-culture assays in vitro and in vivo. AGX148 and AGX148 combined with PH-762 were able to recognize and kill autologous tumor lines in vitro and PH-762-treated cells had increased 4-1BB expression in TIL/tumor cocultures. PH-762 induced knockdown of PD-1 was observed in circulating T cells after ACT in a xenograft model with autologous tumor. Conclusions We have generated a potent tumor-specific ACT TIL product (AGX148) at clinical scale through the isolation and selective expansion of tumor-reactive T cells. Knocking down PD-1 with PH-762 INTASYL™ has the potential to further enhance the function of the AGX148 product and this ACT product will soon be tested in cancer patients. Reference Duhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724. Ethics Approval Human sample collection for this study was approved by the institutional review board, Providence St. Joseph Health IRB (FWA00029175), Study ID: PDX06-108. All patients provided informed consent for participation in this study. Experimental animal studies were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in accordance with the EACRI Institutional Animal Care and Use Committee (Animal Welfare Assurance No. A3913-01) protocol# 51. Summary: Name of IRB: Providence St. Joseph Health IRB (FWA00029175) Study ID: PDX06-108 Most Recent IRB Submission ID: CR2022000115 (Continuing Review) Date of most recent approval: 03/08/2022 The Institutional Animal Care and Use Committee (IACUC) of Providence Portland Medical Center Study Protocol #51 Date of most recent approval: 03/28/202

Subcellular compartmentalization of PKM2 identifies anti-PKM2 therapy response in vitro and in vivo mouse model of human non-small-cell lung cancer.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC

Allogeneic autophagosome-enriched composition for the treatment of disease

A composition, comprising: an enriched population of autophagosomes derived from a non-small cell lung carcinoma cell line, and wherein the enriched population of autophagosomes includes: one or more toll-like receptor agonists; one or more tumor antigens; and one or more damage-associated molecular pattern molecules. In this way, an off-the-shelf vaccine may be available to be administered in order to stimulate a targeted immune response in patients bearing different tumor types