Tarsem Moudgil

OX40 Is a Potent Immune-Stimulating Target in Late-Stage Cancer Patients

OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function, and survival of T cells. Preclinical studies have shown that OX40 agonists increase antitumor immunity and improve tumor-free survival. In this study, we performed a phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4(+) FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, and increased the antitumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in patients with cancer, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells, and intratumoral regulatory T cells.

Signaling Through OX40 Enhances Antitumor Immunity

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.

Vaccination of Women with Metastatic Breast Cancer, Using a Costimulatory Gene (CD80)-Modified, HLA-A2-Matched, Allogeneic, Breast Cancer Cell Line: Clinical and Immunological Results

MDA-MB-231, an HLA-A2(+), HER2/neu(+) allogeneic breast cancer cell line genetically modified to express the costimulatory molecule CD80 (B7-1), was used to vaccinate 30 women with previously treated stage IV breast cancer. Expression of CD80 conferred the ability to deliver a costimulatory signal and thereby improved the antigen presentation capability of the tumor cells to patient T cells in vitro. Patients were vaccinated with 10(7) or 10(8) irradiated gene-modified tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or BCG, three times at 2-week intervals and then monthly until progressive disease developed. GM-CSF-related flulike symptoms and minor injection site reactions were observed frequently. Prolonged disease stabilization was observed in four patients but no objective tumor regressions were seen. Immune responses were measured in matched peripheral blood samples collected before and after treatment from 9 of 15 patients treated at the 10(8) tumor cell dose. Four patients exhibited MHC class I-restricted cytokine production in response to the parental breast cancer cell line. One patient maintained an increased number of circulating tumor-specific, interferon gamma-secreting CD8(+) T cells for 24 months after the last vaccination. One patient exhibited a tumor-specific interleukin 5 response to an autologous tumor cell line. This immunization strategy proved to be safe and feasible, and induced tumor-specific immune responses in a minority of patients; however, no objective tumor regressions were observed.

Cancer Immunotherapy: The Role Regulatory T Cells Play and What Can be Done to Overcome their Inhibitory Effects

Since multiple lines of experimental and clinical data clearly identified regulatory T cells as an integral part of the immune response, these cells have become a major focus of investigation in tumor immunology. Regulatory T cells are in place to dampen ongoing immune responses and to prevent autoimmunity, but they also have profound effects in blocking therapeutic anti-tumor activity. Therefore regulatory T cells are seen as a major hurdle that must be overcome in order for cancer immunotherapy to reach its therapeutic potential. Regulatory T cells are heterogeneous with sub-populations that exhibit distinct functional features. Here we will review the individual sub-populations in regards to their mode of action and their potential impact on blocking anti-tumor immunity. Approaches to measure function and frequency of regulatory T cells in model systems and clinical trails will be discussed. Finally, we will describe possible ways to interfere with regulatory T cell-mediated immune suppression with the focus on recent pre-clinical and clinical findings.

Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory T cells.

BackgroundAutophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles (Defective ribosomal products in blebs), were excellent sources of antigens for efficient cross priming of tumor-specific CD8+ T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported.MethodsDRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus (CMV) pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus (EBV), and Influenza (Flu) epitopes (CEF) were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4+ and CD8+ memory T cells was detected by intracellular staining. The effect of cytokines (GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ) TLR agonists (Lipopolysaccharide, Polyinosinic-polycytidylic acid (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored.ResultsIn this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8+ memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8+ T cell responses.ConclusionsDRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with Poly(I:C), CD40 ligand, and GM-CSF. This study provides proof-of-concept for applying this strategy to activate memory T cells against other antigens, including tumor-specific T cells ex vivo for immunological monitoring and adoptive immunotherapy, and in vivo as vaccines for patients with cancer.

Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation

We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-&ggr; in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non–small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

Developing an immunotherapy strategy for the effective treatment of patients with squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck (HNSCC) arises in the oral cavity, oropharynx, larynx or hypopharynx, and is the 6th leading cause of cancer by incidence worldwide. Approximately 600,000 cases arise each year, and only 40-50% of these patients will survive for 5 years.7 HNSCC can express PD-L1 and suppressive factors that interfere with the differentiation and activation of dendritic cells and effector function of T cells. Similar to observations in other malignancies, CD8 effector T cell infiltration or transcriptional signature consistent with an ongoing T cell response is associated with prolonged survival. This suggests that HNSCC lacking a T cell infiltrate may benefit from immune interventions that induce and/or augment a tumor-specific immune response. To develop a strategy to address this problem we have created a HNSCC tumor bank that is cryopreserving enzymatically isolated viable cells from resected tumors (39 specimens). We are also attempting to develop primary cell lines (4 lines established) and are isolating tumor-infiltrating lymphocytes from these specimens. We are looking to compare immunohistochemical, flowcytometric and functional analyses of these specimens. Consistent with previous studies, results document that isolated TIL secrete IFN-γ when stimulated with their autologous tumor cells (n=2). Since a central aim of these studies is to develop a strategy to create therapeutic immunity in patients lacking T cell infiltrates, we plan to investigate which antigens are recognized and whether, antigens present in a novel DRibble vaccine, that expresses antigens common to HNSCC, are recognized by these TIL. This may provide a strategy to monitor anti-tumor immunity in these patients and possibly provide an antigen source for combination immunotherapy of patients with HNSCC.

Characterizing the immunoprofile and endogenous immune response to squamous cell carcinomas of the head and neck to guide development of effective immunotherapy strategies.

Squamous cell carcinoma of the head and neck (HNSCC) is the 6th leading cause of cancer by incidence worldwide with approximately 600,000 new cases per year. Unfortunately, only 40-50% of these patients will survive for 5 years. In order to study the immune response in patients with this disease we have developed a HNSCC tumor bank to compliment our Oral, Head and Neck Cancer Program. This tumor bank is cryopreserving enzymatically isolated viable cells from resected tumors (n = 128). We are also attempting to develop primary cell lines (10 lines established) and are isolating and assessing tumor-specific function of tumor-infiltrating lymphocytes (TIL) (n = 49). Since HNSCC can express immune inhibitory molecules and secrete suppressive factors, we are developing single stains and multi-spectral imaging protocols to assess the immune contexture of the tumor microenvironment by immunohistochemistry and immunoflouresence and are overlaying these studies with T cell functional activity and ultimately, with clinical outcome. Preliminary analyses suggest that tumor-specific T cells can be detected in 68% (N = 16) of patients evaluated. A goal of these studies is to identify strategies that will allow tailoring of therapy for patients with HNSCC. One component is to identify which inhibitors are present in a given tumor. Since not every tumor appears to contain TIL capable of recognizing autologous tumor, strategies to prime tumor-specific T cells represents another area of interest. DPV-001 is a microvesicle vaccine, DRibbles, that contains an average of at least 66 proteins that are overexpressed by HNSCC (TCGA provisional RNASeq n = 303 pts). The vaccine also contains multiple DAMPs and agonist activity for TLR 2, 3, 4, 7 and 9 packed into stable double membrane microvesicles that are targeted to CLEC9A+ APC. To increase potential activity against HPV positive cancers we have developed a mosaic construct encoding E6 and E7 peptides for a number of HPV strains and are evaluating both protein and gene-based HPV vaccine strategies. We are using the HNSCC TIL lines to evaluate DRibble vaccines and potential for adoptive immunotherapy trials. Support: NCI U43CA165048 and R44 CA121612 (TLH), OMS (HH, RBB), Steve and Cindy Harder, Robert W. and Elsie Franz, Wes and Nancy Lematta, Lynn and Jack Loacker, and The Chiles foundation (BAF).

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.Identifying and enumerating tumor-specific CD8 T cells are important for assessing cancer prognosis and therapy efficacy. Here the authors show that CD39 and CD103 mark a subset of tumor-infiltrating CD8 T cells that are tumor-reactive and exhibit characteristics of exhausted or tissue-resident memory T cells.

Abstract 38: Development of a DC-targeted microvesicle vaccine to intercept the progression of oral preneoplasia to cancer

Background: HPV negative oral squamous cell carcinoma (OSCC) is diagnosed in more than 300,000 people each year worldwide and more than half of these will die within five years despite standard treatment. These cancers are often preceded by the appearance of a preneoplastic lesion, which offers a unique opportunity to identify patients at high risk of developing OSCC and offer them a vaccine that may prevent development of this non-viral cancer. Cancer cell line-derived proteasome-blocked autophagosomes are DC-targeted microvesicles containing tumor-derived short-lived proteins (SLiPs) and defective ribosomal products (DRiPs). SLiPs and DRiPs are short-lived because they are targeted to and degraded by the proteasome, explaining why they are thought to make up the dominant epitopes presented on the surface of cancer cells. This may explain their ability to induce broad anticancer immunity in preclinical models and in patients receiving the vaccine. The human vaccine studied here, DPV-001, was derived from two cancer cell lines. Characterization of the vaccine by mass spectroscopy identified that the vaccine contains more than 100 antigens whose genes are over-expressed by most cancers. Many of these antigens have single amino acid variants that may serve as immunogenic mimetopes, or altered peptide ligands. Further, DPV-001 contains at least 13 antigens from the NCI priority list and multiple toll-like receptor (TLR) agonists packaged into stable double membrane microvesicles that are targeted to CLEC9A+ antigen presenting cells (APCs). We recently reported that every patient receiving vaccine in an NCI-supported phase I/II trial developed or augmented immunity against a large number of putative cancer antigens. Methods: Recently, gene sets developed from a preclinical 4-NQ0 mouse model of oral cancer were shown to enrich for patients with oral preneoplasia that progressed to squamous cell carcinoma (p=0.0495, Foy J-P, 2016). The progressive gene set (PGS) contained 162 genes and we compared this list to results of mass spectroscopy analysis of the DPV-001 vaccine. Results: 94 of the 162 genes contained in the PGS, which enriched for patients with poor oral cancer-free survival, were identified in the DPV-001 vaccine. Conclusions: DPV-001 contains more than 90 proteins for genes (PGS) whose over-expression is associated with progression of oral preneoplasia to OSCC. Based on significant preclinical data documenting induction of protective or therapeutic immunity in animals receiving a DRibble vaccine, coupled with safety data from a phase I/II clinical trial, and evidence that vaccination induces broad immunity against putative cancer antigens in every patient studied to date, we are proposing a pilot clinical trial of DPV-001 in patients with severe oral dysplasia. Citation Format: R. Bryan Bell, Rom Leidner, Carlo B. Bifulco, Christopher Dubay, Tarsem Moudgil, Sam Bookhardt, Glenna McDonnell, Christopher Paustian, Hong-Ming Hu, Walter J. Urba, Traci L. Hilton, Bernard Fox. Development of a DC-targeted microvesicle vaccine to intercept the progression of oral preneoplasia to cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 38.