Christopher Paustian

STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34 pos cells into tumor-competent dendritic cells

The clinical outcomes of dendritic cell (DC)-based immunotherapy remain disappointing, with DCs often displaying a tenuous capacity to complete maturation and DC1 polarization in the tumor host. Surprisingly, we observed that the capacity for successful DC1 polarization, including robust IL12p70 production, could be regulated by STAT-dependent events even prior to DC differentiation. Exposure of CD34(pos) cells to single-agent granulocyte-macrophage colony-stimulating factor (GMCSF) induced multilineage, STAT5-dependent differentiation, including DCs that failed to mature in the absence of further exogenous signals. In contrast, Flt3L induced nearly global differentiation of CD34(pos) cells into spontaneously maturing DCs. IL-6 synergized with Flt3L to produce explosive, STAT3-dependent proliferation of phenotypically undifferentiated cells that nevertheless functioned as committed DC1 precursors. Such precursors not only resisted many tumor-associated immunosuppressants, but also responded to tumor contact or TGFbeta with facilitated DC maturation and IL12p70 production, and displayed a superior capacity to reverse tumor-induced T-cell tolerance. GMCSF preempted Flt3L or Flt3L plus IL-6 licensing by blocking STAT3 activation and promoting STAT5-dependent differentiation. Paradoxically, following overt DC differentiation, STAT5 enhanced whereas STAT3 inhibited DC1 polarization. Therefore, nonoverlapping, sequential activation of STAT3 and STAT5, achievable by sequenced exposure to Flt3L plus IL-6, then GMCSF, selects for multilog expansion, programming, and DC1 polarization of tumor-competent DCs from CD34(pos) cells.

Effect of multiple activation stimuli on the generation of Th1-polarizing dendritic cells

It was originally reported that only a small fraction of total matured dendritic cells (DCs) produced interleukin (IL)-12, but it has never been determined whether different combinations of activating signals now shown to maximize secreted IL-12 do so through increasing output by the same IL-12 producers, or by recruiting additional cytokine-secreting cells. We therefore tested all combinations of bacterial lipopolysaccharide (LPS) (TLR4 ligand), R848 (TLR8 ligand), interferon (IFN)-γ, and CD40L for activating human monocyte-derived dendritic cells (DC), and determined by intracellular flow cytometry that enhanced IL-12 secretion was accomplished in large part by markedly increasing the proportion of cells producing IL-12, with the triple and quadruple combinations recruiting the most DC. This optimization requirement for multiple signals was not reflected in differential Toll-like receptor (TLR) expression by the cells. Interestingly, DCs activated with single TLR ligands plus IFN-γ were capable of responding with a second burst of IL-12 upon later CD40L stimulation, whereas DCs activated with R848 plus LPS were not, despite the trend of the latter for superior polarization of naive T cells toward IFN-γ-secreting Th1. These results have implications for the biology of IL-12-secreting DCs and choice of activation regimen for prospective use in DC-based immunotherapy.

Extracellular ATP and Toll-Like Receptor 2 Agonists Trigger in Human Monocytes an Activation Program That Favors T Helper 17

Strategically-paired Toll-like receptor (TLR) ligands induce a unique dendritic cell (DC) phenotype that polarizes Th1 responses. We therefore investigated pairing single TLR ligands with a non TLR-mediated danger signal to cooperatively induce distinct DC properties from cultured human monocytes. Adenosine triphosphate (ATP) and the TLR2 ligand lipoteichoic acid (LTA) selectively and synergistically induced expression of IL-23 and IL-1β from cultured monocytes as determined by ELISA assays. Flow cytometric analysis revealed that a sizable sub-population of treated cells acquired DC-like properties including activated surface phenotype with trans-well assays showing enhanced migration towards CCR7 ligands. Such activated cells also preferentially deviated, in an IL-23 and IL-1-dependent manner, CD4pos T lymphocyte responses toward the IL-22hi, IL-17hi/IFN-γlo Th17 phenotype in standard in vitro allogeneic sensitization assays. Although pharmacological activation of either ionotropic or cAMP-dependent pathways acted in synergy with LTA to enhance IL-23, only inhibition of the cAMP-dependent pathway antagonized ATP-enhanced cytokine production. ATP plus atypical lipopolysaccharide from P. gingivalis (signaling through TLR2) was slightly superior to E. coli-derived LPS (TLR4 ligand) for inducing the high IL-23-secreting DC-like phenotype, but greatly inferior for inducing IL-12 p70 production when paired with IFN-γ, a distinction reflected in activated DCs’ ability to deviate lymphocytes toward Th1. Collectively, our data suggest TLR2 ligands encountered by innate immune cells in an environment with physiologically-relevant levels of extracellular ATP can induce a distinct activation state favoring IL-23- and IL-1β-dependent Th17 type response.

J-LEAPS vaccines initiate murine Th1 responses by activating dendritic cells.

The Ligand Epitope Antigen Presentation System (LEAPS) converts a peptide containing a T cell epitope as small as 8 amino acids into an immunogen and directs the nature of the subsequent response. Tandem synthesis of the J peptide (a peptide from the beta-2-microglobulin) with peptides of 15 or 30 amino acids from HSV-1 or HIV made them immunogenic and promoted Th1 immune responses. Immunization of A/J or C57BL/6 mice with J-LEAPS heteroconjugates containing an epitope from the HSV-1 glycoprotein D (JgD) or an epitope from the HIV gag protein (JH) emulsified with Seppic ISA51 induced increased levels of IL-12p70 by day 3 and increased levels of interferon gamma (IFN-gamma) on days 10 and 24. Interestingly, levels of IL-10, TNF-alpha, and IL-6 did not change. Neither the H nor the gD peptides alone elicited responses and only weak responses followed immunization with the J peptide. Bone marrow (BM) cells became CD86 and CD11c positive within 48 h of treatment with JgD or JH. JH or JgD treatment promoted IL-12p70 production and expression of CD8 denoting the maturation and activation of a subclass of myeloid DCs. Pure cultures of immature myeloid DCs also responded to JgD treatment, forming clusters, developing dendrites, and producing IL-12p70 within 24 h. The JH or JgD treated bone marrow cells (JgD-DC) were necessary and sufficient to activate splenic T cells to produce IFN-gamma and the JgD-DC provided an antigen specific booster response to T cells from JgD immunized mice. Adoptive transfer of JgD-DC was also sufficient to initiate protective antigen specific immunity from lethal challenge with HSV-1. The J-LEAPS vaccines appear to act as an adjuvant and immunogen on DC precursors in a unique manner to promote activation and maturation into IL-12p70 producing DCs which then can initiate sufficient Th1 immune responses to elicit protection without production of acute phase cytokines.

Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer

ABSTRACT The immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous – single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+ T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85–22% of CD8+ cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+ T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy.

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.

Abstract CT053: Intratumoral coxsackievirus A21 increases immune-cell infiltrates and upregulates immune-checkpoint molecules in the tumor microenvironment of advanced melanoma patients: phase II CALM extension study

Background: CAVATAK, an oncolytic immunotherapy, is a bio-selected oncolytic strain of Coxsackievirus A21 (CVA21). Following intratumoral (IT) injection, CVA21 preferentially infects ICAM-1 expressing tumor cells, resulting in viral replication, cell lysis, and a systemic anti-tumor immune response. The Phase II CALM study investigated the efficacy and safety of IT CVA21 in pts with advanced melanoma with durable responses being observed in both injected and non-injected melanoma metastases, suggesting the generation of significant host anti-tumor responses. Pre-clinical studies in an immune-competent mouse model of melanoma revealed that combinations of intratumoral CAVATAK and anti-PD-1 or anti-CTLA-4 mAbs mediated significantly greater antitumor activity and compared to use of either agent alone. Here we report on an extension study aimed at understanding the impact of CVA21 on immune cell infiltrates and immune checkpoint molecules within treated lesions of advanced melanoma patients. Methods: In the CALM extension study a cohort of 13 advanced melanoma patients received up to 3 × 108 TCID50 CVA21 IT on study days 1,3,5 and 8 and then every three weeks for a further 6 injections. Sequential tumor biopsies of injected lesions (study days 1 and 8) were monitored for evidence of viral-induced changes immune cell infiltrates (Multi-spectral imaging) and immune checkpoint molecules (NanoString® PanCancer Immune profiling panel). Serial serum samples were monitored for viral loads, anti-CVA21 neutralizing antibody (nAb) and levels of immune-inflammatory cytokines. Results: Intratumoral CVA21 administration in a number of cases induced significant increases in immune cell infiltrates within the tumor microenvironment, in particular CD8+ cells (p = 0.044) and increased expression of PD-L1+ cells (p = 0.041) as assessed by multi-spectral imaging. Analysis of a number of pre- and post-treatment biopsy samples by NanoString® digital RNA counting identified notable increases in the levels of immune checkpoint molecules including PD-1, PD-L1, CTLA-4, TIM-3 and LAG-3 and sizable up-regulation of a number of immune modulation elements, including Th1-gene shifts, with increases in interferon-induced genes. Of particular interest was the reconstitution of immune cell infiltrates in a number of CVA21 treated lesions from patients failing previous immune-checkpoint blockade or other immunotherapies. Conclusions: Intratumoral administration of CVA21 can notably influence the dynamics of the tumor micro-environment as evidenced by increases in both immune cell infiltrates and levels of immune-checkpoint genes. Such CVA21 induced immune-changes appear to be beneficial in combination immunotherapy, with preliminary overall response rates in a Phase 1b study CVA21 and anti-CTLA-4 blockade (ipilimumab) in advanced melanoma patients being higher than published response rates for either agent used alone. The use of CVA21 to reconstitute immune cell infiltrates in lesions resistant to immune-checkpoint blockade prior to re-commencing such treatment is an exciting novel therapeutic option. Citation Format: Robert Andtbacka, Brendan Curti, Sigrun Hallmeyer, Zipei Peng, Christopher Paustian, Carlo Bifulco, Bernard Fox, Mark Grose, Darren R. Shafren. Intratumoral coxsackievirus A21 increases immune-cell infiltrates and upregulates immune-checkpoint molecules in the tumor microenvironment of advanced melanoma patients: phase II CALM extension study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT053.

An evaluation of autologous tumor-reactive TIL generation from head and neck squamous cell cancers

Methods Our group formed a multidisciplinary group to study the immunobiology of HNSCC and to develop strategies to effectively treat this disease. Over the past 3 years we have collected and processed more than 192 HNSCC specimens. Starting in June of 2013 we began to set up cultures of tumor-infiltrating lymphocytes (TIL) from specimens with sufficient numbers of cells isolated by triple enzyme digestion. Between June 13, 2013 and May 14, 2015 we processed 120 specimens. Fourty-one were processed but were not set-up for TIL culture. Sixteen were determined to be grossly contaminated within 18-24 hours and excluded from further analysis. Sixty-three TIL cultures were initiated and 33 generated TIL (52%).