Alcide Barberis

Intravesical Toll-like receptor 7 agonist R-837 : Optimization of its formulation in an orthotopic mouse model of bladder cancer

Objective:u2003 To study the immune response caused by the intravesical administration of the immunomodulator R‐837 in various formulations and to estimate its therapeutic potential for bladder cancer.

Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis

Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice. These findings highlight the potential of TMX-306, emphasizing its value in drug development for lung diseases, and particularly silicosis.

Inhibition of keratinocyte proliferation by phospholipid-conjugates of a TLR7 ligand in a Myc-induced hyperplastic actinic keratosis model in the absence of systemic side effects

BACKGROUNDnThe Toll-like receptor 7 (TLR7) activator imiquimod (IMQ) is safe and effective in treating actinic keratosis; however, an intermittent treatment regimen is necessary because of excessive local reactions.nnnOBJECTIVESnTo evaluate in vitro potency, pharmacodynamics/pharmacokinetics, toxicity and efficacy in vivo of the newly developed TLR7 ligand-phospholipid conjugate, TMX-202, in a gel formulation.nnnMATERIAL AND METHODSnThe effects of TMX-202 were assessed both in vitro on a murine macrophage cell line and in primary bone marrow-derived dendritic cells and in vivo on mice (C57BL/6-wild type, Myd88(-/-) and Tlr7(-/-)).nnnRESULTSnTMX-202 was more potent than IMQ in vitro using murine and human cells. In contrast, in vivo it showed less systemic pro-inflammatory activity and better safety than IMQ. Moreover, the TMX-202 gel formulation exhibited at least comparable efficacy to Aldara in a mouse model for skin proliferative diseases.nnnCONCLUSIONnTMX-202 is safe and efficacious without causing excessive adverse effects, suggesting that it may be an alternative to Aldara for the treatment of proliferative skin conditions.

Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist

Objectives: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. Methods: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. Results: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 – 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14+ and DC-SIGN+ DCs. Conclusion: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.

Whole blood targeting and activation of monocytes with TLR7 agonist formulated in cationic liposomes

Monocytes are one of the major phagocytic cells in the periphery that patrols the circulation for invading pathogens, and upon activation differentiates into dendritic cells, capable of migration to lymph nodes eliciting an adaptive immune response. Monocytes has for more than a decade been precursor cell for generation of autologous dendritic cell cancer vaccines, but clinical results have shown limiting benefits for the patients. One way of improving dendritic cell vaccines is targeting the monocytes in vivo with a suitable carrier of adjuvant together with tumor antigens, to boost monocyte differentiation towards tumor antigen presenting DCs. Here we report a novel monocyte targeting liposome technology capable of delivering TLR7 agonist to CD14 positive monocytes in fresh whole human blood. Liposomes with a positive surface charge were able to specifically target monocytes over lymphocytes and granulocytes, and showed association with 90-100 % of the monocytes. Formulation of the TLR7 agonist in monocyte targeting liposomes showed strong activation of the monocytes, with potent induction of proinflammatory cytokines, and differentiation into tissue inflammatory DCs, demonstrating that the liposomes are able to deliver compounds to the endosomes where TLR7 is present. The present monocyte targeting technology may be a promising approach for designing cancer vaccines with suitable adjuvants and cancer antigens.

Abstract 2568: Application of novel phospholipid conjugated Toll like receptor 7 ligands for cancer therapy by topical and systemic administration

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnIntroduction: Toll like receptor 7 (TLR7) activators are effective cancer immunotherapeutic drugs. However, the only clinically approved TLR7 ligand, imiquimod (IMQ), is absorbed into the systemic circulation inducing off-target adverse effects. To overcome this problem, we synthesized phospholipid-conjugated TLR7-specific activators. The objective of this project was to evaluate the safety and the anti-cancer activities of two TLR7 ligand-phospholipid conjugates, designated TMX-201 and TMX-202.nnProcedures: In vitro immune activities of TLR7 agonists were compared in human and mouse leukocytes. Anti-malignant effects were evaluated using Inv-MycERTAM transgenic model of pre-malignant keratinocyte proliferation and murine models of 4T1 breast cancer and B16 melanoma.nnResults: TMX-201 and TMX-202 were more potent cytokine inducers than IMQ in both mouse and human mononuclear cells. TMX-201 and TMX-202 showed less off-target binding compared to IMQ. The maximum tolerated doses of IMQ, TMX-201, and TMX-202 by i.v. administration were 15, 75 and 150 mg/kg, respectively. Topical administration of TMX-202 gel significantly reduced acanthosis in the Inv-mycERTAM model without inducing systemic cytokine production. Systemic administration of TMX-201 (4mg/kg) suppressed the growth of established subcutaneous B16 melanoma by 46%, without discernible adverse effects. Systemic or intra-tumor administration of TMX-201, in combination with anti-CTLA4 antibody, significantly suppressed lung metastasis formation in the 4T1 breast cancer model.nnConclusion: TMX-201 and TMX-202 displayed more potent immune stimulatory activity than IMQ with fewer off-target effects. These phospholipid-conjugated TLR7 agonists showed anti-cancer efficacy in three different mouse models. The new compounds may be useful topical and systemic therapeutic agents for cancer therapy, alone or in combination with checkpoint inhibitors.nnView this table:nnTable 1. nTMX-201 effect on lung metastases in the 4T1 breast cancer modelnnnnnnCitation Format: Dennis A. Carson, Tomoko Hayashi, Brian Crain, Shiyin Yao, Jeffrey Cheng, Nadia Passini, Roberto Maj, Emanuela Mura, Howard Cottam, Johanna Holldack, Alcide Barberis. Application of novel phospholipid conjugated Toll like receptor 7 ligands for cancer therapy by topical and systemic administration. [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 2568. doi:10.1158/1538-7445.AM2014-2568