Cross-Coupling Biarylation of Nitroaryl Chlorides Through High Speed Ball Milling

Solvent-free reaction using a high-speed ball milling technique has been applied to the classical Ullmann coupling reaction. Cross-coupling biarylation of several nitroaryl chlorides was achieved in good yields when performed in custom-made copper vials through continuous shaking without additional copper or solvent. Cross-coupling products were obtained almost pure and NMR-ready. These reactions were cleaner than solution phase coupling which require longer reaction time in high boiling solvents, and added catalysts as well as lengthy extraction and purification steps. Gram quantities of cross biaryl compounds have been synthesized with larger copper vials, a proof that this method can be used to reduce industrial waste and for sustainability

Potentiating Tumor Immunity Using Aptamer-Targeted RNAi to Render CD8+ T Cells Resistant to TGFβ Inhibition

Cancer is one of the leading causes of death in the world. Current treatments for cancer include chemotherapy, radiation therapy, surgery, immunotherapy and transplantation. While these treatments seem successful in some cases, they fail to provide long-term protection. Chemotherapy and radiation therapy lack specificity whereas immunotherapy, although it has the specificity is not very effective due to the ability of cancer cells to invade the host immune system. Over the years, many tumors have developed ways to prevent or escape their immune-based elimination. Therefore, counteracting tumor-associated (derived) immune suppression represents a potential platform for therapeutic intervention. A key mediator of immune suppression is the cytokine, transforming growth factor beta (TGFß) secreted by most tumor cells. The TGFß signaling pathway plays a vital and dual role in the progression of cancer. In the early stages of cancer, TGFß signaling exhibits tumor suppressive effects such as cell cycle arrest, apoptosis and inhibition of cell-cell adhesion. During later stages, TGFß promotes metastasis via evasion of immune surveillance, pro-metastatic cytokine secretion and production of autocrine mitogens. Due to the profound tumor promoting effects of TGFß, many clinical studies in cancer patients have targeted the TGFß pathway using antisense oligonucleotide, antibodies, soluble TGFßRII-Fc fusion decoys, or small molecule TGFßRI and Smad3 inhibitors. Owing to the broad range of roles TGFß plays in tissue homeostasis, these non-specific treatments elicit minimal clinical response and significant side effects. A likely reason, a common limitation of many cancer drugs, is that the physiological roles of TGFß in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFß may be sufficient to inhibit tumor growth. One approach to avoid the broad effects of TGFß therapy is to limit TGFß inhibition to tumor specific CD8+ T cells. Here we propose to develop a clinically feasible and broadly applicable method to render vaccine-activated CD8+ T cells resistant to TGFß inhibition using RNAi to inhibit key steps in the canonical TGFß signaling pathway. The siRNA is targeted to vaccine activated CD8+ T cells in the mouse by conjugation to a 4-1BB binding oligonucleotide (ODN) aptamer ligand. In this work, siRNAs targeting different mediators of the TGFß pathway, namely Smad4, TGFßRII and Foxp1, were conjugated to a murine 4-1BB aptamer. In vitro, all tested candidates knocked down their targets in a 4-1BB dependent delivery manner. When tested for function in vitro, only 4-1BB-Smad4 conjugate rendered T cells partially resistant to TGFß inhibition, and treatment of tumor bearing mice with systemically administered 4-1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFß to tumor-specific T cells will not interfere with its multiple physiological roles and hence reduce the risk of toxicity. TGFß mediated inhibition of tumor immunity is a major impediment in developing effective immune-based treatment for cancer. This approach serves as an adjunct therapy in combination with other forms of immune-based therapy applicable to patients. This study described 4-1BB aptamer-mediated delivery of siRNAs that knock-down key mediators of the canonical TGFß signaling pathway, namely Smad4, TGFßRII and Foxp1, in activated CD8+ T cells to render them insensitive to TGFß signaling, thereby enhancing vaccine-induced antitumor immune response.</p

Abstract 3147: Generation of exhaustion-resistant memory CD8 T-cells by simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells

Abstract CD8 memory T cells play a crucial role in controlling chronic infections and certain malignancies. It is possible to expand the population redirecting early-activated T-cells toward memory differentiation by altering intracellular signaling of mTORC1 pathway. Indeed, treatment with mTORC1 inhibitor rapamycin increased formation of memory cells as published previously; however, animals treated with the drug exhibited signs of immune suppression and rapamycin-generated CD8 memory T-cells failed to protect animals against melanoma challenge. We introduced highly efficient RNAi-based method to downmodulate mTORC1 signaling specifically in target cells. siRNA against raptor, a critical component of the signaling complex, was delivered in vivo into activated T-cells by conjugating to the 4-1BB-binding RNA aptamer resulting in expansion of CD8+ memory T-cells formation. Memory T-cells are generally resistant to immune suppression, yet in vivo, the population suffers from the gradual attrition mediated by PD-1 signalling. We developed the conjugate with two highly effective siRNAs against raptor and PD-1 linked to the same 4-1BB aptamer molecule. In vitro, both siRNAs efficiently dissociated from the conjugate inside target cells and simultaneously downregulated its respective mRNAs. In vivo, using OT-I adoptive transfer model, we demonstrated that treatment with 4-1BB-raptor-PD1 conjugates increased formation of memory CD8+ T-cells (as measured by absolute number of OT-1 cells 30 days after the treatment) in a rate similar to the 4-1BB-raptor conjugate. There was, however, qualitative difference between two populations, as CD8+ memory T-cells expanded with 4-1BB-raptor-PD1 conjugates expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression. Thus, we introduced a new method to generate memory cells with enhanced functional potential and currently exploring its therapeutic usefulness in various models. Citation Format: Alexey Berezhnoy, Agata Levay, Anugraha Rajagopalan, Yvonne Puplampu-Dove, Eli Gilboa. Generation of exhaustion-resistant memory CD8 T-cells by simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2015-3147</jats:p

Aptamer-Targeted Attenuation of IL-2 Signaling in CD8 + T Cells Enhances Antitumor Immunity

Immune responses elicited against cancer using existing therapies such as vaccines or immune stimulatory antibodies are often not curative. One way to potentiate antitumor immunity is to enhance the long-term persistence of anti-tumor CD8 T cells. Studies have shown that the persistence of activated CD8 T cells is negatively impacted by the strength of interleukin 2 (IL-2) signaling. Here, we used small interfering RNAs (siRNAs) against CD25 (IL-2Rα) to attenuate IL-2 signaling in CD8 T cells. The siRNAs were targeted to 4-1BB-expressing CD8 T cells by conjugation to a 4-1BB-binding oligonucleotide aptamer. Systemic administration of the 4-1BB aptamer-CD25 siRNA conjugate downregulated CD25 mRNA only in 4-1BB-expressing CD8 T cells promoting their differentiation into memory cells. Treatment with the 4-1BB aptamer-CD25 siRNA conjugates enhanced the antitumor response of a cellular vaccine or local radiation therapy. Indicative of the generality of this approach, 4-1BB aptamer-targeted delivery of an Axin-1 siRNA, a rate-limiting component of the β-catenin destruction complex, enhanced CD8 T cell memory development and antitumor activity. These findings show that aptamer-targeted siRNA therapeutics can be used to modulate the function of circulating CD8 T cells, skewing their development into long-lasting memory CD8 T cells, and thereby potentiating antitumor immunity

A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice

T cell immunoglobulin-3 (TIM-3) is a negative regulator of interferon-γ (IFN-γ) secreting CD4+ T cells and CD8+ T cytotoxic cells. Recent studies have highlighted the role of TIM-3 as an important mediator of CD8+ T cell exhaustion in the setting of chronic viral infections and cancer. In murine tumor models, antibody blockade of TIM-3 with anti-TIM-3 antibodies as monotherapy has no or minimal antitumor activity, suggesting that TIM-3 signaling exerts an accessory or amplifying effect in keeping immune responses in check. Using a combined bead and cell-based systemic evolution of ligands by exponential enrichment (SELEX) protocol, we have isolated nuclease-resistant oligonucleotide aptamer ligands that bind to cell-associated TIM-3 with high affinity and specificity. A trimeric form of the TIM-3 aptamer blocked the interaction of TIM-3 with Galectin-9, reduced cell death, and enhanced survival, proliferation, and cytokine secretion in vitro. In tumor-bearing mice, the aptamer delayed tumor growth as monotherapy and synergized with PD-1 antibody in prolonging the survival of the tumor-bearing mice. Both in vitro and in vivo, the trimeric aptamer displayed superior activity compared to the currently used RMT3-23 monoclonal antibody. This study suggests that multi-valent aptamers could represent an alternative platform to generate potent ligands to manipulate the function of TIM-3 and other immune modulatory receptors. In murine tumor immunotherapy models, antibody blockade of TIM-3 with anti-TIM-3 antibodies as monotherapy has no or minimal antitumor activity. In this issue of Molecular Therapy, Gefen et al. describe an oligonucleotide TIM-3-binding aptamer that was more effective than anti-TIM-3 antibody in vitro and in vivo

Potentiating tumor immunity using aptamer-targeted RNAi to render CD8 +

TGFβ secreted by tumor cells and/or tumor infiltrating stromal cells is a key mediator of tumor growth and immune suppression at the tumor site. Nonetheless, clinical trials in cancer patients targeting the TGFβ pathway exhibited at best a modest therapeutic benefit. A likely reason, a common limitation of many cancer drugs, is that the physiologic roles of TGFβ in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFβ may be sufficient to inhibit tumor growth. Here we describe an approach to render vaccine-activated CD8 + T cells transiently resistant to TGFβ inhibition using an siRNA against Smad4 to inhibit a key step in the canonical TGFβ signaling pathway. The siRNA was targeted to vaccine activated CD8 + T cells in the mouse by conjugation to a 4-1BB binding oligonucleotide (ODN) aptamer ligand (4-1BB-Smad4 conjugate). In vitro the 4-1BB-Smad4 conjugate rendered T cells partially resistant to TGFβ inhibition, and treatment of tumor bearing mice with systemically administered 4-1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFβ to tumor-specific T cells will not interfere with its multiple physiologic roles and hence reduce the risk of toxicity