Daqing Wang

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.

Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9.

Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2′-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1β and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.

Antitumor Activity and Immune Response Induction of a Dual Agonist of Toll-Like Receptors 7 and 8

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptors 7 and 8 (TLR7 and TLR8). We have reported a novel class of synthetic oligoribonucleotides, referred to as stabilized immune-modulatory RNA compounds, which act as agonists of TLR7, TLR8, or both TLR7 and TLR8 depending on the sequence composition and the presence of specific chemical modifications. In the present study, we evaluated the antitumor activity of a dual TLR7/8 agonist in tumor-bearing mice with peritoneal disseminated CT26.CL25 colon and 3LL-C75 lung carcinomas. Peritoneal administration of dual TLR7/8 agonist in mice bearing CT26.CL25 colon carcinomas had potent dose-dependent antitumor activity, which was associated with a marked decrease in CD4+CD25+Foxp3+ T regulatory cells and a significant increase in tumor antigen–specific IFN-γ–secreting effector cell responses in splenocytes and local tumor-infiltrating cells. In 3LL-C75 lung carcinoma, dual TLR7/8 agonist induced strong immune responses and antitumor effects in C57BL/6 and TLR9−/− mice, but not in TLR7−/− and MyD88−/− mice, indicating that the agonist induces immune responses via TLR7 and through the MyD88-dependent signaling pathway. TLR8 is not functional in mice. Additionally, s.c. administration of TLR7/8 agonist effectively prevented lung metastasis of tumors in the CT26.CL25 pulmonary metastasis model. These studies show that the dual TLR7/8 agonist induced Th1-type immune responses and potent antitumor activity in mice via TLR7 and through the MyD88-dependent pathway. Mol Cancer Ther; 9(6); 1788–97. ©2010 AACR.

Role of toll-like receptors in the pathogenesis of dystrophin-deficient skeletal and heart muscle

Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.

Impact of Secondary Structure of Toll-Like Receptor 9 Agonists on Interferon Alpha Induction

ABSTRACT Oligodeoxynucleotides containing a CpG motif and double- or multistranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of interferon alpha (IFN-α) in addition to other Th1-type cytokines. In the present study, we evaluated three highly effective IFN-α-inducing agonists of TLR9 to determine the type of duplex structures formed and the agonists ability to induce immune responses, including IFN-α induction, in human cell-based assays and in vivo in mice and nonhuman primates. Thermal melting studies showed that two of the agonists evaluated had a single melting transition with similar hyperchromicity in both heating and cooling cycles, suggesting the formation of intermolecular duplexes. A third agonist showed a biphasic melting transition in the heating cycle and a monophasic melting transition with lower hyperchromicity during the cooling cycle, suggesting the formation of both intra- and intermolecular duplexes. All three agonists induced the production of Th1-type cytokines and chemokines, including high levels of IFN-α, in human peripheral blood mononuclear cell and plasmacytoid dendritic cell cultures. Subcutaneous administration of the two intermolecular duplex-forming agonists, but not the intramolecular duplex-forming agonist, induced cytokine secretion in mice. In nonhuman primates, the two agonists that formed intermolecular duplexes induced IFN-α and IP-10 secretion. On the contrary, the agonist that formed an intramolecular duplex induced only low levels of cytokines in nonhuman primates, suggesting that this type of structure formation is less immunostimulatory in vivo than the other structure. Taken together, the present results suggest that oligonucleotide-based agonists of TLR9 that form intermolecular duplexes induce potent immune responses in vivo.

Synthetic oligoribonucleotides containing arabinonucleotides act as agonists of TLR7 and 8

In continuation of our studies with stabilized immune modulatory RNA (SIMRA) compounds, we have synthesized novel SIMRA compounds incorporating arabinonucleotides to study their effects on TLR7 and TLR8 activation. The SIMRA compounds containing ara-G, ara-C, ara-U or ara-A substitutions activated TLR8 in HEK293 cells. Interestingly, the SIMRA compound containing ara-C also activated TLR7 and stimulated immune responses in vivo in mice. In human PBMC and pDC assays, SIMRA compounds containing arabinonucleotides induced Th1-type cytokine profiles. These results suggest that SIMRA compounds containing arabinonucleotides act as agonists of TLR7 and TLR8.

Abstract 2570: IMO-8400, a selective antagonist of TLRs 7, 8 and 9, inhibits MYD88 L265P mutation-driven signaling and cell survival: A potential novel approach for treatment of B-cell lymphomas harboring MYD88 L265P mutation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In studies of B-cell lymphoma, Staudt and colleagues have identified oncogenic mutations in the signaling pathways associated with the B-cell receptor (BCR) and, more recently, the MYD88 L265P mutation (Ngo et al, Nature 2011, 470:115). MYD88 is a key adaptor protein in the Toll-like Receptor (TLR) signaling pathway. It has been shown that the MYD88 L265P mutation leads to the over activation of the TLRs 7 and 9 signaling pathway, whereas blocking of this pathway decreased cell survival, providing a rationale for targeting TLRs 7 and 9 signaling as a therapeutic approach (Lim et al, AACR 2013, #2332). The MYD88 L265P mutation is reported to be present in over 90% of Waldenstroms macroglobulinemia (WM) patients, 29% of patients with activated B-cell-like (ABC) diffuse large B cell lymphoma (DLBCL), and in other B-cell lymphomas. We evaluated IMO-8400, an antagonist for TLRs 7, 8 and 9, in preclinical studies employing three cell lines with the MYD88 L265P mutation (OCI-Ly3, OCI-Ly10 and TMD8), primary bone marrow cells from a WM patient with the mutation, and a control GCB-DLBCL cell line SU-DHL-6 with wild-type MYD88. The presence of MYD88 L265P mutation was confirmed by allele-specific PCR and Sanger sequencing. All cell lines expressed TLRs 7 and 9. Treatment of mutation-positive cell lines with IMO-8400 resulted in dose- and duration-dependent decreases in multiple parameters of cell activation, including cell survival (EC50: 0.95 μM and ∼5 μM, with and without use of lipid, respectively), phosphorylation of BTK, IRAK1, IRAK4, NF-κB, STAT3 and p38 (assayed by Western blot), and secretion of cytokines including IL-10, MIG and IL-2R. Gene array analysis indicated that IMO-8400 inhibited the expression of several genes in the NF-κB and JAK/STAT pathways, including NFKB1, TNFSF10, STAT3 and IL2RA. IMO-8400 also inhibited cell survival and cytokine secretion in cells from the WM patient. In a murine model of disseminated OCI-LY10, IMO-8400 as a single treatment agent showed potent anti-tumor activity in vivo, with dose-dependent increase in animal survival. Treatment was well-tolerated at all dose levels. In a subcutaneous tumor model, growth of even well-established tumor nodules (approximately 500 mm3) was significantly inhibited by IMO-8400 treatment, and this effect correlated with decreased IκBα phosphorylation and IL-10 expression (gene and protein) in tumor cells as well as decreased human IL-10 in the serum of the mice. In contrast, IMO-8400 treatment had no effects on control SU-DHL-6 cells in vitro or in vivo. Our studies show that IMO-8400 inhibits oncogenic MYD88 L265P-mediated cell survival and provides a novel approach for treatment of patients with this mutation. A Phase 1/2 trial of IMO-8400 in patients with WM is now open for enrollment. Citation Format: Lakshmi Bhagat, Daqing Wang, Weiwen Jiang, Sudhir Agrawal. IMO-8400, a selective antagonist of TLRs 7, 8 and 9, inhibits MYD88 L265P mutation-driven signaling and cell survival: A potential novel approach for treatment of B-cell lymphomas harboring MYD88 L265P mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2014-2570

Design of synthetic oligoribonucleotide-based agonists of Toll-like receptor 3 and their immune response profiles in vitro and in vivo

Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment. We report here the results of studies of dsORNs containing varying lengths and compositions of alignment and oligo I/oligo C segments. dsORNs of 50-mer length with a 15-mer alignment segment and a 35-mer oligo I/oligo C segment form stable duplexes under physiological conditions and induce TLR3-mediated immune responses. dsORNs activated the IRF3 signaling pathway in J774 cells, induced production of cytokines, including IFN-β, IFN-α, IP-10, IL-12 and IL-6, in murine and human cell-based assays and also induced multiple cytokines following systemic administration in mice and non-human primates.

Abstract 5652: Translational evidence of reactivated innate and adaptive immunity with intratumoral IMO-2125 in combination with systemic checkpoint inhibitors from a Phase I/II study in patients with anti-PD-1 refractory metastatic melanoma

Background: While checkpoint inhibitor (CPI) therapy has transformed metastatic melanoma (MM) treatment, many patients remain refractory. We reasoned that combining CPI with an agent that activates antigen presenting cells and improves T-cell priming may result in improved response. Our approach is to modulate the tumor microenvironment through image-guided intratumoral (i.t.) injection of the TLR9 agonist, IMO-2125, in combination with either ipilimumab (ipi) or pembrolizumab (pembro). We hypothesize that this will result in dendritic cell (DC) activation and induction of tumor-specific CD8+T-cells which will synergize with ipilimumab or pembrolizumab to overcome immune-escape. Based on this rationale we initiated a phase I/II clinical trial. Study Design/Methods: Adults with refractory MM that have had prior PD-1 blockade therapy (with or without a BRAF inhibitor) are eligible. IMO-2125, in doses escalating from 4mg to 32mg, is given i.t. weeks 1, 2, 3, 5, 8, and 11 along with standard doses of ipilimumab or pembrolizumab. Primary endpoints are safety, tumor response, and PK. Multiple biopsies are obtained in both the injected and distant tumor pre- and on-treatment. Immune analyses include DC subsets and their activation status as well as T cell activation, function and proliferation. T-cell repertoire diversity is evaluated by high-throughput CDR3 sequencing and changes in gene expression signatures are assessed by nanoString. Changes in circulating cytokines are also being assessed during therapy. Results: Enrollment is proceeding on both the IMO-ipilimumab and IMO-pembrolizumab dose-escalation arms of the trial. Safety is acceptable with no DLT recorded to date. Durable clinical responses (including 1 CR) have been observed with IMO-ipilimumab in patients who were refractory to PD-1 inhibitor. Fresh tumor biopsies show maturation (upregulation of HLA-DR) of the myeloid DC1 subset (CD1c+CD303-), upregulation of PD-L1 by malignant cells, as well as an IFNα response gene signature in the IMO-2125 injected tumor lesion 24 hrs post-treatment compared to pre-treatment biopsy. On-treatment biopsy results show a higher expression of CD56+ and Ki67+ effector CD8+ T-cells in responding patients. Initial CDR3 sequencing demonstrates increased diversity in the injected lesions of all patients assessed and expansion of top clones present in the distant lesion in a responding patient. Plasma analysis showed an increase in IFNγ levels in the plasma of responding patients. Conclusions: These results demonstrate that IMO-2125 with a checkpoint inhibitor is a viable strategy to revive the immune response in tumors that are refractory to PD-1 inhibitors. Further clinical evaluation of both the IMO-ipilimumab and IMO-pembrolizumab combination is planned in a Phase 2 expansion of the current trial. Citation Format: Cara Haymaker, Marc Uemura, Ravi Murthy, Marihella James, Daqing Wang, Julie Brevard, Suzanne Swann, James Geib, Mark Cornfeld, Srinivas Chunduru, Sudhir Agrawal, Cassian Yee, Jennifer Wargo, Rodabe Amaria, Sapna Patel, Hussein Tawbi, Isabella Glitza, Scott Woodman, Wen-Jen Hwu, Michael A. Davies, Patrick Hwu, Willem Overwjik, Chantale Bernatchez, Adi Diab. Translational evidence of reactivated innate and adaptive immunity with intratumoral IMO-2125 in combination with systemic checkpoint inhibitors from a Phase I/II study in patients with anti-PD-1 refractory metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5652. doi:10.1158/1538-7445.AM2017-5652