Development of resiquimod-loaded modified PLA-based nanoparticles for cancer immunotherapy: A kinetic study

Resiquimod (R848), a member of the imidazoquinoline family, is a Toll-like receptor 7/8 agonist with high potency for cancer immunotherapy. However, tolerance induction and adverse effects limit its development as a drug. Encapsulation in a polymer matrix can circumvent these limitations, as shown in our formerly published approach where R848 was loaded into polylactic acid (PLA)-based nanoparticles (NP). Although the results were encouraging, low rates of encapsulation and rapid release of the drug were observed. In this study, we present a new strategy using mixed NP from modified linear PLA in order to improve the encapsulation and modulate the release profile of R848. Modified PLA polymers were designed and synthesized by microwave-assisted ring opening polymerization of d,l-lactide, using polyethylene glycol as initiator to increase the hydrophilic properties of the polymer or linear saturated aliphatic chains (C8 or C20) to increase the affinity with hydrophobic R848. NP were prepared by solvent evaporation method, leading to particles of 205–288 nm loaded with either R848 or DiO as a tracking agent. The release profile showed longer retention of R848 at both neutral and acidic pH for NP from grafted polymers. Upon exposure to phagocytic immune cells, NP were actively taken up by the cells and no impact on cell viability was observed, independently of the constitutive polymer. All R848-loaded NP activated macrophages to secrete interleukin-6, demonstrating that the drug cargo was immunologically active. Importantly, macrophage activation by NP- delivered R848 was slower than with free R848, in accordance with the in vitro release profiles. Thus, NP prepared from modified PLA polymers showed no signs of toxicity to immune cells and efficiently delivered their immunoactive cargo in a delayed manner. This delivery strategy may enhance the efficacy and safety of small-molecule immunostimulants

Polymer-based nanoparticles loaded with a TLR7 ligand to target the lymph node for immunostimulation

Small-molecule agonists for the Toll-like receptors (TLR) 7 and 8 are effective for the immunotherapy of skin cancer when used as topical agents. Their systemic use has however been largely unsuccessful due to dose-limiting toxicity. We propose a polymer-based nanodelivery system to target resiquimod, a TLR7 ligand, to the lymph node in order to focus the immunostimulatory activity and to prevent a generalized inflammatory response. We demonstrate successful encapsulation of resiquimod in methoxypoly(ethylene glycol)-b-poly(DL-lactic acid) (mPEG-PLA) and mixed poly(DL- lactic-co-glycolic acid) (PLGA)/mPEG-PLA nanoparticles. We show that these particles are taken up mainly by dendritic cells and macrophages, which are the prime initiators of anticancer immune responses. Nanoparticles loaded with resiquimod activate these cells, demonstrating the availability of the immune-stimulating cargo. The unloaded particles are non-inflammatory and do not have cytotoxic activity on immune cells. Following subcutaneous injection in mice, mPEG-PLA and PLGA/mPEG-PLA nanoparticles are detected in dendritic cells and macrophages in the draining lymph nodes, demonstrating the targeting potential of these particles. Thus, polymer-based nanoparticles represent a promising delivery system that allows lymph node targeting for small-molecule TLR7 agonists in the context of systemic cancer immunotherapy

A Prokaryotic S1P Lyase Degrades Extracellular S1P In Vitro and In Vivo: Implication for Treating Hyperproliferative Disorders

Sphingosine-1-phosphate (S1P) regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7) and colon (HCT 116) carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling

Modern and fossil non-pollen palynomorphs from the Basque mountains (western Pyrenees, France): the use of coprophilous fungi to reconstruct pastoral activity

International audienceThis paper presents results from a modern dataset of non-pollen palynomorphs and its application to aca. 2,000 year peat record from the same area in the western Pyrenees (Basque Country, France). The modern dataset is composed of 35 surface samples (moss polsters) from a mountainous pasture-woodland landscape. Airborne fungal spores (ascospores and conidia), found dominant in the dataset, are linked to the degree of landscape openness and grazing pressure. The complete spectrum of 13 selected spore-types of dung-related Ascomycetes is positively linked with grazing pressure. However, different dung affinities between the spore-types have been identified. These are types clearly related to high grazing pressure and types with no or unclear dung indicative value. The modern dataset is used to aid interpretation of the local fossil pollen record as an independent 'proxy' to assess past pastoral dynamics. This study confirms the utility of modern nonpollen palynomorphs from terrestrial ecosystems in the reconstruction of historical local pastoral activities but also shows their limitation. It may be necessary to extend such study to wetland ecosystems and to investigate the spatial dimension of some fungal spores

Serine Palmitoyltransferase Subunit 3 and Metabolic Diseases

Sphingolipids (SL) are a class of chemically diverse lipids that have important structural and physiological functions in eukaryotic cells. SL entail a long chain base (LCB) as the common structural element, which is typically formed by the condensation of L-serine and long chain acyl-CoA. This condensation is the first and the rate-limiting step in the de novo SL synthesis and catalyzed by the enzyme serine palmitoyltransferase (SPT). Although palmitoyl-CoA is the preferred substrate, SPT can also metabolize other acyl-CoAs, thereby forming a variety of LCBs, which differ in structures and functions. The mammalian SPT enzyme is composed of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to a few specific tissues. The SPTLC1 subunit is essential and can associate with either SPTLC2 or SPTLC3 to form an active enzyme. Depending on the stoichiometry of the SPTLC2 and SPTLC3 subunits, the spectrum of SPT products varies. While SPTLC1 and SPTLC2 primarily form C18 and C20 LCBs, the combination of SPTLC1 and SPTLC3 produces a broader spectrum of LCBs. Genetic and population based studies have shown that SPTLC3 expression and function are associated with an altered plasma SL profile and an increased risk for cardio-metabolic diseases. Animal and in vitro studies showed that SPTLC3 might be involved in hepatic and cardiac pathology and could be a therapeutic target for these conditions.Here we present an overview of the current data on the role of SPTLC3 in normal and pathological conditions. Keywords: Metabolic diseases; Serine palmitoyltransferase; Serine palmitoyltransferase subunit 3 (Sptlc3); Sphingolipid biosynthesis; Sphingolipid

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/β, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/β constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported