Fu-Gang Zhu

A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2′-deoxy-β-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3′-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-κB and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-γ, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

Stabilized immune modulatory RNA compounds as agonists of Toll-like receptors 7 and 8

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptor (TLR)7 and TLR8. However, single-stranded RNA is rapidly degraded by ubiquitous RNases, and the studies reported to date have used RNA with lipid carriers. To overcome nuclease susceptibility of RNA, we have synthesized several RNAs incorporating a range of chemical modifications. The present study describes one pool of RNA compounds, referred to as stabilized immune modulatory RNA (SIMRA) compounds, in which two RNA segments are attached through their 3′ ends. SIMRA compounds showed greater stability in human serum compared with linear RNA and activated human TLR8, but not TLR7, in HEK293 cells without using lipid carriers. Interestingly, another set of SIMRA compounds containing 7-deazaguanosine substituted for natural guanosine activated human TLR7 and TLR8. Additionally, TLR7- and TLR8-activating compounds, but not the compounds that activated only TLR8, stimulated mouse immune cells in vitro and in vivo and produced dose-dependent T helper 1-type cytokines. Both types of compounds activated human peripheral blood mononuclear cells, but only TLR7- and TLR8-activating compounds activated plasmacytoid dendritic cells and produced high levels of IFN-α. In monkeys, s.c. administration of both types of SIMRA compounds induced transient changes in peripheral blood monocytes and neutrophils, and activated T lymphocytes, monocytes, and NK cells. Both types of compounds induced IFN-γ-inducible protein 10, but only the 7-deazaguanosine-containing compound that activated both TLR7 and TLR8 induced IFN-α in monkeys. This is a comprehensive study of RNA-based compounds containing structures and synthetic stimulatory motifs in mouse, monkey, and human systems without using lipid carriers.

A Toll-Like Receptor 7, 8, and 9 Antagonist Inhibits Th1 and Th17 Responses and Inflammasome Activation in a Model of IL-23-Induced Psoriasis

Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1β. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.

Abstract B159: Modulation of checkpoint expression in tumor microenvironment by intratumoral administration of a novel TLR9 agonist: Rationale for combination therapy

Recent advances in the understanding of immune tolerance has led to the targeting of various checkpoints for the treatment of cancer. Several independent clinical studies have pointed out a strong correlation between the clinical activity of checkpoint inhibitors and tumor checkpoint expression. These findings highlight an opportunity to improve cancer immunotherapy by combining checkpoint inhibitors with an immunostimulatory agent that modulates the tumor microenvironment. Our objective was to modulate the tumor microenvironment by activating Toll-like receptor (TLR) 9, a pathogen-associated molecular pattern recognition receptor. In preclinical studies, administration of IMO-2125, a novel TLR9 agonist, led to maturation of dendritic cells and induction of IFN-alpha. We hypothesized that immune activation by a TLR9 agonist administered intratumorally (i.t.) will lead to modulation of immune checkpoints in the tumor microenvironment, allowing these tumors to be targeted with a checkpoint inhibitor. To evaluate the potential of i.t. IMO-2125 therapy and its effect on checkpoint gene expression, we employed three different tumor models: CT26 colon carcinoma, A20 lymphoma, and B16 melanoma mouse models. IMO-2125 was administered at a dose of 50 mcg per treatment, either as a single dose or as multiple doses into a targeted tumor nodule. Blood samples were collected post-dosing for analysis of the cytokine profile. Anti-tumor activity was evaluated by measuring the growth of the injected tumor as well as the distant tumor. Tumor nodules were homogenized to analyze gene expression of selected checkpoints by qPCR. Results showed that a single i.t. injection of IMO-2125 in a CT26 colon carcinoma model induced systemic Th1 type cytokines, and resulted in tumor growth inhibition. Two days post IMO-2125 administration, analysis of gene expression of the treated tumor showed upregulation of IDO1 (13 fold) and PDL1 (2.7 fold), and no changes in expression of PD1 and OX40. Increased IDO1 gene expression correlated with increased IDO protein levels in the treated tumor nodules. In contrast, subcutaneous administration of IMO-2125 showed no substantial anti-tumor effect and no significant changes in checkpoint gene expression despite an induction of similar levels of systemic cytokines as the i.t. treatment. Multiple i.t. injections of IMO-2125 led to inhibition of tumor growth of the injected tumor and the distant tumor in all three tumor models. Tumor growth was correlated with increased CD3+ cells in treated as well as distant tumors. Analysis of the checkpoint gene expression at treated and distant tumor sites was conducted at 1-2 weeks post treatment. There was a significant upregulation of IDO1 and PDL1 gene expression in the treated tumors as well as in distant tumors. In addition to IDO1 and PDL1, depending on the tumor model, other immune checkpoints, including PD1, TIM3, BTLA, LAG3, OX40, and CTLA4, were also modulated. In conclusion, IMO-2125 i.t. treatment led to modulation of the tumor microenvironment by inducing local and systemic Th1 type immune responses, which impacted the levels of checkpoint gene expression. Increased expression of some of these checkpoints may allow tumors to be targeted broadly by checkpoint inhibitor therapy. Citation Format: Wayne Jiang, Daqing Wang, Fugang Zhu, Lakshmi Bhagat, Jillian DiMuzio, Sudhir Agrawal. Modulation of checkpoint expression in tumor microenvironment by intratumoral administration of a novel TLR9 agonist: Rationale for combination therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B159.

Abstract B094: Intratumoral administration of IMO-2125, a novel TLR9 agonist, modulates the tumor microenvironment and exerts systemic antitumor activity alone and in combination with an anti-CTLA-4 mAb

Cancer immunotherapy aims to stimulate host antitumor immune responses or break tumor-related immune tolerance or both with the potential of curative treatment. Potential immunotherapy targets include Toll-like receptors (TLRs), which are key receptors of the innate immune system and play an important role in regulating adaptive immune responses. IMO-2125 is a potent and selective agonist of endosomal TLR9 which significantly induces IFN-α, and the maturation of dendritic cells (DC). In the setting of cancer immunotherapy, we hypothesize that intratumoral (i.t.) administration of IMO-2125 has the potential to stimulate DC maturation and T-cell activation in the tumor microenvironment, leading to increased local and systemic antitumor immune responses and tumor regression, and may potentiate the activity of checkpoint inhibitors. In the present study, we evaluated the antitumor immune activity of i.t. IMO-2125 in murine syngeneic colon carcinoma models. BALB/c mice were s.c. implanted with 3 x 106 CT26 and CT26.CL25 cells, a subclone of CT26 expressing a model antigen beta-galactosidase (beta-gal), on the right and left flanks, respectively. Treatment was initiated when tumor nodules reached 200 mm3. Treated mice received IMO-2125 at doses of 10, 50 and 100 µg per injection, placebo, or a control compound (n=8 each), by i.t. injection only in the CT26 tumor implanted in the right flank twice weekly for two weeks. To evaluate IMO-2125 in combination with an anti-CTLA-4 mAb, 50 µg IMO-2125 and 10 µg anti-mouse CTLA-4 mAb were co-injected into the right tumor. Over two weeks, IMO-2125 was administered five times and the anti-mouse CTLA-4 mAb was administered four times. Results showed that i.t. IMO-2125 treatment led to dose-dependent inhibition of both treated and distant tumor growth. In mice treated with 100 µg, there were reductions in tumor volume of 95.2% (p = 0.0058) and 91.2% (p = 0.0048) in the treated and distant tumors, respectively. At this dose, there was complete tumor regression in 5 out 8 mice (63%). Control compound showed no anti-tumor activity. Antitumor activity was correlated with increased CD8+ T cell infiltration into both treated and distant tumors. In vivo depletion of T cells showed that IMO-2125 i.t mediated antitumor effects depended on CD8+ T cells, while depletion of CD4+ T cells enhanced IMO-2125 mediated tumor regression through elimination of Tregs. Furthermore, treatment elicited tumor-specific cytotoxic T cells not only to CT26 associated antigen AH1 in the injected tumor and also to beta-gal presented only in distant CT26.CL25 tumors. The mice with complete regression were rechallenged with CT26/CT26.CL25 and rejected the implantation; however, they were not protected from rechallenge with A20 lymphoma, indicating IMO-2125 i.t. treatment resulted in persistent tumor memory. Treatment with a combination of i.t. IMO-2125 and i.t. anti CTLA-4 mAb resulted in more potent antitumor activity than either agent alone. In conclusion, i.t. administration of IMO-2125 changes the local tumor microenvironment by inducing Th1 type cytokines, thereby modulating levels of immune checkpoint expression, and exerting potent local and systemic antitumor activity. In addition, IMO-2125 treatment in combination with an anti-CTLA-4 mAb demonstrated more potent antitumor activity compared to either agent alone. IMO-2125 has been well tolerated and showed induction of systemic IFN-α in a human trial. Planning for a clinical trial of a combination of i.t. IMO-2125 and ipilumimab is ongoing. Citation Format: Daqing Wang, Fugang Zhu, Xianzhi Mao, Sudhir Agrawal. Intratumoral administration of IMO-2125, a novel TLR9 agonist, modulates the tumor microenvironment and exerts systemic antitumor activity alone and in combination with an anti-CTLA-4 mAb. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B094.

Abstract 3847: Creating the tumor microenvironment for effective immunotherapy: Antitumor activity of intratumoral IMO-2125, a TLR9 agonist is further enhanced by inhibition of indoleamine-pyrrole 2,3-dioxygenase (IDO)

In recent years, understanding of tumor immunology has led to significant advances in cancer immunotherapy. New approaches include selective blockade of checkpoints, harnessing tumor infiltrating lymphocytes (TILs) and use of intratumoral oncolytic viruses. Evolving data has continued to provide evidence that the tumor microenvironment (TME) is critical for effective immunotherapy. In preclinical studies, intratumoral delivery (i.t.) of the Toll-like receptor (TLR) 9 agonist IMO-2125 resulted in increased IFNα, IL-12, Th1 responses, TIL infiltration and dose-dependent antitumor activity against treated and distant tumors. However, gene expression analysis of the treated tumors showed that levels of IDO1 was increased, thereby hampering the optimal TME. We hypothesized that combination of i.t. IMO-2125 and an IDO1 inhibitor would lead to modulation of the TME and more potent antitumor activity versus either agent alone. In the present study, we evaluated the antitumor activity of i.t. IMO-2125 in combination with an IDO1 inhibitor in a murine syngeneic colon carcinoma CT26 model. Each BALB/c mouse was implanted with two tumors: subcutaneous solid tumor and lung metastatic tumor by s.c. (1×107) and i.v. (3×106) inoculation with CT26 cells. Treatment was initiated when tumor nodules reached 100-200 mm3 with 2.5 mg/kg intratumoral IMO-2125 and 75 mg/kg oral IDO1 inhibitor. All treatments were well tolerated. In the placebo-treated group, the tumor volume was 1039 ± 262 mm3 (mean ± SD). In groups treated with IMO-2125 and IDO1 inhibitor, the tumor volume was 369 ± 216 mm3 (64% tumor growth inhibition, TGI) and 745 ± 252 mm3 (27% TGI), respectively. In the group treated with the combination of IMO-2125 and IDO1 inhibitor, the tumor volume was 45 ± 75 mm3, showing 95% tumor growth inhibition. Similar treatment-related results were observed in the lung nodules, with the combination group showing a 76% reduction in the number of lung tumor nodules over PBS control. Tumors treated with IMO-2125 alone showed 90-fold increase in IDO1 expression which was inhibited 47% in tumors treated with a combination of IMO-2125 and IDO1 inhibitor. The inhibition of IDO1 in combination with i.t. IMO-2125 also resulted in increased TILs. Overall, these preclinical data showed that combination of i.t. IMO-2125 and an IDO1 inhibitor stimulated systemic immune responses and primed the TME to enable potent antitumor activity. In previous preclinical studies IMO-2125 has shown potent antitumor activity in multiple tumor models in combination with anti-CTLA4 and anti-PD1 agents. Citation Format: Daqing Wang, Wayne Jiang, Bhagat Lakshmi, Jillian DiMuzio, Fugang Zhu, Sudhir Agrawal. Creating the tumor microenvironment for effective immunotherapy: Antitumor activity of intratumoral IMO-2125, a TLR9 agonist is further enhanced by inhibition of indoleamine-pyrrole 2,3-dioxygenase (IDO). [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 3847.

Abstract B196: Intratumoral administration of IMO-2125, a novel TLR9 agonist, modulates tumor microenvironment and potentiates antitumor activity of anti-PD-1 mAb in a murine colon carcinoma model

Immunotherapeutic advances in the treatment of cancer by targeting immune checkpoint pathways has shown promising results, however, data for agents currently in development have only shown responses in subgroups of patients. Current research is focused on identifying predictors of response to immunotherapy, including characteristics of the tumor and its microenvironment. It is becoming evident that infiltration of immune cells in the tumor microenvironment is critical to the success of immune-directed strategies. We designed a novel, oligonucleotide-based Toll-like receptor (TLR) 9 agonist to activate immune responses and induce expression of interferon-α. In previously completed preclinical studies, intratumoral (i.t.) administration of IMO-2125 affected the tumor microenvironment by stimulating TIL infiltration, modulated levels of various checkpoints, and exerted anti-tumor activity in injected as well as distant tumors. In the present study, we have evaluated the antitumor activity of i.t. IMO-2125 in combination with anti-murine PD-1 mAb (clone J43, BioXCell) in a murine syngeneic colon carcinoma model. Female BALB/c mice (6 to 8 week-old, n = 8 per group) were subcutaneous (s.c.) implanted with CT26 cells on the right and left flanks. Animals with tumors were randomized in four groups to be treated with placebo, i.t. IMO-2125, intraperitoneal (i.p.) anti-PD-1 mAb, and a combination of i.t. IMO-2125 and i.p. anti-PD-1 mAb. Treated mice received 50 μg IMO-2125 only in their right tumor and 200 μg anti-PD-1 mAb. Treatment was initiated on day 7 when tumor nodules reached 200-300 mm3 and both agents were administered four times on days 7, 8, 11 and 12. All treatments were well tolerated. Treatment with IMO-2125 alone resulted in 59% and 45% growth inhibition in treated and distant tumors respectively compared to placebo. Treatment with anti-PD-1 mAb showed limited antitumor activity with less than 15% tumor growth inhibition in both the tumor lesions compared to placebo. Treatment with a combination of IMO-2125 and anti-PD-1 mAb resulted in increased antitumor activity, leading to 81% growth inhibition in the treated tumors and 65% growth inhibition in the distant tumors compared to placebo. At day 14, the mean tumor volume in the group treated with the combination of IMO-2125 and anti-PD-1 mAb was 180 mm3, whereas mean tumor volume in the IMO-2125 group was 380 mm3 (p Combination treatment with IMO-2125 and an anti-PD-1 mAb showed significant improvements in survival and tumor volume compared to either drug alone. Planning is currently underway for a Phase 1/2 clinical trial with intratumoral IMO-2125 in combination with an anti-CTLA-4 checkpoint inhibitor. Collectively, these data demonstrate additional opportunities to combine intratumoral IMO-2125 with an anti-PD-1 mAb and potentially other checkpoint inhibitors in future clinical trials. Citation Format: Daqing Wang, Fugang Zhu, Jillian DiMuzio, Sudhir Agrawal. Intratumoral administration of IMO-2125, a novel TLR9 agonist, modulates tumor microenvironment and potentiates antitumor activity of anti-PD-1 mAb in a murine colon carcinoma model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B196.

Sa1757 Targeting Innate Immune Receptors to Treat Inflammatory Bowel Disease: Preclinical Activity of IMO-9200, an Antagonist of TLRS 7, 8, and 9 in Mouse Models of Colitis

G A A b st ra ct s recognizing E. coli OmpC. Methods: Tetramer-guided epitope mapping (TGEM) in HLADR*1501 subjects consistently identified a single T cell epitope in OmpC for use in tetramerbased assays. Direct ex vivo characterization was performed by staining with PE-labeled tetramers against OmpC and Flu peptides and subsequent enrichment with anti-PE magnetic beads. Cells were stained for surface markers and analyzed by flow cytometry. Frequency of antigen-specific T cells was calculated as the number of post-enrichment tetramer+ cells/ total pre-enrichment CD4+ cells. Results: The frequency of OmpC-specific T cells was 9.1 (+/-2)/million CD4+ T cells (n=26) compared to 13.7 (+/-2)/million Flu-specific T cells (n= 17), p=.18. The majority of both Flu and OmpC-specific T cells expressed a memory T cell phenotype (CD45RA-). There were no regulatory T cells (CD25+CD127-) identified among OmpC or Flu-specific CD4s. A high percentage of OmpC-specific T cells expressed the guthoming marker, a4b7, compared to Flu-specific T cells (44%+/-2 vs. 13.7% +/-2, p<.0001). Expression of the Th17 marker, CD161, was more frequent on OmpC than Flu-specific T cells (38.5 %+/-5 vs. 13% +/-2, p <.0001). While there was some expression of the Th1 marker, CXCR3, on OmpC-specific T cells, this was less than was expressed by Flu-specific T cells (27%+/-4 vs. 50% +/-5, p= .0015). The current study was underpowered to detect differences in frequency or phenotype among OmpC seropositive and seronegative CD subjects and healthy controls. OmpC-specific T cells identified above were isolated for future RNA transcript analyses to reveal qualitative differences in these cells between patients with and without CD. Conclusions: We have identified T cell epitopes for E.coli OmpC in HLADR *1501 subjects and generated MHC class II tetramers that identify OmpC-specific T cells. OmpC-specific T cells have a frequency similar to Flu-specific T cells and bear a distinct phenotype. They are memory T cells with gut-homing integrin a4b7 expression. They lack Treg markers and express mixed Th1 and Th17 markers. These data confirm that healthy humans and CD subjects harbor commensal-specific T cells and suggest that active suppression of T cell responses is lost in subjects with CD.